@article {pmid42449099,
year = {2026},
author = {Huang, T and He, Y and Wang, X and Liu, Z and Qiao, E and Sun, T and Mei, J and Dai, S and Wang, Z and Feng, C and Cao, H and Wang, Y and Xia, Y and Liu, E and Guo, J and Lu, J},
title = {CRISPR/Cas9 screen identifies DCAF4 as a novel protector of hepatocellular carcinoma against brachytherapy via stress granule-dependent NRF2 activation.},
journal = {Cell death & disease},
volume = {17},
number = {1},
pages = {},
pmid = {42449099},
issn = {2041-4889},
support = {82572339//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82372066//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Humans ; *Carcinoma, Hepatocellular/genetics/radiotherapy/metabolism/pathology ; *Liver Neoplasms/radiotherapy/genetics/metabolism/pathology ; Animals ; *NF-E2-Related Factor 2/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; Kelch-Like ECH-Associated Protein 1/metabolism ; *Stress Granules/metabolism ; Oxidative Stress ; Cell Line, Tumor ; Mice ; Ubiquitin-Protein Ligases/metabolism ; Ubiquitination ; Mice, Nude ; },
abstract = {Hepatocellular carcinoma (HCC) cells sustain viability and radioresistance by actively countering oxidative stress. Understanding the mechanisms regulating reactive oxygen species (ROS) homeostasis is therefore crucial for developing novel therapies. Using integrated genome-wide CRISPR-Cas9 screening coupled with transcriptomic and metabolomic profiling, we identified DDB1 and CUL4-associated factor 4 (DCAF4) as an essential regulator of oxidative stress resistance in HCC. Mechanistically, DCAF4 functions as a CRL4 E3 ligase adapter that promotes KEAP1 ubiquitination and degradation. Notably, under oxidative stress, cytoplasmic stress granules (SGs) form a localized platform that facilitates the DCAF4-KEAP1 interaction, accelerating KEAP1 degradation and leading to NRF2 activation and upregulation of antioxidant genes. We further identified that the transcription factor XBP1 enhances DCAF4 expression. Targeting this axis, we performed computational screening to identify a small-molecule inhibitor that disrupts the DCAF4-KEAP1 interaction. This compound effectively enhanced brachytherapy (BT) sensitivity and inhibited tumor growth in preclinical HCC models.},
}

Humans
*Carcinoma, Hepatocellular/genetics/radiotherapy/metabolism/pathology
*Liver Neoplasms/radiotherapy/genetics/metabolism/pathology
Animals
*NF-E2-Related Factor 2/metabolism/genetics
*CRISPR-Cas Systems/genetics
Kelch-Like ECH-Associated Protein 1/metabolism
*Stress Granules/metabolism
Oxidative Stress
Cell Line, Tumor
Mice
Ubiquitin-Protein Ligases/metabolism
Ubiquitination
Mice, Nude

@article {pmid42745954,
year = {2026},
author = {Nguyen, ANT and Zhang, J and Zhang, S and Pitt, ME and Ganesamoorthy, D and Fritzlar, S and Chang, JJ and Londrigan, SL and Coin, LJM},
title = {Customizable host and viral transcript enrichment using CRISPR-Cas9 long-read sequencing for characterization of low-to-moderate abundance isoforms.},
journal = {NAR genomics and bioinformatics},
volume = {8},
number = {3},
pages = {lqag111},
pmid = {42745954},
issn = {2631-9268},
mesh = {*CRISPR-Cas Systems ; Humans ; Protein Isoforms/genetics ; High-Throughput Nucleotide Sequencing/methods ; *Sequence Analysis, RNA/methods ; },
abstract = {One of the main challenges of whole transcriptome sequencing is the difficulty in detecting and quantifying low-to-moderate abundance transcripts. Methods that address this are either complicated to scale or customize; long-range PCR is problematic to scale, and probe hybridization panels are expensive to customize. In this study, we developed an RNA-guided CRISPR-Cas9 nuclease-based enrichment strategy combined with long-read sequencing, which achieved up to 60-fold enrichment of the target. Our findings demonstrate that the CRISPR-Cas system is a highly effective method for customizable long-read sequencing of target transcripts, which preserves estimation of relative abundance.},
}

*CRISPR-Cas Systems
Humans
Protein Isoforms/genetics
High-Throughput Nucleotide Sequencing/methods
*Sequence Analysis, RNA/methods

@article {pmid42746213,
year = {2026},
author = {Xedzro, C and Shimamoto, T and Ahmed, AM and Yu, L and Sugawara, Y and Sugai, M and Shimamoto, T},
title = {Genomic Insights Into Multidrug-Resistant Foodborne Serratia liquefaciens Strains Carrying mcr-9 and Comparative Genomic Analysis of Novel Biosynthetic Gene Clusters.},
journal = {International journal of food science},
volume = {2026},
number = {},
pages = {5035164},
pmid = {42746213},
issn = {2314-5765},
abstract = {Serratia liquefaciens is an opportunistic nosocomial pathogen with a wide range of antibiotic resistance patterns. This study reports the characterization of the first mcr-9-positive S. liquefaciens strains, 35E-19E1 and CST-066, isolated from meat products in Japan. The strains were screened for the presence of β-lactamases, plasmid-mediated mobile colistin resistance (mcr) genes, and carbapenemase-encoding genes using PCR. Antimicrobial susceptibility was tested using the broth microdilution method. The strains exhibited multidrug resistance (MDR) phenotypes to third-generation cephalosporins, cephamycin, fosfomycin, and other clinically important antimicrobials. Genomic DNA sequencing showed that the genome sizes of CST-066 and 35E-19E1 are 5,529,704 and 5,261,506 bps, respectively. mcr-9 was identified on a chromosome within a genetic environment that included the two-component system qseBC, which plays a key role in the signaling network that triggers colistin resistance in Enterobacterales. Downstream genome analysis revealed a 1695-bp eptB-like kdo2-lipid phosphoethanolamine transferase, which is involved in intrinsic polymyxin resistance mechanisms in Serratia spp. The strain 35E-19E1 carries five CRISPR-Cas enzymes that are essential for adaptive immunity in bacteria, allowing defense against invading elements. Functional analysis using subsystem technology revealed that both strains possess subsystem features responsible for invasion and adhesion within the host biomes. Genome mining using antiSMASH and BAGL4 revealed various biosynthetic gene clusters, responsible for secondary metabolite synthesis. Notably, we identified novel gene clusters, mainly nonribosomal peptide synthetases, in both the strains, indicating their potential to produce bioactive compounds. Although the presence of mcr-9 in Serratia may not be of clinical significance because of natural resistance of the strain to polymyxins, we shed light on the genomic characteristics of this MDR pathogen and the potential spread of mcr-9 among other bacterial species. The emergence of mcr-9 in drug-resistant S. liquefaciens provides significant insights, underscoring the need for increased surveillance of this pathogen.},
}

@article {pmid42747042,
year = {2026},
author = {Stoker, C and Mustafa, Y and Liu, Y and Patrick, T and Perisse, IV and Polejaeva, IA},
title = {Generation of Genetically Engineered Embryos Using Gene Editing and Somatic Cell Nuclear Transfer for Production of Sheep Models of Human Disease.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {235},
pages = {},
doi = {10.3791/72709},
pmid = {42747042},
issn = {1940-087X},
mesh = {Animals ; *Nuclear Transfer Techniques ; Sheep/genetics/embryology ; Humans ; *Disease Models, Animal ; Female ; *Animals, Genetically Modified/genetics ; CRISPR-Cas Systems ; *Gene Editing/methods ; *Genetic Engineering/methods ; },
abstract = {Large animal models are valuable tools for investigating human disease. Sheep, pigs, and goats often better recapitulate the anatomy and physiology of human organs and the complexity of human disease, thereby enhancing their clinical relevance compared to rodents. CRISPR-Cas9 and somatic cell nuclear transfer (SCNT) enable the generation of large animal models with greater precision, versatility, and genetic uniformity. The primary benefit of this approach, compared with zygote microinjection, is the ability to confirm in vitro whether the desired genetic modification and potential off-target mutations are present in gene-edited cells prior to animal production. Moreover, SCNT eliminates the chance of genetic mosaicism, which frequently results from zygote microinjection. Here, we describe the generation of gene-edited ovine cells through non-homologous end-joining (NHEJ) and homology-directed repair (HDR), followed by the production of cloned embryos carrying the mutations of interest. Genetic modifications are introduced by transfecting cultured somatic cells, typically fetal fibroblasts, with the CRISPR-Cas9 system. Mutation efficiency in pooled cells is assessed by polymerase chain reaction (PCR) and Sanger sequencing of edited genes and analyzed using Tracking of Indels by DEcomposition (TIDE)/Tracking of Insertions, Deletions, and Recombination events (TIDER) software. Limiting dilution of the pooled cells is performed to obtain single-cell-derived colonies, which are screened by PCR and DNA sequencing of edited genes. Donor cells with the edit(s) of interest are subsequently expanded and used for the generation of embryos by SCNT. After limiting dilution and cell screening, 22/114 (19.3%) of colonies modified through NHEJ contained knockout (KO) mutations and 4/56 (7.1%) of colonies modified with HDR contained the F508del mutation. A total of 370 genetically modified embryos were created from four colonies. These methods are successfully used for precise gene editing in fetal fibroblasts and generation of genetically engineered embryos to produce ovine models of human disease.},
}

Animals
*Nuclear Transfer Techniques
Sheep/genetics/embryology
Humans
*Disease Models, Animal
Female
*Animals, Genetically Modified/genetics
CRISPR-Cas Systems
*Gene Editing/methods
*Genetic Engineering/methods

@article {pmid42747551,
year = {2026},
author = {Gopukumar, ST and Saha, M and Soni, TK and Alatwi, ES and Khan, GA and Asad, MR and Shamshad, S and Tadakod, S and Sharma, A and Das, U},
title = {CRISPR-Enabled functional genomics in hPSCs-derived neural models for autism spectrum disorder.},
journal = {Metabolic brain disease},
volume = {41},
number = {1},
pages = {},
pmid = {42747551},
issn = {1573-7365},
support = {DBTHRDPMU/JRF/BET-24/I/2024-25/376//Department of Biotechnology, Ministry of Science and Technology, India/ ; 24J/01/00130//Council of Scientific and Industrial Research, India/ ; 3/1/3/BRET-2024/HRD (L1)//Indian Council of Medical Research/ ; },
mesh = {Humans ; *Genomics/methods ; *Autism Spectrum Disorder/genetics/metabolism ; *Pluripotent Stem Cells/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Neurons/metabolism ; *Neural Stem Cells/metabolism ; *CRISPR-Cas Systems ; },
abstract = {Autism Spectrum Disorder (ASD) is a genetically heterogeneous neurodevelopmental condition in which hundreds of individually rare risk variants converge on a small number of shared biological pathways, including synaptic scaffolding, chromatin remodeling, excitation-inhibition balance, and cellular energy metabolism. Translating this genetic heterogeneity into mechanistic insight requires experimental systems capable of interrogating individual gene functions in human-relevant neural contexts at scale. CRISPR-enabled functional genomics in human pluripotent stem cell (hPSC)-derived neural models, spanning neural progenitors, cortical and inhibitory neurons, astrocytes, microglia, and brain organoids, provides precisely this capability. By integrating pooled perturbation screens with multimodal readouts including single-cell and spatial transcriptomics, chromatin accessibility profiling, proximity labeling proteomics, multi-electrode array electrophysiology, and metabolic flux analysis, these platforms enable systematic, causal mapping of ASD gene function at system resolution. Early applications have already revealed convergent mechanisms: BAF complex disruption expands the ventral progenitor pool and biases its fate toward oligodendrocyte and interneuron lineages; ADNP loss impairs microglial synaptic pruning through altered endocytic trafficking; and mTOR pathway dysregulation in PTEN- and TSC2-perturbed models links genetic risk directly to metabolic and mitochondrial dysfunction. Computational frameworks including MIMOSCA and SCEPTRE enable causal network reconstruction and pseudotime inference from these datasets, moving the field from gene lists toward pathway-level models of ASD pathobiology. Translational applications leverage isogenic iPSC panels and variant-level base and prime editing to stratify ASD variants by functional impact, informing gene therapy design for haploinsufficient targets such as CHD8 and SCN2A via AAV or antisense oligonucleotide delivery. Remaining challenges, including model developmental immaturity, batch variability, and the difficulty of modeling polygenic risk, are addressed by a roadmap integrating spatial perturbomics, AI-driven causal inference, and population-scale standardized biobanks. This review synthesizes the current state of CRISPR-based functional genomics in human stem cell neural models as a coherent experimental framework for converting ASD genetic associations into mechanistic understanding and therapeutic opportunity.},
}

Humans
*Genomics/methods
*Autism Spectrum Disorder/genetics/metabolism
*Pluripotent Stem Cells/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*Neurons/metabolism
*Neural Stem Cells/metabolism
*CRISPR-Cas Systems

@article {pmid42747567,
year = {2026},
author = {Hilkmann, M and Welsch, N and Felle, MF and Schweder, T and Appelbaum, M},
title = {An inducer-independent, single-plasmid CRISPR-Cas9 system for genome editing in Bacillus species.},
journal = {Applied microbiology and biotechnology},
volume = {110},
number = {1},
pages = {},
pmid = {42747567},
issn = {1432-0614},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Bacillus/genetics ; *Plasmids/genetics ; Promoter Regions, Genetic ; Genome, Bacterial ; Gene Deletion ; },
abstract = {Advances in molecular biology tools are essential for streamlining and accelerating genetic engineering of cells across industrial and academic applications. While CRISPR-Cas improves genome editing efficiency, current systems have limitations and are often host specific, which restricts their versatility. This study describes a versatile CRISPR-Cas9 system for genome editing in industrially relevant Bacillus species. By adapting the well-established pJOE8999 vector-based CRISPR-Cas9 genome editing system, we constructed an inducer-independent, broad-host-range genome editing system. It maintains the benefits of low toxicity to the target cell and the cloning host as well as the ease to use of a single-plasmid CRISPR-Cas9 system. We utilized the constitutive Sigma70-type promoter from the conserved veg gene of Bacillus, to develop and test the suitability of promoter variants of different strengths for Cas9 expression. Successful gene deletions in three different Bacillus species demonstrated the versatility of the modified system for this industrially important genus. This was further confirmed by the integration of a reporter gene fusion and the introduction of a single point mutation in the genome of Bacillus licheniformis. This one-step CRISPR-based transformation protocol developed in this study enables fast genome editing workflows with minimal hands-on time. KEY POINTS: • Editing and screening of promoter variants for balanced Cas9 expression in Bacillus. • Development of a versatile inducer-independent, single-plasmid CRISPR-Cas-based system. • Verification of the modified CRISPR-based system for genome editing in different Bacilli.},
}

*CRISPR-Cas Systems
*Gene Editing/methods
*Bacillus/genetics
*Plasmids/genetics
Promoter Regions, Genetic
Genome, Bacterial
Gene Deletion

@article {pmid42748215,
year = {2026},
author = {Gou, H and Chen, L and Eick, KL and Bao, D and Miner, TA and Tran, D and Gan, M and Zheng, W and Wang, S and Wu, Z and Huang, Z and Dial, CN and Seo, S and Mollin, S and Miller, MB and Zelazny, AM and Lyon, CJ and Fan, J and Wu, X and Yu, F and Zhu, X and Liu, Q and Ning, B and Hu, T},
title = {A rapid CRISPR-based nanodroplet assay enables direct clinical identification of mycobacteria species.},
journal = {Science translational medicine},
volume = {18},
number = {867},
pages = {eaef2648},
doi = {10.1126/scitranslmed.aef2648},
pmid = {42748215},
issn = {1946-6242},
mesh = {Humans ; *Mycobacterium/genetics/classification/isolation & purification ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Species Specificity ; Mycobacterium tuberculosis/genetics ; CRISPR-Cas Systems/genetics ; *Nanoparticles/chemistry ; },
abstract = {The global incidence and mortality of nontuberculous mycobacterial infections have risen sharply with population aging. In some regions, they are now surpassing Mycobacterium tuberculosis complex infections, imposing a substantial clinical and economic burden. Because nontuberous mycobacteria exhibit species-level heterogeneity and require prolonged culture for identification, their diagnosis remains slow and is frequently inaccurate. Here, we describe a multiplexed clustered regularly interspaced short palindromic repeats (CRISPR)-assisted nanodroplet differential identification (CANDI) diagnostic platform that integrates species-agnostic target amplification with species-specific CRISPR-associated protein 12a (Cas12a) detection in fluorescence-barcoded nanodroplets. By spatially compartmentalizing CRISPR reactions into color-encoded nanodroplets, CANDI overcomes the multiplexing limitations of conventional CRISPR diagnostics and enables simultaneous interrogation of multiple mycobacterial targets in a single assay. We designed a 16-plex panel that distinguishes 15 clinically relevant Mycobacterium species and subspecies. CANDI achieved high analytical sensitivity and accurate discrimination in samples containing coinfections with multiple species or subspecies. When applied to 230 clinical specimens, including sputum, tracheal aspirates, and other respiratory fluids, CANDI delivered subspecies-level results within 3.5 hours, achieving 97.08% sensitivity and 99.7% specificity relative to culture-based identification. By combining multiplexed, high-specificity CRISPR detection with scalable droplet-based engineering, CANDI has the potential to overcome the culture dependency of current diagnostics and enable species- and subspecies-level identification across the genetically complex Mycobacterium genus, offering a clinically adaptable framework for rapid, precision diagnosis of mycobacterial infections.},
}

Humans
*Mycobacterium/genetics/classification/isolation & purification
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Species Specificity
Mycobacterium tuberculosis/genetics
CRISPR-Cas Systems/genetics
*Nanoparticles/chemistry

@article {pmid42749264,
year = {2026},
author = {Rostami, S and Dos Santos, AM and Van, RS and Long, K and Shao, Y and Rajan, R},
title = {Active-site arginines differentially control Cas12a DNA cleavage and specificity.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {113560},
doi = {10.1016/j.jbc.2026.113560},
pmid = {42749264},
issn = {1083-351X},
abstract = {Cas12a is a CRISPR-Cas nuclease with biochemical features that make it useful for genome editing and nucleic acid diagnostics. However, its off-target and non-specific trans and CRISPR RNA-independent DNA cleavages can reduce the accuracy and limit applications requiring high fidelity. Here, we analyzed the role of two conserved arginine residues, R918 and R921, found in the RuvC active site pocket of Francisella novicida Cas12a. Through amino acid substitutions, biochemical assays, kinetic analysis, and computational study, we establish that a positive charge at 921 is required for CRISPR RNA-dependent DNA cleavage (cis cleavage), whereas R918 primarily enhances cleavage efficiency. Replacing R918 with lysine or alanine eliminates trans activity while retaining cis cleavage, whereas replacing R921 with lysine eliminates trans activity and replacing with alanine abolishes cis and trans cleavages. Furthermore, these changes significantly decrease RNA-independent cleavage and improve mismatch discrimination during cis cleavage, especially at PAM-distal sites. Structural analysis shows that R918 assists in the conversion of the lid covering the RuvC active site to an alpha helical form, while R921 stabilizes the DNA in the active site. Molecular dynamics simulations reveal that while R921 is critical in supporting the positioning of scissile phosphate, R918 is essential in maintaining catalytic-site organization through lid's conformational change as well as in positioning DNA through its role in stabilizing the active site framework. Together, our results highlight the importance of R918 and R921 in Cas12a's activity and the potential of modifying active pocket residues to reduce unwanted DNA cleavage while increasing on-target specificity.},
}

@article {pmid42749951,
year = {2026},
author = {Rahbari, M and Lohrasbi, R and Amiri-Yekta, A},
title = {From scissors to editors: how the evolution of precision is redefining therapeutic genome editing.},
journal = {Molecular genetics and genomics : MGG},
volume = {301},
number = {1},
pages = {},
pmid = {42749951},
issn = {1617-4623},
mesh = {*Gene Editing/methods/trends ; Humans ; *CRISPR-Cas Systems/genetics ; *Genetic Therapy/methods ; *Precision Medicine/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; Animals ; },
abstract = {Since its introduction as a genome-editing tool, CRISPR-based technology has undergone rapid refinement, with precision emerging as a central focus of development. Early CRISPR-Cas9 systems demonstrated unprecedented ease and efficiency in targeting specific DNA sequences, but concerns over off-target effects and variable editing outcomes limited their broader application. This review outlines the progression of CRISPR from its discovery in prokaryotes to its application as a versatile tool in precision medicine, where it supports targeted therapies for genetic disorders in various ways. Although technical challenges, including off-target editing and delivery inefficiencies, persist alongside ethical considerations of accessibility and long-term consequences, CRISPR's ongoing refinements and innovations reflect a clear trajectory toward greater specificity, safety, and predictability, positioning CRISPR as an increasingly precise platform for both fundamental research and therapeutic use.},
}

*Gene Editing/methods/trends
Humans
*CRISPR-Cas Systems/genetics
*Genetic Therapy/methods
*Precision Medicine/methods
Clustered Regularly Interspaced Short Palindromic Repeats
Animals

@article {pmid42752133,
year = {2026},
author = {Yoon, PH and Loi, KJ and Zhang, ZT and Docter, TA and Lopez, SC and Langeberg, CJ and Ur-Rehman, MM and Vohra, K and Zhou, Z and Esain-Garcia, I and Trinidad, MI and Shi, H and Boger, R and Wang, PY and Adler, BA and Brohawn, SG and Doudna, JA},
title = {A noncontiguous code for RNA-guided DNA recognition at the origin of CRISPR-Cas.},
journal = {Science (New York, N.Y.)},
volume = {393},
number = {6817},
pages = {1230-1235},
doi = {10.1126/science.aei0498},
pmid = {42752133},
issn = {1095-9203},
mesh = {*Bacteriophages/genetics ; *CRISPR-Cas Systems/genetics ; *DNA/chemistry/metabolism/genetics ; Evolution, Molecular ; *RNA, Guide, CRISPR-Cas Systems/genetics/chemistry/metabolism ; *RNA, Viral/genetics/chemistry ; *Viral Proteins/genetics/chemistry ; *Viral Interference ; Bacteria/virology ; },
abstract = {CRISPR-Cas provides RNA-mediated adaptive immunity, but how its first RNA-guided effector arose is unclear. In this study, we report the discovery of Viral Interference Programmable Repeat (VIPR) systems consisting of a Vipr protein ancestral to the earliest CRISPR-Cas effectors and VIPR RNAs (vrRNAs) comprising alternating GGY/NN motifs. Unlike canonical guide RNAs that pair with target nucleic acids through contiguous complementarity, vrRNAs recognize double-stranded DNA through a noncontiguous code in which the variable NN dinucleotides collectively specify a gapped target sequence. Natural vrRNA targets suggest that VIPR systems act against competing phages, and we demonstrate programmable phage defense by redirecting the complex for transcriptional repression. These results suggest that adaptive immunity originated from ancient warfare between viruses, revealing a previously unidentified logic for encoding information in sequence.},
}

*Bacteriophages/genetics
*CRISPR-Cas Systems/genetics
*DNA/chemistry/metabolism/genetics
Evolution, Molecular
*RNA, Guide, CRISPR-Cas Systems/genetics/chemistry/metabolism
*RNA, Viral/genetics/chemistry
*Viral Proteins/genetics/chemistry
*Viral Interference
Bacteria/virology

@article {pmid42752153,
year = {2026},
author = {Bravo, JPK},
title = {A viral origin for RNA-guided immunity.},
journal = {Science (New York, N.Y.)},
volume = {393},
number = {6817},
pages = {1187-1188},
doi = {10.1126/science.ael0758},
pmid = {42752153},
issn = {1095-9203},
mesh = {*Bacteria/genetics/virology ; *CRISPR-Cas Systems/genetics ; Escherichia coli/genetics/virology ; Viruses/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Ancient viral warfare could be at the root of modern class 1 CRISPR bacterial defense systems.},
}

*Bacteria/genetics/virology
*CRISPR-Cas Systems/genetics
Escherichia coli/genetics/virology
Viruses/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid42753192,
year = {2026},
author = {Jackson, KM and Morales, MM and Szewczyk, E and Homer, CM and Itogawa, AN and Brem, RB and Sil, A and Barker, BM},
title = {Protoplasting and Transformation Using CRISPR/Cas9 in Coccidioides.},
journal = {Current protocols},
volume = {6},
number = {9},
pages = {e70444},
pmid = {42753192},
issn = {2691-1299},
support = {1U19AI166798/AI/NIAID NIH HHS/United States ; U10AI166058/AI/NIAID NIH HHS/United States ; R21AI193607/AI/NIAID NIH HHS/United States ; },
mesh = {*Coccidioides/genetics ; *Protoplasts/metabolism ; *Transformation, Genetic ; *CRISPR-Cas Systems/genetics ; Gene Deletion ; Coccidioidomycosis/microbiology ; },
abstract = {Coccidioides posadasii and C. immitis are human fungal pathogens endemic to the American Southwest. C. posadasii and C. immitis are the causative agents of coccidioidomycosis, or Valley fever. They are pathogens of growing concern, as reported cases of coccidioidomycosis have increased 20-fold in the last two decades. Despite their importance as pathogens, Coccidioides spp. are understudied, especially compared to other human fungal pathogens with similar infectious burdens. The reasons Coccidioides spp. are understudied are multifactorial, including requirement of high biocontainment, technical difficulties in lab-based culture growth, and a historic lack of genetic tools. Previous methods of genetic manipulation have been technically challenging, time consuming, and inefficient. Here, we present protocols for designing gene deletion constructs, generating protoplasts from both genetically engineered biosafety level 2 (BSL2) and wildtype biosafety level 3 (BSL3) strains, and performing transformations with CRISPR/Cas9. The protoplasting protocol described here uses a cell wall digestion enzyme employed in the wine-making industry and results in high-quality protoplasts that have the potential to be used for applications beyond transformations. We also present a high-efficiency transformation method using CRISPR/Cas9. The protocols described here will allow for genetic manipulation of Coccidioides, using both BSL2 and BSL3 strains. This resource can be applied to expand research done in Coccidioides spp., build molecular tools, and expand overall knowledge of these important pathogens. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Construction of gene deletion construct Alternate Protocol 1: Construction of gene deletion construct Basic Protocol 2: Creation of protoplasts in BSL3 Alternate Protocol 2: Creation of protoplasts in BSL2 Basic Protocol 3: CRISPR/Cas9 transformation in BSL3 Alternate Protocol 3: CRISPR/Cas9 transformation in BSL2 Support Protocol: Passaging of mutants and PCR confirmation.},
}

*Coccidioides/genetics
*Protoplasts/metabolism
*Transformation, Genetic
*CRISPR-Cas Systems/genetics
Gene Deletion
Coccidioidomycosis/microbiology

@article {pmid42753721,
year = {2026},
author = {Schargel, RD and Chacon Machado, L and Kumaran, S and Thesier, JE and Krishnan, SS and Guarné, A and Peters, JE},
title = {De novo-engineered guide RNA-directed transposition with TnpB-family proteins.},
journal = {Molecular cell},
volume = {},
number = {},
pages = {},
pmid = {42753721},
issn = {1097-4164},
support = {R35 GM152260/GM/NIGMS NIH HHS/United States ; },
abstract = {Programmable DNA integration using CRISPR-associated transposase elements (CASTs) offers powerful capabilities for genome engineering. The large single effector Cas12k CAST examples evolved from a minimal TnpB nuclease protein. Here, we engineer a de novo RNA-guided transposition systems in bacteria, where the single guide RNA effector components are repurposed nuclease-dead TnpB-family proteins. These compact systems mediate high-efficiency guide-RNA-directed DNA insertion with preserved orientation control, target immunity, and release of a host factor requirement and can be paired with an exonuclease domain to mediate cut-and-paste transposition. In this engineered context, the TnpB derivatives show features not predicted from the original enzymes, suggesting untapped avenues for improvement. In parallel, we show that mutations at the TniQ-TnsC interface in the Cas12k CAST system selectively attenuate off-site insertions while enhancing on-site activity. These results establish how Cas12 proteins and antecedent TnpB proteins can be engineered for high performance and specificity with guide-RNA-directed systems.},
}

@article {pmid42754810,
year = {2026},
author = {Toma, L and Barbălată, T and Hărătău, JIC and Sanda, GM and Fuior, EV and Fenyo, IM and Niculescu, LŞ and Dăian, LM and Sasson, S and Sima, AV and Stancu, CS},
title = {CRISPR/dCas9-induced upregulation of endogenous apolipoprotein A1 and paraoxonase 1 genes reduces the aortic lipid deposits in apoE[-/-] mice.},
journal = {Molecular biomedicine},
volume = {7},
number = {1},
pages = {},
pmid = {42754810},
issn = {2662-8651},
support = {PN-III-P2-2.1-PED-2021-1929//UEFISCDI/ ; 10-CoEx/2026 PN-IV-P6-6.1-CoEx-2024-0029//UEFISCDI/ ; 760059/23.05.2023//PNRR Program/ ; GAR2023 Project No. 83//Academia Româna/ ; },
mesh = {Animals ; *Aryldialkylphosphatase/genetics/metabolism ; *Apolipoprotein A-I/genetics/metabolism ; Humans ; *Up-Regulation/genetics ; *Apolipoproteins E/deficiency/genetics ; *Aorta/metabolism/pathology ; Mice ; Atherosclerosis/genetics/metabolism/pathology ; Tumor Necrosis Factor-alpha ; Hepatocytes/metabolism ; *CRISPR-Cas Systems/genetics ; Mice, Knockout ; Male ; },
abstract = {High-density lipoproteins (HDL) are essential to alleviate the progression of atherosclerosis by mediating reverse-cholesterol transport, antioxidant and anti-inflammatory effects. We aimed to enhance the expression of endogenous HDL components, apolipoprotein A1 (APOA1) and antioxidant enzyme paraoxonase 1 (PON1), and to investigate their athero-protective effects. The CRISPR/dCas9 technology was used to activate the transcription of endogenous APOA1/PON1 in human hepatocytes (Huh7 line) and Apoa1/Pon1 in apoE[-/-] mice. The expression of APOA1/PON1 genes was successfully upregulated in hepatocytes, and their proteins were secreted in the culture medium in the presence/absence of tumor necrosis factor-α (TNFα). APOA1-rich Huh7-derived conditioned medium exerted antioxidant and anti-inflammatory effects in TNFα-activated EA.hy926 endothelial cells. A single dose of the CRISPR/dCas9 plasmids i.v. injected in apoE[-/-] mice increased the expression of hepatic Apoa1/Pon1 and their serum levels up to four weeks. FPLC analysis showed that increased serum APOA1 was distributed between HDL, LDL, and in lipid-free form. These mice also exhibited high levels of hepatic, gallbladder and feces cholesterol, in part due to the upregulation of hepatic scavenger receptor class-B1, cholesterol 7-alpha-hydroxylase, and ATP-binding cassette sub-family-G-member-8 transporter. In apoE[-/-] mice with upregulated Apoa1/Pon1, no increased inflammatory stress or innate immune activation were detected, while lipid peroxides were decreased in PON1 mice. Of major interest, the area of aortic lipid deposits was halved in the treated mice. Our findings demonstrate the successful upregulation of endogenous Apoa1/Pon1 in apoE[-/-] mice by using the CRISPR/dCas9 system, and highlight new mechanisms for APO1/PON1 anti-atherosclerotic action, explaining the reduction of aortic lipid deposits.},
}

Animals
*Aryldialkylphosphatase/genetics/metabolism
*Apolipoprotein A-I/genetics/metabolism
Humans
*Up-Regulation/genetics
*Apolipoproteins E/deficiency/genetics
*Aorta/metabolism/pathology
Mice
Atherosclerosis/genetics/metabolism/pathology
Tumor Necrosis Factor-alpha
Hepatocytes/metabolism
*CRISPR-Cas Systems/genetics
Mice, Knockout
Male

@article {pmid42754893,
year = {2026},
author = {Höijer, I and van Schendel, R and Emmanouilidou, A and Östlund, R and Bunikis, I and Tijsterman, M and den Hoed, M and Ameur, A},
title = {Accurate characterization of CRISPR-Cas9 genome editing outcomes and mosaicism with near-perfect long reads.},
journal = {Genome medicine},
volume = {18},
number = {1},
pages = {},
pmid = {42754893},
issn = {1756-994X},
mesh = {*Mosaicism ; Animals ; Zebrafish/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; High-Throughput Nucleotide Sequencing ; Genome ; },
abstract = {BACKGROUND: Genetic mosaicism is a well-recognized consequence of CRISPR-Cas9 genome editing, yet its characterization remains challenging, especially when it involves low-frequency structural variants. A comprehensive analysis of mosaicism requires deep and unbiased sequencing of the target loci, with accurate single-molecule reads.

METHODS: We performed amplification-free PureTarget PacBio sequencing to investigate CRISPR-Cas9 outcomes at on-target and off-target sites in genome edited zebrafish and their offspring. CRISPR-Cas9 genome editing was performed by micro-injection in fertilized eggs at the single-cell stage.

RESULTS: Thirty samples from pooled larvae and individual zebrafish were successfully sequenced, resulting in > 1100x average target coverage. The PacBio reads reached an exceptional accuracy (QV39) over the target regions, with every read originating from a unique DNA molecule. The two haplotypes of the target loci displayed a balanced depth of coverage, while long-range PCR of the same samples resulted in skewed data. Further analysis of the PureTarget data revealed widespread genetic mosaicism in individual founder (F0) fish, with up to 18 distinct on-target events and 11 off-target events present in a single adult founder. Several CRISPR-Cas9 editing outcomes, including large structural variants and off-target mutations, were inherited to the F1 generation. Notably, as many as seven unique editing events were found among sibling F1 juvenile offspring derived from a single founder pair, thereby confirming the presence of genetic mosaicism in germ cells of founder zebrafish. This implies that some consequences of CRISPR-Cas9 editing may emerge only in the second generation. We also analyzed DNA methylation signals in the PureTarget data but did not observe altered 5mC CpG levels in genome edited samples.

CONCLUSIONS: PureTarget enables efficient, accurate, and unbiased profiling of genetic mosaicism and DNA methylation at pre-defined genomic regions. Our results show that CRISPR-Cas9-induced mosaicism is widespread and represents an important factor to consider in genome editing experiments.},
}

*Mosaicism
Animals
Zebrafish/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
High-Throughput Nucleotide Sequencing
Genome

@article {pmid42755327,
year = {2026},
author = {Wong Castro, DA and Morocho Perugachi, AC and Fuel Herrera, MO},
title = {CRISPR-Based Mediated Reactivation of Fetal Hemoglobin as a Therapeutic Strategy for Hemoglobinopathies: Evidence from Preclinical to Clinical Trials in Sickle Cell Disease and β-Thalassemia.},
journal = {Hemoglobin},
volume = {},
number = {},
pages = {1-10},
doi = {10.1080/03630269.2026.2729189},
pmid = {42755327},
issn = {1532-432X},
abstract = {β-Hemoglobinopathies, including sickle cell disease (SCD) and β-thalassemia, are inherited disorders caused by mutations in the β-globin gene (HBB), leading to defective production of adult hemoglobin (HbA), vaso-occlusive crises, and rapid destruction of erythrocytes as they leave the bone marrow, resulting in hemolytic anemia. In recent years, CRISPR-based genome-editing technologies have emerged as promising therapeutic strategies to reactivate fetal hemoglobin (HbF) expression by targeting key regulatory elements, including the BCL11A enhancer and the HBG1/HBG2 promoters. This review aimed to synthesize available scientific evidence from PubMed, Scopus, Web of Science, and ClinicalTrials.gov on preclinical and clinical studies evaluating CRISPR-based genome-editing approaches for the treatment of SCD and β-thalassemia. This review was conducted and reported in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA 2020) Statement. A total of 247 records were identified; 20 studies met the inclusion criteria and were included in the qualitative synthesis. The included studies investigated various CRISPR-based platforms, including nuclease-mediated editing and base-editing strategies, to reactivate HbF expression. The studies reported durable increases in HbF levels, with therapeutic effects persisting for up to 22 months after treatment. Clinically, these outcomes were associated with the elimination or marked reduction of vaso-occlusive crises in patients with SCD and with transfusion independence in patients with β-thalassemia. Overall, the available evidence suggests that CRISPR-based HbF reactivation is a promising therapeutic approach for β-hemoglobinopathies, although long-term efficacy and safety data remain necessary.},
}

@article {pmid42755359,
year = {2026},
author = {Martella, A and Matreyek, KA and Fisher, DI},
title = {Large serine recombinase-mediated gene insertion for high-throughput screens: advantages, design principles, and applications.},
journal = {Nucleic acids research},
volume = {54},
number = {17},
pages = {},
pmid = {42755359},
issn = {1362-4962},
mesh = {Humans ; Animals ; *DNA Nucleotidyltransferases/metabolism/genetics ; *Mutagenesis, Insertional/methods ; *High-Throughput Screening Assays/methods ; *Recombinases/metabolism/genetics ; CRISPR-Cas Systems ; *Gene Editing/methods ; },
abstract = {High-throughput functional assays, such as multiplexed assays of variant effect (MAVE), increasingly demand stable, precise integration of large DNA libraries into mammalian genomes. While CRISPR-based technologies excel at localized, small-scale edits, they are constrained by payload size limits, heterogeneous editing outcomes, and, depending on the specific modality and repair pathway utilized, potential variability in junction fidelity during multikilobase insertions. In this review, we highlight large serine recombinases (LSRs) as highly efficient, single-enzyme alternatives for unidirectional, site-specific integration of large payloads with deterministic junctions. We survey targeted genomic integration strategies and detail best practices for implementing recombinase-based landing pad architectures. By enforcing single-copy, orientation-fixed integration at defined loci, landing pads decouple variant delivery from local chromatin effects to ensure the uniform, isogenic expression required for quantitative genotype-phenotype mapping. We further outline scalable applications of LSR-mediated integration across pooled and arrayed MAVE, CRISPR screens, and precise gene expression tuning. Finally, we assess current technological bottlenecks, particularly large donor delivery and the requisite pre-installation of canonical att recognition sites, while exploring emerging innovations in virus-like particle delivery, one-step CRISPR-recombinase systems, and computationally engineered programmable recombinases that promise to bypass these limitations and broaden mammalian genome engineering.},
}

Humans
Animals
*DNA Nucleotidyltransferases/metabolism/genetics
*Mutagenesis, Insertional/methods
*High-Throughput Screening Assays/methods
*Recombinases/metabolism/genetics
CRISPR-Cas Systems
*Gene Editing/methods

@article {pmid42758019,
year = {2026},
author = {Boneza, MM and Keller, T and Mostek, J and Smanski, MJ},
title = {Strain-specific outcomes of cytosine-base editing in Streptomyces.},
journal = {Journal of bacteriology},
volume = {},
number = {},
pages = {e0024126},
doi = {10.1128/jb.00241-26},
pmid = {42758019},
issn = {1098-5530},
abstract = {UNLABELLED: The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system has facilitated gene editing of different organisms. Specifically, programmable base editing enables stable conversion of a single nucleotide to another nucleotide without causing DNA double-strand breaks. Cas9-derived base editors, including adenine and cytidine base editors, catalyze the formation of transition mutations with high efficiency. The third-generation cytidine base editors comprise a nickase-Cas9 fused to a cytidine deaminase and uracil DNA glycosylase inhibitor to enable the transition from C:G to T:A. We observed that in certain Streptomyces spp., this cytidine base editor produced C:G to G:C transversions at a surprisingly high rate of 44, while in other strains, it yielded the expected C:G to T:A transition mutations. There was also a notable timing difference for base editing between distinct strains. Bioinformatics analysis revealed differences in DNA mismatch repair and nucleotide excision repair pathways, including the presence of uvrD helicase gene only in the C:G to G:C transversion strain. Expression of uvrD in the C:G to T:A transition strain led to higher rates of C:G to G:C transversions. These discoveries will aid in the development of efficient C:G to G:C base editors.

IMPORTANCE: Base editors are useful tools that allow genetic engineers to precisely change single bases in an organism's genome. Most base editors catalyze transition mutations (e.g., A-to-G or C to-T). Transversion mutations, which convert a purine base to a pyrimidine base, have been described but operate at much lower efficiency. Here, we describe a surprising discovery that a transition base editor produced high-efficiency transversion mutations in a species of Streptomyces. This information was used to engineer a high-efficiency transversion base editor.},
}

@article {pmid42758354,
year = {2026},
author = {Islam, MN and Islam, MM and Feng, H and Bellah, SF and Zaman Miah, MM and Hossain, MS},
title = {CRISPR-Cas9 genome editing: technological advances, delivery strategies and precision engineering of primary cells for therapeutic application in genetic disorders and diabetes mellitus.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42758354},
issn = {1573-4978},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Diabetes Mellitus/genetics/therapy ; Animals ; *Genetic Diseases, Inborn/therapy/genetics ; *Genetic Therapy/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {The CRISPR-Cas9 system has revolutionized modern life sciences, driving a paradigm shift in biomedical research and becoming an indispensable tool in molecular biology due to its remarkable precision, efficiency, and simplicity. Originating from a bacterial adaptive immune mechanism, CRISPR-Cas9 has evolved rapidly, providing a versatile framework for manipulating genetic material and addressing a wide spectrum of human diseases. Recent progress includes the discovery of novel Cas orthologs and the rational engineering of Cas9 variants to enhance editing fidelity, broaden target range, and minimize off-target effects. Structural and functional optimization of single-guide RNAs (sgRNAs) has further improved target binding affinity, stability, and Cas9-sgRNA complex formation, thereby increasing overall editing performance. The development of high-fidelity Cas9 derivatives and next-generation platforms such as base editors and prime editors has enabled precise single-nucleotide substitutions and small insertions or deletions without generating double-stranded DNA breaks. Advanced delivery systems-including viral vectors, lipid nanoparticles, and ribonucleoprotein electroporation-have facilitated efficient CRISPR-mediated editing across in vitro, ex vivo, and in vivo models. Remarkable therapeutic milestones have been achieved in treating monogenic disorders such as sickle cell disease, β-thalassemia, and cystic fibrosis, where long-term clinical benefits have been documented. Furthermore, CRISPR-Cas9 technology is redefining diabetes research by enabling precise modeling of disease mechanisms, uncovering molecular pathways involved in glucose homeostasis, and opening new avenues for cellular and gene-based therapies. This review highlights recent advances in CRISPR-Cas9-mediated genome editing, emphasizing breakthroughs in primary cell editing and the development of translational models for genetic diseases and diabetes mellitus.},
}

Humans
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Diabetes Mellitus/genetics/therapy
Animals
*Genetic Diseases, Inborn/therapy/genetics
*Genetic Therapy/methods
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid42758820,
year = {2026},
author = {Diepstraten, ST and Deng, Y and Potts, MA and Heidersbach, A and König, C and Dorighi, KM and Tai, L and Kueh, AJ and Whelan, L and Chang, C and Brown, F and Kelly, GL and Fortin, JP and Haley, B and La Marca, JE and Herold, MJ},
title = {A high-density CRISPR activation platform for mapping cancer dependencies and resistance pathways ex vivo and in vivo.},
journal = {Science advances},
volume = {12},
number = {38},
pages = {eaec0722},
pmid = {42758820},
issn = {2375-2548},
mesh = {Animals ; Mice ; *Drug Resistance, Neoplasm/genetics ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Expression Regulation, Neoplastic ; Humans ; Lymphoma/genetics/drug therapy ; Proto-Oncogene Proteins c-myc/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Neoplasms/genetics ; },
abstract = {CRISPR activation (CRISPRa) enables precise up-regulation of gene expression for ex vivo and in vivo applications. However, a lack of scalable, high-coverage tools has limited comprehensive genetic screening in murine models. Here, we introduce Partita, a next-generation mouse whole-genome CRISPRa sgRNA platform, designed for unparalleled efficiency in gene activation studies. Partita uses a high-density targeting strategy, deploying 10 sgRNAs per transcriptional start site, structured into five gene class-specific sublibraries to maximize transcriptional induction. To demonstrate the capabilities of Partita, we performed a series of large-scale screens: an in vitro enrichment/depletion screen, whole-genome CRISPRa screens in a double-hit lymphoma model to uncover resistance factors to proapoptotic drugs (venetoclax, nutlin-3a, and etoposide) and an in vivo screen to identify accelerators of MYC-driven lymphomagenesis. Each experiment revealed both expected and unexpected regulators, with high validation rates. By enabling robust gain-of-function screening, Partita unlocks new avenues for functional genomics and expands the toolkit for discovering key drivers of biological processes across diverse research fields.},
}

Animals
Mice
*Drug Resistance, Neoplasm/genetics
*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
Gene Expression Regulation, Neoplastic
Humans
Lymphoma/genetics/drug therapy
Proto-Oncogene Proteins c-myc/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
*Neoplasms/genetics

@article {pmid42760354,
year = {2026},
author = {Jáquez-Gutiérrez, M and Bretones, S and Martin-Vásquez, C and Fonseca, R and Aguiar, A and Pineda, B and Lozano, R and Moreno, V and Yuste-Lisbona, FJ and Atarés, A},
title = {Tomato DOR encodes a signal peptide peptidase-like protein required for adventitious organogenesis and rooting.},
journal = {Plant cell reports},
volume = {45},
number = {10},
pages = {},
pmid = {42760354},
issn = {1432-203X},
support = {PID2023-151867OB-C32//MICIU/AEI/10.13039/501100011033 and ERDF/EU/ ; PID2023-151867OB-C31//MICIU/AEI/10.13039/501100011033 and ERDF/EU/ ; CIGRIS/2022/116//Generalitat Valenciana and the European Social Fund (ESF+)/ ; },
mesh = {*Solanum lycopersicum/genetics/growth & development/enzymology ; *Plant Roots/growth & development/genetics/metabolism ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; *Organogenesis, Plant/genetics ; Mutation/genetics ; Phenotype ; Plants, Genetically Modified ; },
abstract = {The identification of the tomato DOR gene that is essential for adventitious organogenesis provides a genetic target for improving regeneration efficiency and root system performance in tissue culture and plant breeding. Plant rooting and adventitious organogenesis represent major bottlenecks in the application of plant tissue culture techniques. In this study, we characterize the dor mutant (defective in organogenesis and rooting) identified in our collection of tomato T-DNA lines. This mutant exhibits normal callus proliferation but fails to differentiate adventitious buds. Additionally, it displays underdeveloped embryonic roots, and its adventitious roots derived from various explants are also altered. Grafting experiments revealed compromised in vivo development primarily due to its abnormal root system. Upon identifying an allelic mutant in another tomato line, we observed no co-segregation between a T-DNA insert and the phenotype in either of the identified allelic mutants. Through mapping-by-sequencing, we identified Solyc12g098670 as the gene responsible for this mutation, which is homologous to SIGNAL PEPTIDE PEPTIDASE-LIKE (SPPL) genes from Arabidopsis thaliana, particularly SPPL3 and SPPL5. The expression pattern of DOR in tomato is nearly ubiquitous, similar to SPPL3 in Arabidopsis, yet the regeneration and rooting of sppl3 Arabidopsis mutants resemble the wild-type. In both tomato and Solanum pennellii (Correll) D'Arcy, RNAi lines and plants edited by CRISPR/Cas exhibit a dor mutant phenotype. However, by increasing the expression level of DOR, the plants become indistinguishable from the wild-type in terms of in vitro and in vivo development. Overexpression of this gene in the mutant has enabled the regeneration of plants with a wild-type phenotype. These results demonstrate that DOR is the first member of the tomato SPPL gene family known to be associated with root development and adventitious organogenesis.},
}

*Solanum lycopersicum/genetics/growth & development/enzymology
*Plant Roots/growth & development/genetics/metabolism
*Plant Proteins/genetics/metabolism
Gene Expression Regulation, Plant
*Organogenesis, Plant/genetics
Mutation/genetics
Phenotype
Plants, Genetically Modified

@article {pmid42760426,
year = {2026},
author = {Sun, Y and Xu, W and Chen, K and Hong, X and Sun, X and Ma, W and Wang, X and Cao, Q and Xue, Z and Zhou, B and Zhang, Y and Liu, Z and Cui, Z and Wang, D and Dong, Z and Zhang, Y},
title = {The phosphate specific transport system protein PhoU modulates persister cell formation of Klebsiella pneumoniae.},
journal = {Molecular genetics and genomics : MGG},
volume = {301},
number = {1},
pages = {},
pmid = {42760426},
issn = {1617-4623},
support = {202410440038//2024 National College Student Innovation and Entrepreneurship Training Program/ ; 202410440009//2024 National College Student Innovation and Entrepreneurship Training Program/ ; QDZK112023003//Doctoral Projects of Lishui University/ ; },
mesh = {*Klebsiella pneumoniae/genetics/drug effects/metabolism ; Biofilms/drug effects/growth & development ; *Bacterial Proteins/genetics/metabolism ; Gene Expression Regulation, Bacterial/drug effects ; Anti-Bacterial Agents/pharmacology ; Phosphates/metabolism ; CRISPR-Cas Systems ; },
abstract = {The indiscriminate use of antibiotics has exacerbated the clinical challenge posed by Klebsiella pneumoniae, with persisters often evading antibiotic treatment. To investigate the mechanisms underlying persister formation, we identified the phosphate transporter gene phoU as differentially expressed during persister emergence and recovery. Using CRISPR-Cas9 gene editing, we created a ∆phoU knockout strain in K. pneumoniae ATCC 700603, along with its complemented (CphoU) and various overexpression strains (OE phoU, OE metE, and OE phoA). While growth rates and antibiotic susceptibility remained unchanged in the ∆phoU strain, its capacity to form persisters under levofloxacin and tobramycin stress was significantly reduced. Metabolomic and transcriptomic analyses linked phoU deletion to downregulation of metE (involved in methionine synthesis) and phoA (encoding alkaline phosphatase). ∆phoU also exhibited impaired biofilm formation and reduced extracellular polymeric substance (EPS) production compared to WT, CphoU, and the OE metE and OE phoA overexpression strains. Complementation of metE or phoA partially restored both biofilm formation and persister levels. Importantly, the reduction in persisters is primarily attributed to metabolic defects in planktonic cells-specifically, impaired methionine synthesis and stress response-rather than to reduced biofilm mass, which is considered a parallel phenotype. We conclude that PhoU promotes K. pneumoniae persistence by transcriptionally upregulating metE and phoA, thereby supporting the stress response capacity of planktonic cells. This study elucidates a novel pathway for persister formation and identifies PhoU and its downstream targets as potential therapeutic vulnerabilities.},
}

*Klebsiella pneumoniae/genetics/drug effects/metabolism
Biofilms/drug effects/growth & development
*Bacterial Proteins/genetics/metabolism
Gene Expression Regulation, Bacterial/drug effects
Anti-Bacterial Agents/pharmacology
Phosphates/metabolism
CRISPR-Cas Systems

@article {pmid42761838,
year = {2026},
author = {Sahaja, MSCG and Sahana, MSCG and Thottathil, R and Rekha, C and Priyada, P and Babu, VS},
title = {Graft Biology in the CRISPR Era: From Tissue Fusion to Genome Compatibility.},
journal = {Plant-environment interactions (Hoboken, N.J.)},
volume = {7},
number = {5},
pages = {e70208},
pmid = {42761838},
issn = {2575-6265},
abstract = {Plant lineage has traditionally constrained grafting compatibility, with monocots generally considered incompatible because of their dispersed vascular bundles and limited secondary growth. Recent studies have shown that embryonic grafting can establish successful graft unions in selected monocot systems by exploiting early developmental plasticity before anatomical constraints become fully established. Experimental evidence from cereals and orchids has demonstrated callus adhesion, vascular reconnection, and early tissue integration under controlled conditions, indicating that embryonic grafting represents a promising developmental approach distinct from conventional grafting. However, successful graft union formation does not necessarily ensure long-term functional integration. Current evidence indicates that distant grafts may exhibit developmental desynchronization, endoplasmic reticulum stress-associated reproductive defects, genomic dosage imbalance, and sterility, while reproducibility across taxa and translation beyond controlled environments remains poorly understood. Genome-editing studies have identified sterility-associated genes (ORF3/4/5, Ms1, and S-RNase), flowering regulators (Hd1 and FT homologs), and meristem-vascular identity genes (WUS, CUC/LOB, and MADS-box family members) as candidate targets for investigating mechanisms underlying compatibility and reproductive stability. However, the application of these molecular approaches to embryonic graft-derived systems remains largely unexplored. This review synthesizes current advances in embryonic grafting, discusses the molecular basis of graft compatibility and reproductive stability, and highlights key challenges and future research directions for integrating developmental biology with genome editing to improve graft success across wider taxonomic boundaries.},
}

@article {pmid42762688,
year = {2026},
author = {Osada, M and Aung, MS and Nishida, M and Kawamura, K and Ito, M and Urushibara, N and Kawaguchiya, M and Ohashi, N and Suzuki, S and Higuchi, T and Hirose, M and Kobayashi, N},
title = {Molecular epidemiology, genetic characteristics, and antimicrobial resistance of Staphylococcus capitis clinical isolates: First identification of the NRCS-A and proto-NRCS-A clones in Japan.},
journal = {Journal of infection and public health},
volume = {19},
number = {11},
pages = {103365},
doi = {10.1016/j.jiph.2026.103365},
pmid = {42762688},
issn = {1876-035X},
abstract = {BACKGROUND: A pathogenic clone of Staphylococcus capitis, NRCS-A, possesses specific virulence factors and shows broader antimicrobial resistance (AMR). The present study aimed to analyze the genetic characteristics and AMR profiles of S. capitis clinical isolates and determine the prevalence of the NRCS-A clone in Japan.

METHODS: S. capitis isolates from clinical specimens were analyzed for the presence of virulence factors and AMR-associated genes by PCR and sequencing. Genetic classification of S. capitis isolates was performed by an in-house multilocus sequence typing (MLST) based on four loci (arcC-rpoB-gap-tuf).

RESULTS: A total of 194 S. capitis isolates (44 subsp. capitis and 150 subsp. urealyticus) were collected for 7 months. mecA was detected in 33% (63/194) of isolates (only subsp. urealyticus), while the most common AMR determinants identified were fosSC (77%, n = 150) and blaZ (59%, n = 114). Virulence factors associated with the NRCS-A clone, nsr and tarJ, were found in 20% (n = 39) and 37% (n = 71) of isolates, respectively. Both nsr and tarJ were present in 13 isolates (7%), among which three isolates had the traits of the NRCS-A clone (SCCmec-V with CRISPR/Cas-IIIA; SCCcad/ars/cop in two isolates), whereas the remaining 10 isolates were considered presumptive proto-NRCS-A clone (nsr+, tarJ+, CRISPR-Cas-IIIA/SCCmec-negative). By the MLST scheme, all the S. capitis isolates were differentiated into 48 STs and six major clonal complexes (CCs). Isolates assigned to the NRCS-A clone were classified as ST13 or ST14 in CC1, which was a minor lineage of S. capitis subsp. urealyticus. Similarly, most isolates of proto-NRCS-A clone were grouped into CC1, with ST13 being dominant.

CONCLUSION: The present study first revealed the presence of NRCS-A and proto-NRCS-A clones among S. capitis clinical isolates in Japan. Careful monitoring and attention to these clones may be necessary in clinical settings.},
}

@article {pmid41208519,
year = {2026},
author = {Qiu, Y and Shimada, K and Ikawa, M},
title = {CRISPR/Cas9-mediated Genome-editing Reveals 10 Testis-enriched Genes and One Non-testis-enriched Gene are Dispensable for Male Fecundity in Mice.},
journal = {Andrology},
volume = {14},
number = {7},
pages = {2150-2159},
pmid = {41208519},
issn = {2047-2927},
support = {R01 HD088412/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; Male ; *Fertility/genetics ; *Testis/metabolism ; *CRISPR-Cas Systems ; Mice ; Mice, Knockout ; *Gene Editing ; Spermatogenesis/genetics ; Sperm Motility/genetics ; },
abstract = {BACKGROUND: More than 1000 genes have been identified as predominantly expressed in the human testis. Advances in gene editing technologies have enabled the rapid and efficient generation of genetically engineered mice. This approach facilitates the screening of genes essential for spermatogenesis by analyzing knockout mouse models.

OBJECTIVES: This study aimed to elucidate the essential genes in male reproductive function by generating knockout mouse models.

MATERIALS AND METHODS: We selected 11 target genes that may have potential roles in the male reproductive system based on a public database. Knockout mouse lines of these target genes were generated using the CRISPR/Cas9 system to elucidate their functions in male reproduction. Also, we conducted natural mating tests to elucidate fecundity and analyzed the phenotype of the knockout males.

RESULTS: Natural mating tests revealed that all 11 gene-deficient mouse lines maintained normal male fertility. The phenotypic analysis, including testis appearance and weight, histology of testis and epididymis, and sperm motility and morphology, showed no apparent abnormalities.

DISCUSSION AND CONCLUSION: These results suggest that each gene is not essential for male reproductive function.},
}

Animals
Male
*Fertility/genetics
*Testis/metabolism
*CRISPR-Cas Systems
Mice
Mice, Knockout
*Gene Editing
Spermatogenesis/genetics
Sperm Motility/genetics

@article {pmid42052931,
year = {2026},
author = {Bartylla, MM and Düthorn, ELR and Müller, JT and Wirtz, M and van Veen, H and Sasidharan, R and Mustroph, A},
title = {Rorippa islandica: a genetically accessible dicot model system to study flooding tolerance.},
journal = {Plant physiology},
volume = {202},
number = {1},
pages = {},
doi = {10.1093/plphys/kiag254},
pmid = {42052931},
issn = {1532-2548},
support = {//DFG Priority Programme 1529 "Adaptomics"/ ; 496871662//DFG projects/ ; 544882710//DFG projects/ ; /NWO_/Dutch Research Council/Netherlands ; //NWO/ ; },
mesh = {*Floods ; *Rorippa/genetics/physiology ; *Adaptation, Physiological/genetics ; Gene Expression Regulation, Plant ; Plant Proteins/genetics/metabolism ; Transcriptome ; Plants, Genetically Modified ; Photosynthesis ; CRISPR-Cas Systems ; },
abstract = {Most crop species cannot survive prolonged flooding events. Within the Cardamineae tribe of the Brassicaceae family, several wild species display high flooding tolerance and are therefore attractive study systems to unravel tolerance mechanisms. However, the genetic recalcitrance of many of these species has prevented detailed mechanistic studies of observed tolerance traits. Here, Rorippa islandica was identified as a genetically accessible diploid species with high submergence tolerance. Comparison of its submergence transcriptome with that of another diploid species from the same genus, the submergence-sensitive R. stylosa, revealed a strong and partially overlapping transcriptomic response to 48 h submergence. It also revealed RiBCA3 as a potential tolerance gene contributing to the higher submergence survival of R. islandica. Successful CRISPR-Cas9-mediated knockout of RiBCA3 confirmed the suitability of this species for genetic transformation. However, although it was hypothesized that RiBCA3 might have an important function in carbon fixation under water, no differences in submergence survival or underwater photosynthesis were observed between wild-type and bca3 knockout lines. The molecular mechanisms of submergence tolerance of Rorippa islandica are therefore not yet understood. This work demonstrates the suitability of Rorippa islandica for molecular genetics studies and presents a promising dicot model for investigation of underlying tolerance mechanisms, especially for comparative studies with Arabidopsis. This species might contribute to knowledge on flood tolerance in dicots, particularly Brassica oilseed crops and vegetables, while current knowledge is based primarily on rice (a monocot) studies.},
}

*Floods
*Rorippa/genetics/physiology
*Adaptation, Physiological/genetics
Gene Expression Regulation, Plant
Plant Proteins/genetics/metabolism
Transcriptome
Plants, Genetically Modified
Photosynthesis
CRISPR-Cas Systems

@article {pmid42743106,
year = {2026},
author = {Grüschow, S and Wotherspoon, P and Hilton-Balfe, E and Graham, S and White, MF},
title = {Cyclic tri-adenylate controls a CARF-TM effector in type II Panoptes anti-phage systems.},
journal = {PLoS biology},
volume = {24},
number = {9},
pages = {e3003934},
pmid = {42743106},
issn = {1545-7885},
mesh = {*Bacteriophages ; Nucleotides, Cyclic/metabolism ; *Bacterial Proteins/metabolism/genetics ; CRISPR-Cas Systems ; Second Messenger Systems ; Viral Proteins/metabolism ; *Cyclic AMP/metabolism ; Signal Transduction ; },
abstract = {Cyclic nucleotide second messengers are used in all domains of life to amplify viral infection signals and activate cellular defences. In prokaryotes, CBASS (cyclic nucleotide-based antiphage signalling system) and type III CRISPR-Cas systems generate a range of cyclic nucleotides which bind and allosterically activate effector proteins to mount an anti-viral response. Viruses have evolved counter measures to antagonise these signalling pathways in the form of cyclic nucleotide sponges and phosphodiesterases that sequester or degrade these molecules to subvert immunity. Recently, the Panoptes system was shown to function as a guard against these viral tactics. The type I Panoptes polymerase, mCpol, generates cyclic dinucleotides as decoy molecules that, when sequestered by phage proteins, results in the activation of the membrane-permeabilising effector 2TMβ to halt the phage infection cycle. Here, we investigate the type II Panoptes system, demonstrating that it generates cyclic tri-adenylate (cA3) to maintain a CRISPR-associated Rossmann fold-transmembrane (CARF-TM) effector in an inactive, dimeric state. When cA3 is sequestered or degraded, the CARF protein undergoes conformational changes. In vivo, the absence of cA3 results in membrane disruption and growth arrest. Type II Panoptes provides defence against phages that express the cA3-degrading enzyme Acb1; phage escapers introduce mutations into the acb1 gene to avoid triggering the Panoptes system. These findings expand our understanding of the guard systems that constitute a fascinating component of the bacterial immune system.},
}

*Bacteriophages
Nucleotides, Cyclic/metabolism
*Bacterial Proteins/metabolism/genetics
CRISPR-Cas Systems
Second Messenger Systems
Viral Proteins/metabolism
*Cyclic AMP/metabolism
Signal Transduction

@article {pmid42744140,
year = {2026},
author = {Nihaluddin, S and Rengasamy, K and Sivadoss, SR and Rajendran, L and Manivannan, N and Sankareswari, RU and Kumar, PS},
title = {Molecular diagnostics and integrated management challenges of tobacco streak virus: Current status and future perspectives.},
journal = {Journal of microbiological methods},
volume = {250},
number = {},
pages = {107710},
doi = {10.1016/j.mimet.2026.107710},
pmid = {42744140},
issn = {1872-8359},
abstract = {Tobacco streak virus (TSV) is an economically important viral pathogen causing severe yield and quality losses in several agricultural, horticultural and medicinal crops worldwide. Its complex epidemiology involving sap transmission, infected pollen and pollen-feeding thrips, together with symptom similarity to other necrosis-inducing pathogens, frequently results in misdiagnosis and delayed disease management. This review critically evaluates recent advances in TSV diagnostics and integrated disease management strategies. Particular emphasis is placed on the transition from conventional biological and serological assays to advanced molecular diagnostics including reverse transcription polymerase chain reaction (RT-PCR), quantitative real-time PCR, multiplex PCR and emerging isothermal amplification technologies such as recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP). The review also highlights emerging innovations including CRISPR/Cas-based diagnostics in addition, integrated management approaches involving phytosanitation, weed reservoir management, vector ecology-based, host resistance breeding, RNA interference (RNAi) and genome editing technologies are critically analysed. Major challenges including inadequate field validation, limited multiplex capability, poor assay standardization and scarcity of resistant cultivars are discussed. Future objectives to develop quick, field-adaptable and durable TSV detection and management methods are additionally discussed.},
}

@article {pmid42745070,
year = {2026},
author = {Mehmood, MA and Iqbal, MM and Yin, Y and Ashfaq, M and Chen, S and Shao, X and Wang, J},
title = {Transforming Plant Viruses into Vectors for Next-Generation Agriculture-A Review.},
journal = {Current microbiology},
volume = {83},
number = {11},
pages = {},
pmid = {42745070},
issn = {1432-0991},
support = {2024530000241011//Yunnan Tobacco Company Program/ ; 32260038//National Natural Science Foundation of China/ ; 202301AS070050//Yunnan Fundamental Research Projects/ ; 202405AS350009//the Innovation Team of Xingdian Scholar Funding of Yunnan/ ; },
mesh = {*Plant Viruses/genetics ; *Genetic Vectors/genetics ; *Agriculture/methods ; Gene Editing ; CRISPR-Cas Systems ; Plants, Genetically Modified/genetics/virology ; Crops, Agricultural/genetics/virology ; Genetic Engineering ; },
abstract = {Plant viral vectors have evolved from tools for transient gene expression into a versatile platform for precise genetic intervention, offering a rapid, transgene-free alternative to conventional crop transformation. This review critically assesses their engineering for scalable field application, moving beyond foundational techniques like virus-induced gene silencing (VIGS). We highlight how advanced vector design, including deconstructed genomes and synthetic regulatory circuits enhances cargo capacity, specificity, and biosafety. The integration of viral delivery with CRISPR-Cas systems has unlocked virus-induced genome editing (VIGE), base editing, and prime editing, enabling heritable trait modification without tissue culture. However, the transition from proof-of-concept in model plants to robust field technology hinges on overcoming critical bottlenecks: expanding host range through chimeric vectors, ensuring environmental containment, and developing scalable delivery methods such as nano-formulations or adjusted agroinfiltration protocols. We evaluate these delivery routes and emerging synergies with nanobiotechnology for targeted and efficient applications. While challenges in regulation, public perception, and large-scale production persist, the strategic engineering of viral vectors for stability, specificity, and safety positions them as a transformative, next-generation biotechnological input for achieving sustainable crop improvement and protection under changing climatic conditions.},
}

*Plant Viruses/genetics
*Genetic Vectors/genetics
*Agriculture/methods
Gene Editing
CRISPR-Cas Systems
Plants, Genetically Modified/genetics/virology
Crops, Agricultural/genetics/virology
Genetic Engineering

@article {pmid42742639,
year = {2026},
author = {Zhang, Y and Wang, Y and Li, Y and Hao, Y and Zheng, P and Liu, F and Long, J and Yang, H},
title = {The application of the CRISPR-Cas system in Pseudomonas aeruginosa infections.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42742639},
issn = {1573-4978},
support = {2025ZD01900202//Prevention and Control of Emerging and Major Infectious Diseases-National Science and Technology Major Project/ ; 82273696//National Natural Science Foundation of China/ ; 25IRTSTHN038//Program for Innovative Research Team (in Science and Technology) in University of Henan Province/ ; },
mesh = {*Pseudomonas aeruginosa/genetics/pathogenicity ; *CRISPR-Cas Systems/genetics ; Humans ; *Pseudomonas Infections/genetics/microbiology/therapy/diagnosis ; Gene Editing/methods ; Drug Resistance, Bacterial/genetics ; },
abstract = {Due to the extensive drug resistance of Pseudomonas aeruginosa (P. aeruginosa), it is still a great clinical challenge. The clustered regularly interspaced short palindromic repeats and associated proteins (CRISPR-Cas) system has become a promising strategy against this pathogen. This review critically evaluates the multifaceted applications of CRISPR-Cas technology in P. aeruginosa, including its role in antimicrobial resistance, diagnostics, genome editing, and emerging therapeutic and vaccine strategies. In addition to conducting a comprehensive analysis of various studies, we also compared the performance and limitations of various CRISPR platforms, and discussed the main technologies and transformation obstacles in this field. Finally, we look forward to the direction of applying these experimental tools to clinical research in the future.},
}

*Pseudomonas aeruginosa/genetics/pathogenicity
*CRISPR-Cas Systems/genetics
Humans
*Pseudomonas Infections/genetics/microbiology/therapy/diagnosis
Gene Editing/methods
Drug Resistance, Bacterial/genetics

@article {pmid42692283,
year = {2026},
author = {Chowdhury, D and Singh, N and Garai, S and Pandey, DP and Bodakhe, SH},
title = {Precision genome editing strategies for enduring lipid lowering in atherosclerosis.},
journal = {European journal of pharmacology},
volume = {1034},
number = {},
pages = {179312},
doi = {10.1016/j.ejphar.2026.179312},
pmid = {42692283},
issn = {1879-0712},
mesh = {Humans ; Animals ; *Gene Editing/methods ; *Atherosclerosis/genetics/therapy/blood ; Proprotein Convertase 9/genetics ; *Genetic Therapy/methods ; Lipids ; CRISPR-Cas Systems ; Lipid Metabolism/genetics ; },
abstract = {Atherosclerosis continues to be a primary contributor to global cardiovascular mortality, influenced by intricate lipid and inflammatory mechanisms. Despite the efficacy of conventional pharmacotherapies, ongoing issues of patient non-adherence and residual risk have prompted the exploration of more enduring therapeutic alternatives. This review examines the significant transition in cardiovascular research from conventional, wide knockout models to the utilization of advanced precision genome editing methods, particularly emphasizing CRISPR-Cas9, base editing, and prime editing. These sophisticated molecular tools allow for the accurate insertion and rectification of single-nucleotide variants without causing double-strand breaks, marking a significant shift from rudimentary gene disruption to precise variant engineering. By specifically targeting essential lipid-regulating genes like proprotein convertase subtilisin/kexin type 9 (PCSK9) and angiopoietin-like 3 (ANGPTL3), precision editing presents an exceptional opportunity for lasting, one-shot lipid-lowering treatments. Additionally, we examine the advancement of preclinical modeling, emphasizing humanized models that precisely represent population genetics. This review highlights the essential obstacles to clinical translation, focusing on the optimization of delivery systems such as adeno-associated viruses (AAVs) and lipid nanoparticles (LNPs), as well as the thorough assessment of off-target effects and ethical implications.},
}

Humans
Animals
*Gene Editing/methods
*Atherosclerosis/genetics/therapy/blood
Proprotein Convertase 9/genetics
*Genetic Therapy/methods
Lipids
CRISPR-Cas Systems
Lipid Metabolism/genetics

@article {pmid42736446,
year = {2026},
author = {Safaei, Z and Khalili, K},
title = {HIV cure: from early therapies to cutting-edge strategies.},
journal = {Journal of neurovirology},
volume = {32},
number = {5},
pages = {},
pmid = {42736446},
issn = {1538-2443},
mesh = {Humans ; *HIV Infections/therapy/immunology/virology/genetics ; Receptors, CCR5/genetics/immunology ; *HIV-1/genetics/drug effects/immunology ; CRISPR-Cas Systems ; *Gene Editing/methods ; *Genetic Therapy/methods ; Virus Latency/drug effects ; Animals ; Immunotherapy/methods ; Host-Directed Therapy ; *Anti-HIV Agents/therapeutic use ; },
abstract = {HIV has challenged humanity for decades, quietly establishing latent reservoirs that evade even the most potent antiretroviral therapies (ART). While ART transformed HIV from a fatal disease into a manageable condition, the virus's hidden persistence continues to fuel the global epidemic. This review narrates the story of HIV treatment, tracing the evolution of therapeutic strategies from conventional ART to innovative gene-editing and immunotherapeutic approaches. Central to this journey is the CRISPR-Cas system, a molecular scalpel that has enabled precise excision of integrated proviral DNA, disruption of entry co-receptors like CCR5, and modulation of host restriction factors to bolster innate defenses. Preclinical studies reveal that CRISPR interventions can reduce viral reservoirs, prevent reactivation, and restore antiviral immunity, offering a glimpse of a future beyond lifelong ART. Challenges remain, notably efficient delivery to infected cells and minimizing off-target effects, but advances in vector design and computational prediction tools are steadily overcoming these barriers. By weaving experimental findings with translational perspectives, this review highlights CRISPR's potential to provide durable, scalable, and cost-effective HIV control, particularly in low-resource settings. Ultimately, this narrative underscores a hopeful vision: that precise gene editing may transform HIV therapy from lifelong suppression into a pathway toward functional cure, changing the story of the virus and the lives it touches.},
}

Humans
*HIV Infections/therapy/immunology/virology/genetics
Receptors, CCR5/genetics/immunology
*HIV-1/genetics/drug effects/immunology
CRISPR-Cas Systems
*Gene Editing/methods
*Genetic Therapy/methods
Virus Latency/drug effects
Animals
Immunotherapy/methods
Host-Directed Therapy
*Anti-HIV Agents/therapeutic use

@article {pmid42737512,
year = {2026},
author = {Yako, H and Takahashi, M and Tada, S and Waragai, M and Miyamoto, Y and Yamauchi, J},
title = {Modeling NDD-Associated NLGN2 Depletion Using CRISPR/Cas13 Reveals Exaggerated Process Elongation Mediated by the CCDC88A-G Protein-ELMO Axis.},
journal = {International journal of molecular sciences},
volume = {27},
number = {17},
pages = {},
pmid = {42737512},
issn = {1422-0067},
support = {//Japan Science and Technology Agency/ ; //Ministry of Education, Culture, Sports, Science and Technology/ ; //Ministry of Health Labour and Welfare/ ; //Takeda Science Foundation/ ; },
mesh = {*Neuroligins/genetics/metabolism ; Animals ; *Cell Adhesion Molecules, Neuronal/genetics/metabolism ; *CRISPR-Cas Systems ; Neurons/metabolism ; *Microfilament Proteins/metabolism/genetics ; Signal Transduction ; Humans ; *Neurodevelopmental Disorders/metabolism/genetics ; Mice ; Gene Knockdown Techniques ; Neurodevelopment ; Cell Differentiation ; Nerve Tissue Proteins ; },
abstract = {Neuroligin-2 (NLGN2) is a cell adhesion molecule implicated in neurodevelopmental disorders (NDDs), including autism spectrum disorder (ASD) and intellectual disability (ID). While NLGN2 is well known as a postsynaptic organizer of predominantly inhibitory synapses, accumulating evidence suggests that NLGN family proteins are also involved in neuronal morphogenesis during early developmental stages. However, a gap remains in our understanding of how loss of function in NLGN2 potentially leads to abnormal neuronal morphogenesis. Here, we investigated the molecular basis of excessive neuronal process formation induced by depletion of NLGN2 using the N1E-115 cell line, an established model of neuronal differentiation. Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas13-mediated knockdown of NLGN2 promoted neuronal process elongation and neuronal differentiation marker expression. Mechanistically, NLGN2 knockdown resulted in activation of coiled-coil and hook domain-containing protein 88A (CCDC88A, also known as Girdin or GIV), a non-receptor guanine nucleotide exchange factor for heterotrimeric G proteins. Transfection of the regulator of G protein signaling (RGS) domain of RGS3, a negative regulator of G proteins, or the G protein-binding domain of engulfment and cell motility 1 (ELMO1) effectively decreased the excessive process elongation phenotype. Similar effects were observed in primary cortical neurons. Furthermore, these interventions normalized elevated Rac1 activity induced by NLGN2 knockdown. Collectively, our findings identify the CCDC88A-G protein-ELMO signaling pathway as a key mediator of excessive neuronal morphogenesis following NLGN2 knockdown. These results provide valuable insight into the mechanisms by which NLGN2 dysfunction may contribute to abnormal neuronal morphogenesis and suggest potential recovery strategies.},
}

*Neuroligins/genetics/metabolism
Animals
*Cell Adhesion Molecules, Neuronal/genetics/metabolism
*CRISPR-Cas Systems
Neurons/metabolism
*Microfilament Proteins/metabolism/genetics
Signal Transduction
Humans
*Neurodevelopmental Disorders/metabolism/genetics
Mice
Gene Knockdown Techniques
Neurodevelopment
Cell Differentiation
Nerve Tissue Proteins

@article {pmid42737922,
year = {2026},
author = {Kojima, KK},
title = {When Homing Endonuclease Meets Transposon: The OMEGA System.},
journal = {Biology},
volume = {15},
number = {17},
pages = {},
pmid = {42737922},
issn = {2079-7737},
abstract = {Sequence-specific DNA endonucleases have made significant contributions to biology, biotechnology, and medicine; restriction enzymes and homing endonucleases are among classic examples. The demonstration of programmable genome editing using Cas9 in the CRISPR-Cas system, in which the target DNA sequence is recognized by base pairing with a guide RNA, revolutionized the field of genome engineering, making target selection more flexible and convenient. The OMEGA (Obligate Mobile Element-Guided Activity) system, considered a precursor to Cas12, and likely to Cas9, in the CRISPR-Cas system, is an RNA-guided DNA endonuclease composed of a TnpB, IscB, IsrB, or Fanzor protein, and a structural RNA designated reRNA or ωRNA. The OMEGA system is present in the three domains of life as an auxiliary component of transposons. The OMEGA system cuts DNA in an allele from which a transposon is excised and triggers recombination to reinstate the transposon at the same position. This "transposon restorative homing" redefines the OMEGA system as a homing endonuclease. In this review, the selfish aspects of the OMEGA system are discussed in the historical context of homing endonuclease research.},
}

@article {pmid42738810,
year = {2026},
author = {Cai, J and Zhao, B and Fan, A and Chen, Y and Wu, X},
title = {CRISPR-Mediated POSTN Editing Modulates Proliferation, Apoptosis, and Molecular Profiles of Primary Rabbit Hair Follicle Stem Cells via the cAMP/PKA/CREB Signaling Pathway.},
journal = {Cells},
volume = {15},
number = {17},
pages = {},
pmid = {42738810},
issn = {2073-4409},
support = {Grant No. 32072724//National Natural Science Foundation of China/ ; CARS-43-A-1//China Agriculture Research System/ ; KYCX25_4054//Government of Jiangsu Province/ ; },
mesh = {Animals ; *Periostin/metabolism/genetics ; Cell Proliferation/genetics ; *Hair Follicle/cytology/metabolism ; *Apoptosis/genetics ; *Stem Cells/metabolism/cytology ; *Signal Transduction ; *Cyclic AMP/metabolism ; *Cyclic AMP Response Element-Binding Protein/metabolism ; *Cyclic AMP-Dependent Protein Kinases/metabolism ; Rabbits ; *CRISPR-Cas Systems/genetics ; Cells, Cultured ; *Cell Adhesion Molecules/genetics/metabolism ; },
abstract = {Hair follicle stem cells (HFSCs) are critical for hair follicle (HF) morphogenesis and cyclic hair regeneration. Periostin (POSTN), an extracellular matrix protein involved in tissue development and skin cell regulation, remains poorly characterized in HFSCs. In this study, we constructed CRISPR/Cas9 vectors targeting POSTN and validated their editing efficiency in primary HFSCs. POSTN editing significantly suppressed cell proliferation and promoted apoptosis. Transcriptomic analysis identified 988 differentially expressed genes (DEGs) enriched in immune responses and MAPK, PI3K-Akt, and cAMP pathways. Metabolomic analysis revealed 98 differential metabolites (DMs) associated with nucleotide metabolism and FoxO, AMPK, and cAMP pathways. Integrated multi-omics analysis showed extensive correlations between DEGs and DMs, and highlighted the cAMP pathway as the core regulatory axis. Western blot (WB) validation confirmed that POSTN editing reduced PKA and CREB phosphorylation, indicating inhibition of the cAMP/PKA/CREB signaling. These findings demonstrate that POSTN regulates HFSCs' proliferation and apoptosis partially via the cAMP/PKA/CREB pathway, providing novel insights into the functional regulation of HFSCs.},
}

Animals
*Periostin/metabolism/genetics
Cell Proliferation/genetics
*Hair Follicle/cytology/metabolism
*Apoptosis/genetics
*Stem Cells/metabolism/cytology
*Signal Transduction
*Cyclic AMP/metabolism
*Cyclic AMP Response Element-Binding Protein/metabolism
*Cyclic AMP-Dependent Protein Kinases/metabolism
Rabbits
*CRISPR-Cas Systems/genetics
Cells, Cultured
*Cell Adhesion Molecules/genetics/metabolism

@article {pmid42739482,
year = {2026},
author = {Thakur, V and Vats, AK and Kumar, R and Kumar, A and Vyas, S and Chakravarty, S and Bardhan, K and Paul, M and More, A and Johar, V and Kumar, V and M S, N and Teja, RR},
title = {Genome Editing in Solanaceae: Harnessing CRISPR-Cas Technology for Precision Crop Improvement.},
journal = {Plants (Basel, Switzerland)},
volume = {15},
number = {17},
pages = {},
pmid = {42739482},
issn = {2223-7747},
abstract = {Malnutrition and climate-induced stress remain major constraints to global food and nutritional security despite the yield gains of the Green Revolution. Solanaceae crops such as tomato, potato, brinjal, and pepper are key sources of vitamins, minerals, and bioactive compounds. Yet, their genetic improvement has been limited by narrow diversity and complex polygenic traits. The advent of CRISPR/Cas-mediated genome editing provides a transformative platform for precision crop improvement by enabling targeted modification of genes controlling stress tolerance, yield, and nutritional quality. In Solanaceae, CRISPR/Cas applications have successfully enhanced resistance against major pathogens (SlMlo1, SlPelo, SlDCL2), improved abiotic stress tolerance through editing of SlMAPK3, SlCBF1, and SlBZR1, and optimized fruit quality traits via modulation of Psy1, CrtR-b2, and fiAD2/3. Emerging innovations, such as base and prime editing, and RNP-mediated transgene-free delivery, are expanding the precision and scope of editing. However, challenges persist, including genotype-dependent transformation, low HDR efficiency, and incomplete understanding of off-target and epigenetic effects. Integrating CRISPR with omics-guided gene discovery, efficient transformation systems, and regulatory harmonization can accelerate the development of nutritionally enriched, stress-resilient, and sustainable Solanaceae varieties. This review synthesizes recent advances, identifies critical limitations, and outlines future opportunities for deploying CRISPR/Cas technology to achieve next-generation breeding and food system resilience.},
}

@article {pmid42740595,
year = {2026},
author = {Bozzetti, M and Raneri, M and Cortimiglia, C and Milani, G and Volontè, F and Lucchini, F and Bassi, D and Cocconcelli, PS},
title = {Endogenous CRISPR-Based Removal of Tetracycline Resistance in Bifidobacterium animalis subsp. lactis Through a Safe-by-Design Approach.},
journal = {Microbial biotechnology},
volume = {19},
number = {9},
pages = {e70443},
pmid = {42740595},
issn = {1751-7915},
support = {PE00000003//National Recovery and Resilence Plan Next generation EU, OnFoods/ ; //Fondazione Romeo ed Enrica Invernizzi/ ; },
mesh = {*Tetracycline Resistance/genetics ; *Bifidobacterium animalis/genetics/drug effects ; Tetracycline/pharmacology ; Anti-Bacterial Agents/pharmacology ; *CRISPR-Cas Systems ; Microbial Sensitivity Tests ; *Gene Editing/methods ; Probiotics ; Humans ; Genome, Bacterial ; Whole Genome Sequencing ; },
abstract = {Bifidobacterium animalis subsp. lactis is widely used as a probiotic; however, the presence of the tetracycline resistance gene tetW raises safety and regulatory concerns due to its potential mobility within the gut microbiome. Here, we applied a Safe-by-Design strategy using the endogenous CRISPR-Cas system of B. animalis subsp. lactis BLC01 to inactivate tetW through the introduction of premature stop codons. Whole-genome sequencing confirmed the intended editing and excluded relevant off-target effects. tetW inactivation markedly reduced the tetracycline minimum inhibitory concentration, restoring susceptibility below the tetracycline cut-off value for bifidobacteria (8 μg/mL). Comparative phenotypic analyses demonstrated that the edited strain (BLC01-2F3G10) retained key probiotic traits, including tolerance to acid, bile, and osmotic stress, exopolysaccharide production, aggregation capacity, survival during simulated gastrointestinal digestion and adhesion to intestinal epithelial cells. Importantly, no reversion to tetracycline resistance was observed after prolonged exposure to sub-inhibitory minimal selective antimicrobial concentration, indicating genetic stability of the edited phenotype. Collectively, these findings demonstrate that endogenous CRISPR-based genome editing can be leveraged to selectively remove antimicrobial resistance determinants from probiotic strains while preserving functionality, supporting the development of next-generation probiotics with an improved safety profile and reduced potential for antimicrobial resistance dissemination in the human gut.},
}

*Tetracycline Resistance/genetics
*Bifidobacterium animalis/genetics/drug effects
Tetracycline/pharmacology
Anti-Bacterial Agents/pharmacology
*CRISPR-Cas Systems
Microbial Sensitivity Tests
*Gene Editing/methods
Probiotics
Humans
Genome, Bacterial
Whole Genome Sequencing

@article {pmid42740853,
year = {2026},
author = {Wang, D and Sun, M and Meng, Q and Kang, Q and Bai, L},
title = {An efficient endogenous type I-E CRISPR-Cas genome-editing platform for producing transglutaminase in Streptomyces mobaraensis.},
journal = {Engineering microbiology},
volume = {6},
number = {4},
pages = {100302},
pmid = {42740853},
issn = {2667-3703},
abstract = {Streptomyces mobaraensis is an industrially important actinomycete capable of producing transglutaminase (TGase), a valuable crosslinking enzyme that is widely used in the food, pharmaceutical, and textile industries. However, its genetic manipulation remains challenging owing to the lack of efficient genome-editing tools. Here, we characterized an endogenous type I-E CRISPR-Cas system in S. mobaraensis IPIO2 through bioinformatics analysis and plasmid interference assays, identifying the protospacer adjacent motif as 5'-AAC-3'. We engineered an artificial editing plasmid, pCRISPR, by inserting a mini-CRISPR array (repeat-spacer-repeat) and homologous recombination repair templates into the replicative plasmid pJTU1278. This system exhibited high editing efficiencies, achieving 70% for single-gene deletions and 75-80% for large DNA fragment deletions ranging from 10 to 40 kb. Based on this system, deletion of four genes consistently downregulated during TGase production, identified through comparative proteomics, enhanced TGase production by 8.5-18.5%. Furthermore, deleting the pseudouridimycin and piericidin A1 biosynthetic gene clusters using this system significantly improved the safety profile of TGase production, resulting in a 17% increase in TGase yield. This study established a robust and efficient endogenous CRISPR-Cas-based genome-editing platform in S. mobaraensis, providing a powerful tool for strain engineering and industrial optimization of TGase production.},
}

@article {pmid42741839,
year = {2026},
author = {Noble-Molnar, C and Garrett, SC and Catchpole, RJ and Johnson, KA and Graveley, BR and Terns, MP},
title = {Type III CRISPR-Cas systems preferentially acquire spacers from early-expressed phage genes in a transcription-dependent manner.},
journal = {Nucleic acids research},
volume = {54},
number = {17},
pages = {},
pmid = {42741839},
issn = {1362-4962},
support = {R35 GM118140/GM/NIGMS NIH HHS/United States ; R35 GM118160/GM/NIGMS NIH HHS/United States ; R35GM118140/NH/NIH HHS/United States ; R35GM118160/NH/NIH HHS/United States ; },
mesh = {*Streptococcus thermophilus/genetics/virology ; *Transcription, Genetic ; Promoter Regions, Genetic ; *CRISPR-Cas Systems ; *Genes, Viral ; *Bacteriophages/genetics ; },
abstract = {CRISPR-Cas immunity depends on acquiring spacers that generate functional CRISPR RNA (crRNA) guides. For most RNA-targeting type III systems, this poses a paradox: spacers are captured from DNA but defense requires RNA interaction, thus the system must infer transcriptional relevance of DNA fragments. We compared spacer acquisition profiles for co-occurring type III (RNA-recognizing) and type II (DNA-recognizing) systems in Streptococcus thermophilus during lytic phage infection. Type III spacer acquisition was highly enriched for early phage genes whereas type II spacers were distributed genome wide. High-throughput spacer sequencing showed that the type III early gene bias arose during adaptation, was independent of interference, and peaked immediately downstream of a conserved early phage promoter. This bias for early phage promoters was also observed in natural type III spacers identified in publicly available S. thermophilus genomes. Cloning this early phage promoter and downstream gene into a plasmid recreated the type III-acquisition hotspot. Mutation to disrupt the promoter eliminated the spacer hotspot, while mutation to disrupt translation enhanced it. Thus, type III-A adaptation preferentially samples DNA associated with the earliest phage transcriptional program, enriching for spacers that generate crRNAs targeting early phage RNA.},
}

*Streptococcus thermophilus/genetics/virology
*Transcription, Genetic
Promoter Regions, Genetic
*CRISPR-Cas Systems
*Genes, Viral
*Bacteriophages/genetics

@article {pmid42742450,
year = {2026},
author = {Karvounis, IG and Daskalakis, V},
title = {A dual gating mechanism controls target-strand cleavage in Cas12j: Implications for engineering efficient nickases.},
journal = {Protein science : a publication of the Protein Society},
volume = {35},
number = {10},
pages = {e70792},
pmid = {42742450},
issn = {1469-896X},
support = {EHPC-REG-2025R01-105//EuroHPC Joint Undertaking/ ; EHPC-EXT-2025E01-070//EuroHPC Joint Undertaking/ ; 014775//Hellenic Foundation for Research and Innovation/ ; },
mesh = {Molecular Dynamics Simulation ; *CRISPR-Associated Proteins/chemistry/metabolism/genetics ; *Deoxyribonuclease I/chemistry/metabolism/genetics ; *DNA/chemistry/metabolism ; *Bacterial Proteins/chemistry/genetics/metabolism ; Protein Engineering ; Catalytic Domain ; CRISPR-Cas Systems ; *Endodeoxyribonucleases/chemistry/metabolism/genetics ; },
abstract = {The rapid expansion of CRISPR technologies has unveiled a diverse repertoire of RNA-guided endonucleases, among them the compact Cas12j, which has emerged as a promising genome-editing tool. Cas12j cleaves the two DNA strands sequentially; however, the molecular mechanism that regulates this cleavage remains incompletely understood. Here, we combine extensive all-atom molecular dynamics simulations with well-tempered metadynamics, totaling approximately 175 μs of cumulative sampling, to investigate how target-strand accessibility to the catalytic site is controlled. Our results are in agreement with previous experimental observations, and furthermore reveal new mechanistic details that are difficult to access experimentally, namely a coordinated dual-barrier mechanism governing target-strand accessibility that can be fine-tuned through targeted mutations in the α7-helix and/or the REC2 loop. These findings provide a mechanistic basis for tuning Cas12j activity along the nuclease-to-nickase spectrum, supporting the rational engineering of genome-editing tools with controlled target-strand cleavage kinetics, and offering a path toward applications that bypass dependence on Non-Homologous End Joining (NHEJ) or Homology-Directed Repair (HDR).},
}

Molecular Dynamics Simulation
*CRISPR-Associated Proteins/chemistry/metabolism/genetics
*Deoxyribonuclease I/chemistry/metabolism/genetics
*DNA/chemistry/metabolism
*Bacterial Proteins/chemistry/genetics/metabolism
Protein Engineering
Catalytic Domain
CRISPR-Cas Systems
*Endodeoxyribonucleases/chemistry/metabolism/genetics

@article {pmid42380742,
year = {2026},
author = {Fazeli, SA and Javanmard, AR},
title = {Integrative machine learning and CRISPR/Cas9 analysis reveals the role of APOE in lipid metabolism-associated kidney fibrosis.},
journal = {Molecular medicine (Cambridge, Mass.)},
volume = {32},
number = {1},
pages = {},
pmid = {42380742},
issn = {1528-3658},
mesh = {Humans ; *Lipid Metabolism/genetics ; *Apolipoproteins E/genetics/metabolism ; Fibrosis ; *CRISPR-Cas Systems ; *Machine Learning ; *Kidney Diseases/metabolism/pathology/etiology/genetics ; *Kidney/pathology/metabolism ; Gene Expression Profiling ; Animals ; Gene Editing ; },
abstract = {Kidney fibrosis, characterized by excessive extracellular matrix deposition and progressive renal tissue remodeling, frequently culminates in End-stage kidney disease (ESKD). Emerging evidence links dysregulated lipid metabolism to renal fibrogenesis; however, the precise molecular mechanisms connecting these processes remain elusive. Apolipoprotein E (APOE), a central coordinator of lipid transport and metabolic homeostasis, is implicated in various metabolic and inflammatory diseases, yet its specific contribution to renal fibrotic signaling is poorly defined. In this study, we utilized an integrative framework comprising machine learning-assisted transcriptomic analysis, clinical validation, and CRISPR/Cas9 gene editing to elucidate the role of APOE in kidney fibrosis. Machine learning analysis of public transcriptomic datasets pinpointed APOE as a top-ranked gene highly correlated with renal fibrotic signatures and metabolic pathways. Consistently, APOE expression was significantly altered in clinical blood samples from Maintenance hemodialysis patients compared to healthy controls. To mechanistically validate these findings, we generated an in vitro APOE-knockout model using CRISPR/Cas9, followed by Angiotensin II stimulation to induce renal fibrogenesis. APOE ablation profoundly impacted the expression of canonical pro-fibrotic markers (COL1A1, FN1, and α-SMA) alongside crucial lipid metabolism genes (FASN and ACC), as confirmed by quantitative real-time PCR and Western blot analyses. Pathway enrichment further corroborated APOE's role as a critical regulatory node bridging lipid homeostasis and fibrotic cascades in the kidney. Overall, our findings establish APOE as a pivotal regulator of renal fibrotic remodeling, highlighting a direct mechanistic interplay between lipid metabolism and kidney fibrosis. Consequently, therapeutic modulation of APOE-dependent pathways may offer a promising strategy for treating chronic kidney disease and associated fibrotic pathologies.},
}

Humans
*Lipid Metabolism/genetics
*Apolipoproteins E/genetics/metabolism
Fibrosis
*CRISPR-Cas Systems
*Machine Learning
*Kidney Diseases/metabolism/pathology/etiology/genetics
*Kidney/pathology/metabolism
Gene Expression Profiling
Animals
Gene Editing

@article {pmid42419216,
year = {2026},
author = {Jung, KM and Mony, SI and Chen, PR and Lee, K and Lee, HJ},
title = {Sex chromosome-targeted Cas9 knock-in and functional validation in chicken primordial germ cells.},
journal = {Poultry science},
volume = {105},
number = {10},
pages = {107384},
pmid = {42419216},
issn = {1525-3171},
mesh = {Animals ; *Chickens/genetics ; *Gene Knock-In Techniques/veterinary/methods ; *Sex Chromosomes/genetics ; *Germ Cells/metabolism ; *CRISPR-Cas Systems ; Female ; Male ; },
abstract = {Sex-specific control of genome editing remains a significant challenge in birds. Chickens exhibit a ZW sex-determination system in which the Z and W chromosomes encode genes essential for sex differentiation and germline development, providing a rationale for sex-linked genome engineering. In this study, we established the sex chromosome-linked knock-in system for Cas9 in chicken primordial germ cells (PGCs). Donor constructs carrying Cas9-GFP were engineered to integrate into either the Z chromosome (DMRT1-DMRT3 intergenic region) or the W chromosome (5' region of HINTW locus). The targeting strategy was validated in DF-1 fibroblasts and PGCs, where site-specific integration was confirmed by junction PCR and sequencing. Functionality of the integrated Cas9 was verified by targeting two different loci, demonstrating efficient genome cleavage at NHEJ1 loci and indel-associated loss of GFP fluorescence following GFP targeting. The knock-in PGCs expressed Cas9 protein while retaining germ cell markers and migration capacity, demonstrating preservation of germline identity. Collectively, our findings establish a sex chromosome-linked Cas9 knock-in system in chicken PGCs and demonstrate that these sites support stable Cas9 expression without compromising germline characteristics, thereby providing a practical foundation for controlled, sex-specific genome engineering in avian research.},
}

Animals
*Chickens/genetics
*Gene Knock-In Techniques/veterinary/methods
*Sex Chromosomes/genetics
*Germ Cells/metabolism
*CRISPR-Cas Systems
Female
Male

@article {pmid42501624,
year = {2026},
author = {Cao, Z and Chen, C and He, Y and Wei, W and Song, J and Han, W and Xue, Q and Chen, G and Li, B and Jin, K},
title = {Optimization of genome editing strategies for targeted integration at chicken sex chromosome loci.},
journal = {Poultry science},
volume = {105},
number = {10},
pages = {107397},
pmid = {42501624},
issn = {1525-3171},
mesh = {Animals ; *Chickens/genetics ; *Gene Editing/veterinary/methods ; *Sex Chromosomes/genetics ; CRISPR-Cas Systems ; Male ; Female ; },
abstract = {The precise targeted integration of large exogenous genes into chicken sex chromosomes is of particular interest for sex-linked trait manipulation, sex-control breeding, and the development of avian bioreactor models. However, efficient targeted integration at sex chromosome loci remains technically challenging, and optimized editing strategies for these loci are still lacking. To improve targeted genome editing at two previously identified chicken sex chromosome safe-harbor loci, EE0.6 and NC_006127.4, this study systematically evaluated and optimized key parameter affecting editing efficiency. First, we evaluated the effects of different sgRNA combinations on targeted knockout efficiency, establishing the advantage of a dual-sgRNA/Cas9 architecture, which achieved knockout efficiencies of 86.67% and 75.00% at the EE0.6 and NC_006127.4 loci, respectively. We next introduced the Cas9 nickase (Cas9n) system, which has previously been reported to exhibit improved editing specificity, and evaluated its performance at both target loci. Quantitative analysis showed that the dual-sgRNA/Cas9 targeting system successfully mediated the precise targeted integration of a 1.1-kb SV40-mCherry reporter cassette, reaching 100% (28/28) at the EE0.6 locus and 80.00% (20/25) at the NC_006127.4 locus. Based on this result, this study further investigated the effects of donor homology arm (HA) lengths (200 bp, 600 bp, and, 1000 bp) and vector topologies (circular and linearized) on targeted knock-in efficiency. The results revealed that in the circular donor system, the optimal HA lengths for the EE0.6 and NC_006127.4 loci were 200 bp (50.4% ± 4.4%) and 600 bp (30.1% ± 1.2%), respectively. However, upon the introduction of linearized donors with free ends, the knock-in efficiency of the exogenous target fragment was significantly enhanced, and its HA length preference underwent a significant reversal. The optimal HA length for EE0.6 was extended to 600 bp (78.9% ± 1.0%), whereas that for NC_006127.4 was shortened to 200 bp (49.9% ± 0.4%). In summary, this study established an efficient targeted integration strategy for chicken sex chromosome loci. The optimized system provides a foundation for future applications in sex-linked breeding and avian bioreactor development.},
}

Animals
*Chickens/genetics
*Gene Editing/veterinary/methods
*Sex Chromosomes/genetics
CRISPR-Cas Systems
Male
Female

@article {pmid42725884,
year = {2026},
author = {Marconcini, M and Cruchet, S and Goswami, S and Viswanatha, R and Butnaru, M and De, J and Roselli, C and Hadjieconomou, D and Perrimon, N and Mohr, SE and Benton, R},
title = {Intersecting experimental evolution and CRISPR screens to identify novel toxin resistance loci.},
journal = {eLife},
volume = {15},
number = {},
pages = {},
pmid = {42725884},
issn = {2050-084X},
support = {10.3030/101117267/ERC_/European Research Council/International ; P40 OD018537/OD/NIH HHS/United States ; 3200-0-239882//Schweizerische Nationalfonds zur Förderung der wissenschaftlichen Forschung/ ; 10.3030/833548/ERC_/European Research Council/International ; 310030_219185//Schweizerische Nationalfonds zur Förderung der wissenschaftlichen Forschung/ ; },
mesh = {Animals ; *Caprylates/toxicity ; *Drosophila/genetics/drug effects ; Drosophila Proteins/genetics/metabolism ; Drosophila melanogaster/genetics/drug effects ; Morinda/chemistry ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Drosophila simulans/genetics/drug effects ; CRISPR-Cas Systems ; *Drug Resistance/genetics ; },
abstract = {Understanding toxin resistance in insects is key to appreciating niche adaptations but remains challenging due to its often-polygenic basis. A well-known example is the specialized association of Drosophila sechellia with noni fruit (Morinda citrifolia), which is toxic to other insects, including Drosophila simulans and Drosophila melanogaster. The main noni toxin is octanoic acid (OA), but the mechanisms that determine sensitivity or resistance to OA in different species remain unclear. Here, we experimentally evolved D. simulans with increased OA resistance, identifying multiple loci under selection. Cross-referencing these with a genome-wide, OA resistance CRISPR screen in a D. melanogaster cell line highlighted two proteins: Kraken, a putative detoxification enzyme expressed in digestive and renal tissues, and Alkbh7, a mitochondrial protein linked to fatty acid metabolism. Both genes show elevated expression in D. sechellia and OA-resistant D. simulans. In D. melanogaster, kraken mutants are more OA-sensitive, while Alkbh7 overexpression increased OA resistance. Mutation of these genes in D. sechellia reduced OA tolerance. Our identification of genes contributing to OA resistance in laboratory and natural contexts demonstrates how complementary selection approaches can provide insights into complex mechanisms of toxin susceptibility and adaptation. Such methods could have practical applications in the characterization of natural and artificial insecticides.},
}

Animals
*Caprylates/toxicity
*Drosophila/genetics/drug effects
Drosophila Proteins/genetics/metabolism
Drosophila melanogaster/genetics/drug effects
Morinda/chemistry
*Clustered Regularly Interspaced Short Palindromic Repeats
*Drosophila simulans/genetics/drug effects
CRISPR-Cas Systems
*Drug Resistance/genetics

@article {pmid42730668,
year = {2026},
author = {Huang, Y and Wentink, M and Fueyo, R},
title = {Genetic Deletion of Cis-Regulatory Elements to Dissect the Function of the Non-coding Genome in human Preimplantation Models.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {235},
pages = {},
doi = {10.3791/73225},
pmid = {42730668},
issn = {1940-087X},
mesh = {Humans ; *Blastocyst/physiology ; CRISPR-Cas Systems ; Endogenous Retroviruses/genetics ; Pluripotent Stem Cells/physiology ; *Regulatory Sequences, Nucleic Acid/genetics ; *Gene Deletion ; },
abstract = {Cis-regulatory elements coordinate gene expression in a spatially and temporally controlled manner and contribute to the establishment of distinct cellular states during development. A substantial proportion of transcriptionally active cis-regulatory elements in primate embryos originated from ancient retroviral integrations into the germline. These endogenous retroviruses, also known as long terminal repeat retrotransposons, retain intrinsic regulatory activity and are often species-specific, making them strong candidates for regulating species-divergent aspects of embryonic development. Ethical and legal restrictions on human embryo research have historically limited direct investigation of gene regulation during human embryogenesis. Human naive pluripotent stem cells and three-dimensional stem cell-based blastocyst models provide alternative systems for studying early developmental processes. This protocol describes the CRISPR-Cas9-mediated deletion of endogenous retrovirus-derived cis-regulatory elements in human naive pluripotent stem cells. Preassembled Cas9 and single-guide RNA ribonucleoprotein complexes are delivered by nucleofection, followed by single-cell cloning, PCR-based genotyping, Sanger sequencing, expansion, cryopreservation, and genomic stability assessment of the edited lines. The resulting wild-type, heterozygous, and homozygous or hemizygous deletion clones provide a platform for investigating the contribution of individual endogenous retrovirus-derived elements to gene regulation in human preimplantation models. This method enables direct functional interrogation of species-specific non-coding regulatory sequences and supports the study of transcriptional mechanisms involved in early human development.},
}

Humans
*Blastocyst/physiology
CRISPR-Cas Systems
Endogenous Retroviruses/genetics
Pluripotent Stem Cells/physiology
*Regulatory Sequences, Nucleic Acid/genetics
*Gene Deletion

@article {pmid42731613,
year = {2026},
author = {Lin, X and Zhang, Y and Cheng, J and Chen, J and Liu, X},
title = {Advances in CRISPR-based genetic engineering of Bifidobacterium.},
journal = {Biochimie},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.biochi.2026.09.003},
pmid = {42731613},
issn = {1638-6183},
abstract = {Bifidobacterium plays a vital role in gut health, immunity, and metabolism, yet natural strains show limited native functional capabilities and genetic manipulability. Recent advances in genome editing, especially CRISPR-based systems, have provided novel approaches for engineering Bifidobacterium, though strain-specific variability and restriction-modification barriers remain substantial constraints. This review outlines the evolution from traditional homologous recombination to CRISPR/Cas technologies, highlighting applications of endogenous and exogenous systems, including CRISPRi/a and base editing. In preclinical and food biotechnology contexts, genetically engineered strains have shown potential as targeted delivery vehicles and functional probiotics. Despite these achievements, challenges such as strain variability, restriction-modification barriers, and biosafety concerns persist. Where genetic accessibility can be established, the integration of CRISPR technology with synthetic biology may enable more precise gene regulation and could support the development of next-generation engineered Bifidobacterium-based platforms.},
}

@article {pmid42733042,
year = {2026},
author = {Liang, F and Wu, BY and Liu, JY and Wang, X and Liang, MX and Wang, CY and Zhang, JY and Wang, JJ and Miao, YT and Mai, KS and Zhao, J and Wang, XG},
title = {Inheritance of the epigenetic signature and reduced intermuscular bone phenotype acquired via DNA methylation editing of the runx2 b promoter in zebrafish.},
journal = {Zoological research},
volume = {47},
number = {5},
pages = {1769-1779},
doi = {10.24272/j.issn.2095-8137.2025.494},
pmid = {42733042},
issn = {2095-8137},
mesh = {Animals ; *Zebrafish/genetics ; *DNA Methylation ; *Promoter Regions, Genetic ; *Epigenesis, Genetic ; *Core Binding Factor Alpha 1 Subunit/genetics/metabolism ; Gene Editing ; Phenotype ; *Zebrafish Proteins/genetics/metabolism ; Epigenetic Memory ; CRISPR-Cas Systems ; },
abstract = {The presence of intermuscular bones (IBs) can directly affect the economic value of aquaculture fish. Although genome editing can create IB-free fish by knocking out key IB-related genes, such as runx2b, the associated DNA sequence alterations raise food safety and health concerns, limiting its breeding applications. In this study, we used CRISPR/dCas9-mediated epigenome-editing technology targeting the runx2 b promoter in zebrafish to alter DNA methylation patterns without changing the DNA sequence. Our results showed that higher runx2 b promoter methylation patterns significantly inhibited eGFP mRNA expression levels in the recombinant plasmid. Using the CRISPR/dCas9-Dnmt7 system to enhance methylation of the zebrafish runx2b promoter, we observed a significant decrease in runx2 b mRNA expression levels in the F0 generation. The IBs in the 11 [th]-16 [th] muscle segments of the adult F0 fish were significantly shorter compared with the controls. Inbreeding of fish was used to produce F1 and F2 offspring that retained these high promoter methylation levels, along with persistent runx2b expression suppression and IB development inhibition. Transcriptome sequencing analysis suggested that increasing runx2 b promoter methylation levels may synergistically induce additional epigenetic modifications, potentially affecting the PPAR signaling pathway and FoxO transcription factor regulation, which appears to inhibit osteoblast proliferation and differentiation. Overall, this study demonstrates an innovative application of epigenetic editing technology for aquaculture breeding. By precisely regulating the expression patterns of key genes for economically important traits while preserving genomic DNA integrity, this approach provides a theoretical foundation and technical support for improving fish economic traits.},
}

Animals
*Zebrafish/genetics
*DNA Methylation
*Promoter Regions, Genetic
*Epigenesis, Genetic
*Core Binding Factor Alpha 1 Subunit/genetics/metabolism
Gene Editing
Phenotype
*Zebrafish Proteins/genetics/metabolism
Epigenetic Memory
CRISPR-Cas Systems

@article {pmid42734752,
year = {2027},
author = {Zhang, J and Brennand, KJ and Zhang, B and Wang, M and Li, A},
title = {Systematic Dissection of Key Driver Perturbation Signatures in Single Cells via ECCITE-seq.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3074},
number = {},
pages = {193-208},
pmid = {42734752},
issn = {1940-6029},
mesh = {Humans ; Induced Pluripotent Stem Cells/metabolism/cytology ; *Single-Cell Analysis/methods ; Alzheimer Disease/genetics/metabolism ; Single-Cell Gene Expression Analysis ; CRISPR-Cas Systems ; Neurons/metabolism/cytology ; *Transcriptome ; *Gene Expression Profiling/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; High-Throughput Nucleotide Sequencing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {CRISPR screens, such as expanded CRISPR-compatible cellular indexing of transcriptomes and epitopes by sequencing (ECCITE-seq), enable the simultaneous measurement of transcriptomes, gRNA identity, and cell-surface protein expression at single-cell resolution to systematically interrogate gene function. This platform provides a powerful and scalable experimental approach for validating disease-associated regulators identified by large-scale association studies and other computational methods, including network-based analyses of multi-omics data. Here, as an example application, we describe an ECCITE-seq framework to characterize the transcriptomic consequences of perturbing multiple neuronal key driver genes associated with Alzheimer's disease (AD) in human-induced pluripotent stem cell (hiPSC)-derived neurons. More broadly, by integrating customized pooled gRNA libraries with different CRISPR effectors across multiple cell types, this approach allows for the assessment of the regulatory impact of candidate genes implicated in development and disease processes.},
}

Humans
Induced Pluripotent Stem Cells/metabolism/cytology
*Single-Cell Analysis/methods
Alzheimer Disease/genetics/metabolism
Single-Cell Gene Expression Analysis
CRISPR-Cas Systems
Neurons/metabolism/cytology
*Transcriptome
*Gene Expression Profiling/methods
Clustered Regularly Interspaced Short Palindromic Repeats
High-Throughput Nucleotide Sequencing/methods
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid42734786,
year = {2027},
author = {Bhardwaj, P and Dwivedi, GR and Joshi, HS and Singh, R},
title = {RT-RPA-Assisted CRISPR/Cas12a-Based Isothermal Detection of Chikungunya Virus.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3067},
number = {},
pages = {19-30},
pmid = {42734786},
issn = {1940-6029},
mesh = {*Chikungunya virus/genetics/isolation & purification ; *CRISPR-Cas Systems ; Humans ; Animals ; *Nucleic Acid Amplification Techniques/methods ; *Chikungunya Fever/diagnosis/virology ; RNA, Viral/genetics ; *Molecular Diagnostic Techniques/methods ; Rapid Diagnostic Tests ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Chikungunya virus (CHIKV) is transmitted through the bite of Aedes mosquitoes, specifically A. aegypti and A. albopictus. CHIKV belongs to the alphavirus with a positive-sense ssRNA genome of 11-12 kb size. The virus has been reported from various geographical regions across the globe. Chikungunya fever is an acute febrile illness, which, if left untreated, may develop into chronic arthralgia that may persist for several months or acute encephalitis syndrome. Therefore, early diagnosis of CHIKV is crucial to initiate prompt supportive treatment. Laboratory diagnosis of CHIKV typically relies on serological tests such as IgM antigen capture ELISA and molecular methods including RT-PCR or qRT-PCR. However, both these methods are not viable in peripheral settings. This chapter highlights recent advancements in molecular detection techniques for CHIKV, specifically isothermal detection methods that eliminate the requirement for complex instruments. The detection is facilitated by RT-RPA and CRISPR/Cas12a endonuclease. The assay offers advantages over existing methods such as rapid and early detection, and eliminates cross-over contamination, ultra-sensitivity, high specificity, and ease of result interpretation.},
}

*Chikungunya virus/genetics/isolation & purification
*CRISPR-Cas Systems
Humans
Animals
*Nucleic Acid Amplification Techniques/methods
*Chikungunya Fever/diagnosis/virology
RNA, Viral/genetics
*Molecular Diagnostic Techniques/methods
Rapid Diagnostic Tests
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid42734876,
year = {2026},
author = {Qiu, L and Ungerbäck, JT and Bennett, EP and Bak, RO},
title = {Biologic Therapies for Alleviating Neurodegeneration in Lysosomal Storage Diseases.},
journal = {BioDrugs : clinical immunotherapeutics, biopharmaceuticals and gene therapy},
volume = {},
number = {},
pages = {},
pmid = {42734876},
issn = {1179-190X},
abstract = {Lysosomal storage diseases (LSDs) are a group of rare inherited metabolic disorders characterized by lysosomal dysfunction and progressive accumulation of undegraded substrates, leading to multisystem involvement and, in many cases, severe neurodegeneration. Because the blood-brain barrier (BBB) restricts central nervous system (CNS) access for most therapeutic modalities, neurological manifestations remain the major unmet need across LSDs. In this review, we summarize current and emerging strategies aimed at correcting CNS pathology, including enzyme replacement therapy (ERT), adeno-associated virus (AAV)-mediated gene therapy, allogeneic hematopoietic stem cell transplantation (HSCT), and autologous HSCT with gene-modified hematopoietic stem cells. While ERT provides limited CNS benefits and allogeneic HSCT mitigates neurodegeneration only partially, their overall impact on CNS outcomes remains restricted. Newer approaches, such as BBB-shuttling ERTs, CNS-tropic AAV capsids, and genetically modified autologous hematopoietic stem and progenitor cells capable of sustained supraphysiological enzyme production, offer promising avenues for enhanced CNS delivery and cross-correction. Together, these advances underscore a shift toward integrated therapeutic strategies that combine systemic and CNS-directed interventions, with the potential to transform outcomes for patients with LSDs and other neurodegenerative disorders amenable to cross-correction.},
}

@article {pmid42365126,
year = {2026},
author = {Choi, I and Kim, S and Baek, I},
title = {Advances and clinical potential of epigenome editing.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {83},
number = {1},
pages = {},
pmid = {42365126},
issn = {1420-9071},
support = {RS-2023-00278378//Ministry of Science and ICT, South Korea/ ; RS-2025-02217909//Ministry of Science and ICT, South Korea/ ; RS-2026-25478918//Ministry of Science and ICT, South Korea/ ; },
mesh = {Humans ; *Epigenome Editing/methods ; Animals ; CRISPR-Cas Systems/genetics ; *Epigenesis, Genetic ; *Epigenome ; Precision Medicine/methods ; Chromatin/genetics/metabolism ; Neoplasms/genetics/therapy ; },
abstract = {Epigenome editing has emerged as a powerful platform to modulate gene expression in a precise and reversible manner. Recent advances have significantly improved the efficiency, specificity, and durability of epigenome editing systems, enabling fine-tuned transcriptional control. Building on these developments, epigenome editing platforms are now being explored for therapeutic applications. In this review, we summarize the evolution of clustered regularly interspaced short palindromic repeats (CRISPR)-based epigenome editing technologies, highlighting key improvements in effector modules. We then discuss the disease models in which epigenome editing has been applied, including monogenic disorders, cancer, neurological diseases, and chronic diseases. These examples demonstrate the broad therapeutic promise of targeted epigenetic modulation across diverse pathological contexts. Finally, we tackle key barriers to clinical translation, including cell-type and chromatin context-specific design, in vivo delivery, and multi-gene targeting for complex disease. Collectively, this review underscores the potential of epigenome editing as a versatile platform for precision medicine.},
}

Humans
*Epigenome Editing/methods
Animals
CRISPR-Cas Systems/genetics
*Epigenesis, Genetic
*Epigenome
Precision Medicine/methods
Chromatin/genetics/metabolism
Neoplasms/genetics/therapy

@article {pmid42420452,
year = {2026},
author = {Al-Zahrani, KN and Langille, ER and Nurtanto, J and Obersterescu, A and Teng, K and Lowden, C and Dessapt, J and Chiu, CH and Caldwell, LV and Cook, DP and Pérez-Castro, MA and Berman, JM and Tsai, R and Bahcheli, AT and Mbamalu, G and Wu, S and Narimatsu, M and Lopes, AG and Fotiadou, I and Chan, K and Zhang, L and Bang, KWA and Parsons, MJ and Mourao, L and Temel, EI and McCulla, L and Sravya, P and Zhang, L and Sajjakulnukit, P and Lyssiotis, CA and Borowsky, AD and Scheele, CLGJ and Wahl, DR and Jackson, HW and Stewart, KS and Fuchs, E and Egan, SE and Pujana, MA and Reimand, J and Wrana, JL and Schramek, D},
title = {Aneuploidy selects for the acquisition of driver genes in breast cancer.},
journal = {Nature},
volume = {657},
number = {8132},
pages = {800-810},
pmid = {42420452},
issn = {1476-4687},
mesh = {Animals ; *Aneuploidy ; Female ; *Breast Neoplasms/genetics/pathology ; Humans ; Mice ; *Oncogenes/genetics ; CRISPR-Cas Systems/genetics ; DNA Copy Number Variations/genetics ; Signal Transduction/genetics ; Disease Models, Animal ; Reactive Oxygen Species/metabolism ; Tumor Suppressor Protein p53/genetics ; *Genes, Neoplasm/genetics ; Mitochondria/metabolism ; },
abstract = {Chromosome instability is highly prevalent in cancer and drives large-scale chromosomal imbalances, known as aneuploidies[1-4]. How aneuploidy contributes to tumorigenesis remains difficult to study due to the vast numbers of genes affected. Here we established a CRISPR knockout- and activation-linked assay (CRISPR-KOALA), enabling high-throughput bidirectional genetic screens in immunocompetent mouse models of cancer. We developed a compendium of the ten most frequent human chromosome-arm-level alterations in basal-like breast cancer (BLBC), a disease type that is driven by large copy-number alterations (CNAs)[5-8]. Using CRISPR-KOALA, we screened the mouse orthologues of 3,752 genes on these arms and identified 90 cancer driver genes, the function of the vast majority of which is unknown. These genes drive distinct signalling pathways including MAPK, HIPPO and WNT, reflecting the high degree of BLBC heterogeneity. Manipulating the identified cancer driver genes overcomes the need for CNAs in Trp53-mutant BLBC mouse models. Mechanistically, we identify that PLGRKT is a potent oncogene that lies on chromosome 9p and show that its tumour-promoting activity is associated with highly stress-resistant mitochondria and an increased ability to detoxify reactive oxygen species. Together, our findings reveal that arm-level CNAs can function to select specific driver genes to promote heterogeneous biological processes.},
}

Animals
*Aneuploidy
Female
*Breast Neoplasms/genetics/pathology
Humans
Mice
*Oncogenes/genetics
CRISPR-Cas Systems/genetics
DNA Copy Number Variations/genetics
Signal Transduction/genetics
Disease Models, Animal
Reactive Oxygen Species/metabolism
Tumor Suppressor Protein p53/genetics
*Genes, Neoplasm/genetics
Mitochondria/metabolism

@article {pmid42552655,
year = {2026},
author = {Wang, Y and Xu, H and Lu, W and Wan, Z and Xu, Z and Wang, W and Chen, C and Pan, E and Jiang, F and Liu, T and Li, Y and Xiao, D and Yang, X and Li, F and Hou, X and Zhang, C},
title = {Establishment of an efficient and PAM-relaxed LbCas12a genome editing tool in plants.},
journal = {The New phytologist},
volume = {252},
number = {2},
pages = {936-949},
pmid = {42552655},
issn = {1469-8137},
support = {PAPD//Priority Academic Program Development of Jiangsu Higher Education Institutions/ ; CARS-23-A-16//National vegetable industry technology system/ ; HUNONGKE AI-A [2025] 004//Shanghai Academy of Agricultural Sciences AI-Empowered Agriculture Special Project/ ; 2025B02008//Key R&D Program of Xinjiang Uygur Autonomous Region/ ; CAAS-BRC-CB-2025-02//The Agricultural Science and Technology Innovation Program, the Basic Research Center/ ; JBGS[2021]064//Jiangsu Seed Industry Revitalization Project/ ; 2022YFD1200502//National Key Research and Development Program of China/ ; },
mesh = {*Genome, Plant/genetics ; Oryza/genetics ; Solanum lycopersicum/genetics ; Brassica/genetics ; Base Sequence ; *Nucleotide Motifs/genetics ; Mutation/genetics ; *CRISPR-Cas Systems/genetics ; Plants, Genetically Modified ; Genetic Engineering/methods ; *Gene Editing/methods ; *CRISPR-Associated Proteins/metabolism ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Cas12a is widely used in plant genome editing, but its targeting scope is constrained by stringent protospacer adjacent motif (PAM) requirements and variable activity across species, limiting its application at diverse genomic loci. LbCas12a-RRV-based editing system was established in nonheading Chinese cabbage, and T5exo-PF-LbCas12a was generated by introducing a triple mutation (D535G/S551F/D665N) and fusing with T5 exonuclease. This engineered system recognizes an expanded PAM sequence from 5'-VTTV-3' to 5'-NYHV-3'. The system exhibited efficient editing at noncanonical PAM sites in cabbage, tomato, and rice. Additionally, it successfully mediated large-fragment deletions via microhomology-mediated end joining (MMEJ) in plants. This study expands Cas12a targeting scope in plants and provides the first evidence for Cas12-mediated MMEJ-based large-fragment deletion. The toolkit facilitates functional genomics and crop improvement, and the methodology is readily adaptable to other plant species.},
}

*Genome, Plant/genetics
Oryza/genetics
Solanum lycopersicum/genetics
Brassica/genetics
Base Sequence
*Nucleotide Motifs/genetics
Mutation/genetics
*CRISPR-Cas Systems/genetics
Plants, Genetically Modified
Genetic Engineering/methods
*Gene Editing/methods
*CRISPR-Associated Proteins/metabolism
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid42557315,
year = {2026},
author = {Neiswender, JV and Maffa, S and Brenan, L and ElHarouni, D and Yun, Y and Boyle, I and Wienand, K and Inam, H and Bertea, T and Anderson, A and Wong, M and Enriquez, M and Lenz, E and Villafranca, B and Shanks, N and Hager, M and Lloyd, N and Shadmany, H and Wie, SJ and Liang, H and Yunghans, K and Zhang, X and Golden, L and Harris, H and Day, S and Montgomery, P and Stokes, S and Giglio, RM and Hajal, C and Whittle, JR and Garcia, G and Mills, CE and Touat, M and Pelton, K and Li, H and Prabhakar, PS and Herter, S and Kamrat, ZH and Gui, D and Dilly, J and Wong, CK and Guo, JA and Pal, S and Baidi, Y and Johnston, R and Brown, DD and Bhatia, S and Winter, PS and Raghavan, S and Beroukhim, R and Colas, E and Spector, DL and Bass, AJ and Sorger, PK and Chen, Y and Hill, SJ and Oesterreich, S and Lee, AV and Beltran, H and Boehm, JS and Tseng, YY and Root, DE and Hahn, WC and Aguirre, AJ and Campbell, CD and Ligon, KL and Dempster, JM and Shibue, T and Vazquez, F},
title = {A dependency map enhanced with next-generation 3D cancer models.},
journal = {Nature},
volume = {657},
number = {8132},
pages = {789-799},
pmid = {42557315},
issn = {1476-4687},
mesh = {Humans ; *Neoplasms/genetics/pathology/classification/drug therapy ; Spheroids, Cellular/metabolism/pathology ; Animals ; Cell Line, Tumor ; *Models, Biological ; Multiomics ; Genomics ; CRISPR-Cas Systems/genetics ; Biomarkers, Tumor/genetics ; Gene Expression Regulation, Neoplastic ; },
abstract = {Despite advances in precision oncology, effective personalized treatments are still lacking for most patients with cancer[1]. The Cancer Dependency Map (DepMap) accelerates this field by systematically identifying cancer vulnerabilities in diverse preclinical models. Data from over 1,300 cell lines have led to the discovery of new therapeutic strategies across multiple tumour types[2]. However, mapping cancer vulnerabilities using traditional cell lines has limitations, including insufficient cancer subtype representation and the impact of culture conditions on perturbation responses. Here we perform 147 genome-scale CRISPR screens and multi-omic characterizations of next-generation (NextGen) cancer models (organoids and spheroids) across 10 cancer types. This strategy enables the expansion of DepMap to cover new genomic and molecular subtypes and to identify new biomarker-associated vulnerabilities. These new models also preserve transcriptional programs that are silenced in traditional cell lines and facilitate the discovery of specific gene dependencies associated with these programs. Comparisons of traditional and NextGen cancer models enable further identification of distinct effects of growth format and culture medium on gene essentiality. The integrated dataset combines data from both model types to offer a valuable, expansive resource for exploring cancer vulnerabilities and is accessible via the DepMap portal.},
}

Humans
*Neoplasms/genetics/pathology/classification/drug therapy
Spheroids, Cellular/metabolism/pathology
Animals
Cell Line, Tumor
*Models, Biological
Multiomics
Genomics
CRISPR-Cas Systems/genetics
Biomarkers, Tumor/genetics
Gene Expression Regulation, Neoplastic

@article {pmid42557320,
year = {2026},
author = {Herranz-Ors, C and Bhosle, SG and Beck, AE and Gilbert, JGR and Picco, G and Espejo Valle-Inclan, J and Muyas, F and Valentini, S and Andres, AE and Ansari, R and Barthorpe, S and Battarbee, G and Beaver, CM and Brocklesby, S and Cantwell, J and Collins, CA and Davis, J and Dimitrova, HG and Doran, J and Efendi, E and Evans, K and Fekry, M and Fowler, TA and Garcia-Casado, M and Griffiths, JAT and Hall, C and Hamer, R and Hardy, C and Hewitson, Z and Hitch, E and Holland, L and Jackson, DA and Joshi, N and Kavasakali, A and Letchford, L and Lightfoot, HB and Lingala, H and Mali, I and May, K and Mironenko, T and Morris, J and Pacini, C and Price, S and Robert-Tissot, G and Rogers, HA and Smith, JV and Smith, K and Souster, E and Spence, WJ and Thomas, F and Vieira, SF and Walker, S and Alfonsin, G and Bermingham, H and Coles, H and Ennis, DP and Freeman, A and Giannone, G and Grehan, N and Griffiths, EA and Hall, J and Lee, SL and Leung, EYL and Loreno, C and Millington, C and Mirnezami, A and Nutzinger, B and Orzechowska, K and Pinna, CMA and Redmond, AM and Roberts, K and Roy, S and Sanders, DA and Taniere, P and Vias, M and Wanigasooriya, K and , and Stratton, MR and Staudt, LM and McDermott, U and Brenton, JD and McNeish, IA and Biankin, A and Sansom, OJ and Cortes-Ciriano, I and Underwood, TJ and Fitzgerald, RC and Beggs, AD and Francies, HE and Garnett, MJ},
title = {A tumour-derived organoid biobank maps cancer gene dependencies.},
journal = {Nature},
volume = {657},
number = {8132},
pages = {765-774},
pmid = {42557320},
issn = {1476-4687},
mesh = {Humans ; *Organoids/metabolism/pathology ; *Neoplasms/genetics/pathology/drug therapy ; *Biological Specimen Banks ; Female ; *Genes, Neoplasm/genetics ; Proto-Oncogene Proteins p21(ras)/genetics ; Genes, Essential/genetics ; Precision Medicine ; ErbB Receptors/metabolism ; CRISPR-Cas Systems/genetics ; Alleles ; Colorectal Neoplasms/genetics/pathology ; ras Proteins/metabolism ; },
abstract = {Cancer cell lines remain foundational for research and drug discovery, yet they incompletely capture tumour diversity, lack linked patient context, and have undergone adaptation to culture. Tumour organoids are three-dimensional cultures derived from patient tissue that offer a powerful complement to cell lines[1]. Here we derived and characterized 256 clinically annotated tumour organoids directly from colorectal, oesophageal, ovarian, pancreatic and gastric cancers as renewable, genetically stable models. Extensive characterization of each model and matched patient tumour samples included whole-genome and transcriptome sequencing, and genome-wide CRISPR-Cas9 screens across 162 organoids mapped gene dependencies. Integrative analyses revealed genomic and clinical markers of dependency across common and rare subtypes, identified organoid-specific essential genes, and revealed targetable vulnerabilities following tumour evolution in paired pre- and post-treatment samples. In colorectal cancer, functional and pharmacological interrogation of the EGFR-RAS-MAPK axis uncovered differential effects of KRAS variant alleles. This open, publicly available resource provides a systematic map of gene dependencies in patient-derived organoids, expanding the model diversity and mechanistic insight needed to advance precision oncology.},
}

Humans
*Organoids/metabolism/pathology
*Neoplasms/genetics/pathology/drug therapy
*Biological Specimen Banks
Female
*Genes, Neoplasm/genetics
Proto-Oncogene Proteins p21(ras)/genetics
Genes, Essential/genetics
Precision Medicine
ErbB Receptors/metabolism
CRISPR-Cas Systems/genetics
Alleles
Colorectal Neoplasms/genetics/pathology
ras Proteins/metabolism

@article {pmid42557325,
year = {2026},
author = {Lee, JH and Li, Z and Soto, JS and Kim, AR and Illouz, T and Polonio, CM and Kilian, M and Kenison, JE and Schüle, AM and Akl, CF and Lee, HG and Andersen, BM and Ye, JJ and Rone, JM and Piester, G and Srun, L and Martinez, J and Danko, A and Lee, J and Heo, TH and Chung, EN and Oetjen, LK and Duart-Abadia, P and Koo, N and Perrimon, N and Wheeler, MA and Khakh, BS and Zandee, SEJ and Prat, A and Quintana, FJ},
title = {Antigen presentation by CD40[+]MHC-II[+] astrocytes promotes CNS autoimmunity.},
journal = {Nature},
volume = {657},
number = {8132},
pages = {755-764},
pmid = {42557325},
issn = {1476-4687},
mesh = {Animals ; *Astrocytes/immunology/metabolism/cytology ; *CD40 Antigens/immunology/metabolism ; Encephalomyelitis, Autoimmune, Experimental/immunology/pathology ; *Autoimmunity/immunology ; Mice ; Female ; *Antigen Presentation/immunology ; CD40 Ligand/metabolism/immunology ; *Central Nervous System/immunology/cytology ; CD4-Positive T-Lymphocytes/immunology ; *Histocompatibility Antigens Class II/immunology/metabolism ; Th17 Cells/immunology ; Humans ; Male ; Multiple Sclerosis/immunology/pathology ; Coculture Techniques ; Mice, Inbred C57BL ; CRISPR-Cas Systems ; },
abstract = {Astrocytes contribute to the pathology of multiple neurological disorders, including the T cell-driven autoimmune disease of the central nervous system (CNS) multiple sclerosis and its mouse model, experimental autoimmune encephalomyelitis[1]. However, little is known about functional interactions between astrocytes and CD4[+] T cells. Here using rabies barcode interaction detection followed by sequencing[2], in combination with single-cell RNA sequencing, in vitro co-culture systems and cell-specific in vivo CRISPR-Cas9-based genetic perturbation studies, we established that astrocytes expressing CD40 and MHC-II promote CNS T cell autoimmunity. We harnessed universal labelling immune partnerships by SorTagging intercellular contacts[3] to analyse astrocyte-interacting CD4[+] T cells, finding that direct astrocyte-CD4[+] T cell interactions enhance pathogenic T helper 17 cell responses in experimental autoimmune encephalomyelitis. In addition, we studied the effect of these interactions on astrocytes. Using in vivo subproteomic approaches[4] and AlphaFold-Multimer predictions[5], we established that CD40 activation in astrocytes by CD40L expressed by CD4[+] T cells induces the accumulation of PLIN4-positive lipid droplets, which provide acetyl-CoA to promote p65 acetylation-dependent NF-κB activation and antigen presentation. Finally, we detected CD40[+]MHC-II[+]LD[+] astrocytes in multiple sclerosis samples by single-nucleus RNA sequencing and immunohistochemistry. In summary, these studies define a previously unrecognized mechanism by which astrocytes promote CNS autoimmunity.},
}

Animals
*Astrocytes/immunology/metabolism/cytology
*CD40 Antigens/immunology/metabolism
Encephalomyelitis, Autoimmune, Experimental/immunology/pathology
*Autoimmunity/immunology
Mice
Female
*Antigen Presentation/immunology
CD40 Ligand/metabolism/immunology
*Central Nervous System/immunology/cytology
CD4-Positive T-Lymphocytes/immunology
*Histocompatibility Antigens Class II/immunology/metabolism
Th17 Cells/immunology
Humans
Male
Multiple Sclerosis/immunology/pathology
Coculture Techniques
Mice, Inbred C57BL
CRISPR-Cas Systems

@article {pmid42723509,
year = {2026},
author = {Tsuda, H},
title = {Whole-genome safety assessment of Loigolactobacillus coryniformis WBB05 and identification of a candidate gene for aerobic reuterin production.},
journal = {The Journal of dairy research},
volume = {},
number = {},
pages = {1-7},
doi = {10.1017/S0022029926102659},
pmid = {42723509},
issn = {1469-7629},
abstract = {This study reports on the safety profile of Loigolactobacillus coryniformis WBB05 for food industry applications and identifies glycerol-3-phosphate oxidase (GlpO) as a candidate gene associated with aerobic reuterin production. The safety of L. coryniformis WBB05 was evaluated through whole-genome sequencing, phenotypic analysis of haemolytic activity and determination of minimum inhibitory concentrations (MICs) of antibiotics. Comparative genomic analysis was performed to identify candidate genetic determinants for aerobic reuterin production. The draft genome (2.83 Mb, 179 contigs) harboured no known virulence factors, acquired antimicrobial resistance (AMR) genes or biogenic amine biosynthetic genes. Prophage analysis identified only one incomplete prophage region, and four CRISPR-Cas systems (212 spacers) were consistent with phage defence capacity. Secondary metabolite analysis revealed biosynthetic gene clusters encoding a coagulin-like bacteriocin. No β-haemolytic activity was observed. The MICs of all antibiotics tested were below the European Food Safety Authority cut-off values except for kanamycin (128 mg/L), although no acquired AMR genes were detected. Comparative genomic analysis revealed that L. coryniformis WBB05 possesses two putative copies of GlpO, a gene not detected in publicly available genomes of Limosilactobacillus reuteri, which produces reuterin only under anaerobic conditions. These findings support the use of L. coryniformis WBB05 as a safe adjunct culture for dairy applications and highlight GlpO as a candidate determinant of aerobic reuterin production. Further studies comparing GlpO-positive and GlpO-negative strains under aerobic and anaerobic conditions are warranted to confirm the role of GlpO.},
}

@article {pmid42725110,
year = {2026},
author = {Nilova, O and Zajakina, A},
title = {Next-generation macrophage engineering in cancer therapy: From TAM reprogramming to CAR-macrophages.},
journal = {Molecular therapy. Nucleic acids},
volume = {37},
number = {3},
pages = {103060},
pmid = {42725110},
issn = {2162-2531},
abstract = {Macrophages are central regulators of the tumor microenvironment (TME), shaping immune suppression, angiogenesis, metabolism, and therapeutic resistance in solid cancers. While early strategies sought to deplete tumor-associated macrophages (TAMs) or block monocyte recruitment, limited efficacy and compensatory mechanisms revealed the need for functional reprogramming rather than elimination. Recent advances in viral vectors, CRISPR-Cas genome editing, and RNA-based delivery platforms have enabled precise genetic modification of macrophages, giving rise to chimeric antigen receptor macrophages (CAR-Ms) and related engineered products. Beyond antigen targeting, effective macrophage engineering requires stabilization of pro-inflammatory identity, resistance to tumor-induced repolarization, metabolic reinforcement, and integration of checkpoint modulation pathways. This review synthesizes current strategies across DNA, mRNA, and siRNA-based platforms, highlighting convergent design principles that connect TAM reprogramming with CAR-M development. We discuss reshaping phagocytosis checkpoints, metabolic and transcriptional stabilization, cytokine augmentation, and synthetic receptor architecture, emphasizing combinatorial and context-aware engineering, while proposing new candidate gene targets. Engineered macrophages are thus evolving from simple effector cells into programmable immune coordinators capable of converting immunologically "cold" tumors into inflamed, therapy-responsive niches.},
}

@article {pmid42725430,
year = {2026},
author = {Ding, W and Yang, X and Yang, Y and Liu, M and Liu, C and Wang, J and Liu, X},
title = {Repeat region engineering of Cas13a crRNA enables conformational gating-based autocatalytic CRISPR biosensing.},
journal = {Nucleic acids research},
volume = {54},
number = {17},
pages = {},
pmid = {42725430},
issn = {1362-4962},
support = {22474111//National Natural Science Foundation of China/ ; 22104077//National Natural Science Foundation of China/ ; 2026JC-YXQN-058//Natural Science Basic Research Program/ ; 2025RS-CXTD-058//Innovation Capability Support Program/ ; 25ZQRC00009//Xi'an Science and Technology Project/ ; //Northwest A&F University/ ; },
mesh = {*CRISPR-Associated Proteins/genetics/metabolism/chemistry ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *Repetitive Sequences, Nucleic Acid ; Nucleic Acid Conformation ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {CrRNA engineering has emerged as a pivotal strategy for extending CRISPR-Cas13a biosensing. However, structural modulation of the direct repeat (DR) region remains exceptionally challenging due to its intricate architecture and the high energetic barrier of the Cas13a-crRNA interface, which is conventionally viewed as a rigid and immutable scaffold. Here, we demonstrate that the DR region is instead a programmable topological element with unexpected structural plasticity. By systematically engineering the DR through sequence insertion and structural splitting, we identified multiple DR variants that retain robust catalytic activity. Crucially, this topological reconfiguration enables Cas13a activity to be precisely gated by unmodified nucleic acid blockers, a level of regulation unattainable with the wild-type crRNA. Building on this flexible modulation, we developed Dre-CRISPR, a DR-engineered platform that couples target-triggered DR restoration to a self-reinforcing autocatalytic loop. This self-amplifying system provides a 2 × 106-fold sensitivity enhancement over nonamplified systems. Furthermore, the Dre-CRISPR platform extends the diagnostic scope of Cas13a to a broader spectrum of analytes, ranging from microRNAs to enzymatic activities and heavy metal ions. Our findings redefine the crRNA scaffold as a versatile signaling node and provide a generalizable framework for developing high-sensitivity, self-amplifying CRISPR biosensors through topology-driven guide RNA engineering.},
}

*CRISPR-Associated Proteins/genetics/metabolism/chemistry
*Biosensing Techniques/methods
*CRISPR-Cas Systems
*Repetitive Sequences, Nucleic Acid
Nucleic Acid Conformation
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42725481,
year = {2026},
author = {Li, Z and Li, F and Jiang, X},
title = {Confinement-enabled nanobiosensing: from chemical signal amplification to programmable single-molecule diagnostics.},
journal = {Chemical communications (Cambridge, England)},
volume = {},
number = {},
pages = {},
doi = {10.1039/d6cc04236h},
pmid = {42725481},
issn = {1364-548X},
abstract = {For rare targets in complex samples, ultrasensitive biosensing requires not only strong signal amplification but also reliable discrimination of target-derived signals from the background. Confinement-enabled nanobiosensing addresses this challenge by organizing molecular recognition, amplification and readout across nanoscale interfaces and microscale compartments. In this Feature Article, we review how chemical amplification strategies, from plasmonic nanogold probes and enzyme-triggered cascades to CRISPR-Cas signal networks, are being coupled with droplets and microwell arrays to convert ensemble signals into digital molecular counts. We further highlight programmable microfluidics, particularly digital microfluidics, as an automation layer for sample handling, washing, reagent exchange and compartment sealing. Recent dual-digital immunoassay platforms illustrate how fluidic digitization and molecular digitization can be integrated for single-molecule diagnostics. We conclude by outlining challenges in interface stability, scalable fabrication, multiplexed readout and clinical translation.},
}

@article {pmid42725609,
year = {2026},
author = {Meka, SG and A, SS and Dasari, V and Ali, OAI and Yeddhu, Y and Kuragala, N and Gudisinti, V and E, B},
title = {Nanocarrier-enabled Gene and Nucleic Acid Therapeutics for Rheumatoid Arthritis: Advances, Translational Evidence, and Regulatory Challenges.},
journal = {Recent advances in inflammation & allergy drug discovery},
volume = {},
number = {},
pages = {},
doi = {10.2174/0127722708482830260826121447},
pmid = {42725609},
issn = {2772-2716},
abstract = {Rheumatoid arthritis (RA) is a chronic autoimmune disorder characterized by persistent synovial inflammation, cartilage degradation, and bone erosion. Conventional diseasemodifying antirheumatic drugs and biologics, although effective in many patients, are limited by systemic toxicity, variable responsiveness, and loss of efficacy over time. Recent advances in nanomedicine have enabled the development of non-viral and viral gene delivery platforms designed to modulate pathogenic pathways at the molecular level. Non-viral nanocarriers, including liposomes, polymeric nanoparticles, lipid nanoparticles, and hybrid systems, have demonstrated efficient delivery of siRNA, antisense oligonucleotides, and mRNA targeting key inflammatory mediators such as TNF-α and NF-κB, resulting in significant disease attenuation in preclinical RA models. Viral vectors, particularly adeno-associated viruses and lentiviruses, provide sustained intra-articular gene expression but raise concerns related to immunogenicity and long-term safety. Emerging gene-editing strategies, notably CRISPR/Cas-based approaches, offer the potential to precisely and durably modulate disease-driving genes. But clinical translation is restricted by off-target effects, ethical considerations, and regulatory barriers. Collectively, the available evidence underscores both the therapeutic potential and inherent complexity of nanocarrier-mediated gene and nucleic acid therapies for RA, emphasizing the necessity for optimized delivery strategies, rigorous safety assessments, and biomarker-guided patient stratification to achieve successful clinical translation. This review comprehensively covered various gene delivery modalities, including non-viral and viral vectors, and gene editing (CRISPR/Cas) approaches. At the end, preclinical and clinical evidence, along with extensive case studies, for RA treatment were also highlighted. This review also outlined challenges and future directions in the management of arthritis.},
}

@article {pmid41152569,
year = {2026},
author = {Whittaker, MN and Testa, LC and Quigley, A and Brooks, DL and Grandinette, SA and Said, H and Dwivedi, G and Jindal, I and Volpp, D and Hacker, JL and Qu, P and Wang, JZ and Levine, MA and Ahrens-Nicklas, RC and Li, Q and Musunuru, K and Alameh, MG and Peranteau, WH and Wang, X},
title = {Improved specificity and efficiency of in vivo adenine base editing therapies with hybrid guide RNAs.},
journal = {Nature biomedical engineering},
volume = {10},
number = {9},
pages = {1943-1955},
pmid = {41152569},
issn = {2157-846X},
support = {Predoctoral Fellowship//American Heart Association (American Heart Association, Inc.)/ ; R01-HD110733//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; Graduate Research Fellowship//National Science Foundation (NSF)/ ; U19 NS132301/NS/NINDS NIH HHS/United States ; U01-TR005355//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R01 HD110733/HD/NICHD NIH HHS/United States ; R21-AR084212//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; Career Development Award//American Heart Association (American Heart Association, Inc.)/ ; U19-NS132301//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; },
mesh = {Humans ; Animals ; *Adenine/metabolism ; Mice ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Tyrosinemias/genetics/therapy ; *Genetic Therapy/methods ; },
abstract = {Phenylketonuria (PKU), pseudoxanthoma elasticum (PXE) and hereditary tyrosinemia type 1 (HT1) are autosomal recessive disorders linked to the PAH, ABCC6, and FAH and HPD genes, respectively. Here we evaluate the off-target editing profiles of clinical lead guide RNAs (gRNAs) that, when combined with adenine base editors (ABEs), correct the recurrent PAH P281L variant, PAH R408W variant or ABCC6 R1164X variant, or disrupt either of two sites in the HPD gene (a modifier gene of HT1) in human hepatocytes. To mitigate off-target mutagenesis, we systematically screen hybrid gRNAs with DNA nucleotide substitutions. Comprehensive and variant-aware specificity profiling of these hybrid gRNAs reveals dramatically reduced off-target editing and reduced bystander editing in cells. In humanized PAH P281L and ABCC6 R1164X mouse models of PKU and PXE, we show that when formulated in lipid nanoparticles with ABE messenger RNA, selected hybrid gRNAs revert disease phenotypes, reduce off-target editing, increase on-target editing and reduce bystander editing in vivo. These studies highlight the use of hybrid gRNAs to improve the safety and efficiency of adenine base-editing therapies.},
}

Humans
Animals
*Adenine/metabolism
Mice
*RNA, Guide, CRISPR-Cas Systems/genetics
Tyrosinemias/genetics/therapy
*Genetic Therapy/methods

@article {pmid42336896,
year = {2026},
author = {Novoa, JJ and Gaykema, LH and de Klerk, JA and Peerlings, JHD and van Nieuwland, RY and van der Slik, A and van den Hil, FE and Orlova, VV and Gonçalves, MAFV and van den Berg, CW and Rabelink, TJ and Zaldumbide, A},
title = {CRISPR-Cas9-mediated knock-in of cytomegalovirus US2 provides an alternative strategy for generating hypoimmunogenic hiPSC lines.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {42336896},
issn = {2045-2322},
support = {2020-01//LUF/Stichting Prof. Jaap de Graeff-Lingling Wiyadharma Fonds/ ; NNF21CC0073729//Novo Nordisk Foundation Center for Stem Cell Medicine (reNEW)/ ; },
mesh = {Humans ; *Cytomegalovirus/genetics ; *CRISPR-Cas Systems ; HLA-A2 Antigen/immunology/genetics/metabolism ; *Induced Pluripotent Stem Cells/immunology/metabolism/cytology ; Killer Cells, Natural/immunology ; *Viral Proteins/genetics ; Cell Line ; T-Lymphocytes/immunology ; Lymphocyte Activation ; Viral Envelope Proteins ; },
abstract = {Mismatches in HLA haplotypes between donors and recipients significantly increase the risk of graft failure due to immune rejection. Knockout (KO) of beta-2 microglobulin (B2M) is the current standard for eliminating HLA class I (HLA-I) surface expression and protecting allogeneic products from T-cell-mediated rejection; however, complete HLA-I ablation can trigger natural killer (NK) cell "missing-self" responses and disrupt critical immune-regulatory interactions. To address these limitations, we introduced a cytomegalovirus-derived US2 encoding sequence into the AAVS1 safe-harbor locus of a human induced pluripotent stem cell (hiPSC) line. Flow cytometry showed that US2 selectively abrogates HLA-A2 surface expression while retaining low levels of total HLA-I. In coculture assays, US2 expression abolished HLA-A2 alloreactive T-cell activation without increasing NK cell degranulation, indicating preserved inhibitory signaling. Together, these findings establish US2-mediated immune evasion as a refined single-edit alternative to B2M KO, enabling selective HLA-I modulation while preserving critical immune-regulatory interactions.},
}

Humans
*Cytomegalovirus/genetics
*CRISPR-Cas Systems
HLA-A2 Antigen/immunology/genetics/metabolism
*Induced Pluripotent Stem Cells/immunology/metabolism/cytology
Killer Cells, Natural/immunology
*Viral Proteins/genetics
Cell Line
T-Lymphocytes/immunology
Lymphocyte Activation
Viral Envelope Proteins

@article {pmid42544503,
year = {2026},
author = {Zou, G and Xie, J and Wu, M and Wei, X and Li, J},
title = {An electrochemiluminescence biosensor for sulfadimethoxine detection based on enzymatic DNA walker-activated CRISPR/Cas12a cascade for signal enhancement.},
journal = {The Analyst},
volume = {151},
number = {18},
pages = {5335-5343},
doi = {10.1039/d6an00344c},
pmid = {42544503},
issn = {1364-5528},
mesh = {*Biosensing Techniques/methods ; *Sulfadimethoxine/analysis ; Luminescent Measurements/methods ; *Electrochemical Techniques/methods ; *CRISPR-Cas Systems ; *DNA/chemistry/genetics ; Limit of Detection ; Gold/chemistry ; Aptamers, Nucleotide/chemistry/genetics ; Electrodes ; Ferrous Compounds/chemistry ; Metallocenes/chemistry ; },
abstract = {Sulfadimethoxine (SDM) is a widely used veterinary antibiotic. Its residues in food and the environment may cause bacterial resistance and threaten human health. In this study, a novel electrochemiluminescence (ECL) biosensor with signal amplification was developed for the sensitive detection of SDM. A g-C3N4@Au composite was modified on the electrode surface, generating a strong ECL signal with K2S2O8 as the coreactant. Hairpin DNA terminated with ferrocene carboxylic acid (FcA) was self-assembled onto g-C3N4@Au via thiol-gold bonds, causing Fc to quench the electrochemiluminescence signal of g-C3N4. Upon SDM addition, it specifically bound to the aptamer strand, releasing the DNA Walker. The DNA walker then continuously hybridized with the activator strand S1 and was cleaved by the nicking endonuclease Nt.BsmAI, generating a large number of S1. S1 further activated the trans-cleavage activity of CRISPR/Cas12a, which cleaves the hairpin DNA on the electrode, releasing Fc and recovering the ECL signal. By monitoring the ECL intensity change during the "signal-off" to "signal-on" transition, quantitative detection of SDM was indirectly realized. The detection range for SDM is 1.0 × 10[-14] to 1.0 × 10[-7] mol L[-1], with a detection limit of 7.15 × 10[-15] mol L[-1]. The sensor was successfully applied to the detection of SDM in food and water samples. Benefiting from the catalytic cascade amplification involving multiple enzymes, the developed method demonstrated high ultrahigh sensitivity.},
}

*Biosensing Techniques/methods
*Sulfadimethoxine/analysis
Luminescent Measurements/methods
*Electrochemical Techniques/methods
*CRISPR-Cas Systems
*DNA/chemistry/genetics
Limit of Detection
Gold/chemistry
Aptamers, Nucleotide/chemistry/genetics
Electrodes
Ferrous Compounds/chemistry
Metallocenes/chemistry

@article {pmid42601452,
year = {2026},
author = {Pecori, R and Casati, B and Merdler-Rabinowicz, R and Landesman, N and Sanghvi, K and Zens, S and Bilgin, E and Topouzi, M and Kipfstuhl, K and Pinamonti, V and Arnold, A and Abukhalaf, M and Gomez-Zepeda, D and Tenzer, S and Lindner, JM and Platten, M and Offringa, R and Carretero, R and Ruppin, E and Levanon, EY and Papavasiliou, FN},
title = {Engineering editopes through programmable RNA editing toward tumor neoantigen generation.},
journal = {The EMBO journal},
volume = {45},
number = {18},
pages = {6655-6678},
pmid = {42601452},
issn = {1460-2075},
support = {2039/20 and 231/21//Israel Science Foundation (ISF)/ ; 2021//HI-TRON seed funding program 2021/ ; 439669440 TRR319 RMaP TP A04//Deutsche Forschungsgemeinschaft (DFG)/ ; Project "ENIGMA"//DKFZ-Hector Seed Funding/ ; Core Funding DKFZ//Helmholtz Association ()/ ; },
mesh = {*RNA Editing ; Humans ; Animals ; Adenosine Deaminase/genetics/metabolism ; *Antigens, Neoplasm/immunology/genetics ; RNA-Binding Proteins/genetics/metabolism ; Mice ; MART-1 Antigen/immunology/genetics ; Cell Line, Tumor ; Immunotherapy/methods ; *Epitopes/immunology/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Neoplasms/immunology/genetics/therapy ; T-Lymphocytes/immunology ; },
abstract = {Neoepitope-based therapies hold great promise for cancer immunotherapy because they target tumor-specific mutations and elicit potent anti-tumor T-cell responses. However, their clinical implementation remains limited by the complexity of neoepitope discovery and uncertainty regarding presentation by tumor cells. A potential alternative is the generation of immunogenic neoepitopes directly within cancer cells through programmable RNA editing. Here, we develop Short Precise-Encodable ADAR Recruiting (SPEAR) gRNAs that harness endogenous ADAR1 to direct precise adenosine-to-inosine (A-to-I) editing at selected transcript sites. Using these gRNAs, we demonstrate the generation of immunogenic neoepitopes through RNA editing at the transcript level, termed editopes. In a proof-of-concept model based on the melanoma antigen MART-1, SPEAR-mediated RNA editing restored antigen-specific T cell recognition and enabled tumor control in vivo. Finally, we developed a computational pipeline to identify candidate tumor-selective neoepitopes across multiple cancer types amenable to guided RNA editing. Our findings establish programmable RNA editing as a strategy for engineering immunogenic editopes and provide a framework for neoepitope-directed cancer immunotherapy.},
}

*RNA Editing
Humans
Animals
Adenosine Deaminase/genetics/metabolism
*Antigens, Neoplasm/immunology/genetics
RNA-Binding Proteins/genetics/metabolism
Mice
MART-1 Antigen/immunology/genetics
Cell Line, Tumor
Immunotherapy/methods
*Epitopes/immunology/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
*Neoplasms/immunology/genetics/therapy
T-Lymphocytes/immunology

@article {pmid42657609,
year = {2026},
author = {Lin, X and Ye, P and Ding, Y and Cao, Y and Wang, Y and He, J and Zhou, Y},
title = {A cascaded CHA-CRISPR/Cas12a photoelectrochemical platform enabled by S-scheme Bi2WO6/BiOBr for piR-823 detection.},
journal = {The Analyst},
volume = {151},
number = {18},
pages = {5510-5517},
doi = {10.1039/d6an00974c},
pmid = {42657609},
issn = {1364-5528},
mesh = {Bismuth/chemistry ; *Electrochemical Techniques/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *Biosensing Techniques/methods ; Limit of Detection ; *RNA, Small Interfering/analysis/genetics ; Tungsten Compounds/chemistry ; Exosomes/chemistry ; Photochemical Processes ; *Biomarkers, Tumor/analysis ; },
abstract = {Exosomal piR-823 is a promising specific biomarker for colorectal cancer, yet its ultrasensitive detection remains challenging due to its extremely low abundance and high sequence complexity. Herein, we developed a cascaded CHA-CRISPR/Cas12a photoelectrochemical (PEC) sensing platform enabled by an S-scheme Bi2WO6/BiOBr heterojunction for the reliable detection of piR-823. The flower-like spherical Bi2WO6/BiOBr heterojunction exhibits enhanced visible-light absorption and efficient charge transfer arising from the S-scheme structure, which establishes a strong photoactive basis for the PEC platform. By integrating catalytic hairpin assembly with CRISPR/Cas12a trans-cleavage activity, a cascaded signal amplification strategy is established, which significantly improves the detection sensitivity. Under optimized conditions, the platform shows a distinct negative correlation between transient photocurrent and the logarithm of piR-823 concentration (1.0-1.0 × 10[6] fM), with an ultralow detection limit of 0.34 fM (S/N = 3). It also demonstrates high specificity, good reproducibility, and satisfactory recovery (96.87%-103.41%) in exosome lysate. This work not only presents an efficient PEC biosensing platform for piRNA analysis but also offers guidance for the rational construction of S-scheme heterojunctions in high-performance PEC biosensors, holding great promise for early colorectal cancer diagnosis and prognostic monitoring.},
}

Bismuth/chemistry
*Electrochemical Techniques/methods
*CRISPR-Cas Systems/genetics
Humans
*Biosensing Techniques/methods
Limit of Detection
*RNA, Small Interfering/analysis/genetics
Tungsten Compounds/chemistry
Exosomes/chemistry
Photochemical Processes
*Biomarkers, Tumor/analysis

@article {pmid42677820,
year = {2026},
author = {Chen, J and He, M and Yin, F and Pan, H and Chao, M and Cheng, W and Sun, H and Wang, Z and Xiang, Y},
title = {A rapid and straightforward detection of the novel obesity gene LGR4 A750T point mutation by the single-tube Cas12a system with split crRNA.},
journal = {Journal of materials chemistry. B},
volume = {14},
number = {35},
pages = {11039-11045},
doi = {10.1039/d6tb01366j},
pmid = {42677820},
issn = {2050-7518},
mesh = {*Point Mutation/genetics ; Humans ; *Receptors, G-Protein-Coupled/genetics ; *Obesity/genetics ; *CRISPR-Cas Systems/genetics ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {With the increasing number of obesity cases globally, immediate screening for obesity-related mutations has become increasingly important. LGR4 is a novel obesity gene. Here, we report a dual-mode detection strategy for the LGR4 p.A750T genetic variant by integrating asymmetric recombinase polymerase amplification (aRPA) with a split CRISPR-Cas12a system. Through isothermal aRPA amplification, the system directly generates abundant single-stranded DNA products that activate the split Cas12a complex, enabling ultra-sensitive and PAM-independent detection without spatial constraints on mutation sites. By employing a split crRNA design rather than conventional CRISPR-Cas12a, the method demonstrates exceptional specificity in distinguishing DNA point mutations. The assay achieves a detection limit of 13.5 fM, exhibits excellent linearity from 100 fM to 1 nM, and completes analysis within 30 minutes. Furthermore, when coupled with lateral flow test strip visualization, the system eliminates the need for sophisticated instrumentation and enables rapid single-tube amplification for interpretation of results. Based on the high mutation discrimination capability of the SCas12a system, the method achieves highly sensitive and specific LGR4 point mutation detection.},
}

*Point Mutation/genetics
Humans
*Receptors, G-Protein-Coupled/genetics
*Obesity/genetics
*CRISPR-Cas Systems/genetics
Nucleic Acid Amplification Techniques/methods

@article {pmid42716490,
year = {2026},
author = {Qu, Y and Wang, Y and Wang, Y and Tang, H and Chen, Q},
title = {SpacerScope: binary-vectorized, genome-wide off-target profiling for RNA-guided nucleases without prior candidate-site bias.},
journal = {Briefings in bioinformatics},
volume = {27},
number = {5},
pages = {},
pmid = {42716490},
issn = {1477-4054},
support = {32572957//National Natural Science Foundation of China/ ; },
mesh = {*CRISPR-Cas Systems ; *Software ; Humans ; Algorithms ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Genome, Plant ; Genomics/methods ; Computational Biology/methods ; },
abstract = {The precision of CRISPR/Cas systems is fundamental to their application in plant and animal biotechnology. However, comprehensive sequence-based off-target candidate discovery remains a computational bottleneck, particularly in large and complex genomes. Here we developed SpacerScope, an off-target candidate discovery framework that enables unbiased, genome-wide discovery by leveraging binary vectorization, bitwise filtering, and right-end-anchored alignment. Benchmarking against human CIRCLE-seq data demonstrated that SpacerScope recovered 100% of validated off-target sites (6142/6142), matching the sensitivity of exhaustive algorithms. Crucially, SpacerScope achieved this maximum candidate recovery while substantially reducing computational overhead. In large-genome evaluations, SpacerScope maintained low peak memory usage of 2.20 GiB and achieved substantial runtime improvements over indel-aware comparator tools, including more than 50-fold speedup relative to Cas-OFFinder 3 (544 s versus 29 185 s). Furthermore, comparative analyses in polyploid species, such as the octoploid strawberry, revealed that SpacerScope identified larger sequence-compatible candidate burdens than standard web-based design platforms. Our results establish SpacerScope as a high-speed framework for sequence-based genome-wide off-target candidate discovery across diverse and highly repetitive genomic landscapes. The source code and program was publicly available at https://github.com/charlesqu666/SpacerScope. Short Abstract CRISPR/Cas sequence-based off-target candidate discovery remains computationally challenging in large, repetitive, and polyploid genomes. Existing tools either miss indel-containing candidate sites or incur prohibitive runtime and memory costs. We developed SpacerScope, a binary-vectorized framework that enables unbiased, genome-wide off-target candidate discovery without pre-selected candidate sites. By integrating bitwise filtering with right-end-anchored alignment, SpacerScope recovered 100% of validated off-target sites in human CIRCLE-seq data while using only 2.20 GiB of memory and achieving more than 10-fold speedup over indel-aware alternatives. Evaluation in plant genomes, including rice and octoploid strawberry, further demonstrated SpacerScope's capacity to identify larger sequence-compatible candidate burdens overlooked by standard tools. SpacerScope thus provides a high-speed framework for sequence-based genome-wide off-target candidate discovery across diverse and highly repetitive genomic landscapes, supporting downstream prioritization.},
}

*CRISPR-Cas Systems
*Software
Humans
Algorithms
*RNA, Guide, CRISPR-Cas Systems/genetics
Genome, Plant
Genomics/methods
Computational Biology/methods

@article {pmid42716927,
year = {2026},
author = {Cong, T and Xu, R and Chen, X and Yuan, J and Lin, Z and Yang, W and Ju, A and Wang, L and Wang, Q and Zhang, J and Li, Y and Li, D and Zhu, M},
title = {Ultra-sensitive profiling of CRISPR-Cas off-target effects with Tracking-seq2.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42716927},
issn = {2041-1723},
support = {32571678//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82322054//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32025023//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32230064//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Hematopoietic Stem Cells/metabolism ; T-Lymphocytes/metabolism ; DNA End-Joining Repair ; },
abstract = {Accurate detection of off-target activity in primary human cells is crucial for ensuring the safety of gene therapies, yet existing methods often lack sufficient sensitivity. To address this limitation, we develop Tracking-seq2, an advanced technology that integrates exogenous 5' → 3' exonuclease treatment and non-homologous end joining (NHEJ) pathway inhibitors with the original Tracking-seq. Tracking-seq2 exhibits enhanced sensitivity in profiling off-target sites of diverse genome editors-including Cas9, Cas12a, cytosine base editors (CBEs), adenine base editors (ABEs), and prime editors (PEs). Critically, Tracking-seq2 is directly applicable to clinically relevant primary human cell types, such as T cells and CD34[+] hematopoietic stem and progenitor cells (HSPCs). Furthermore, our findings reveal that genomic variations drive distinct off-target heterogeneity across different individuals, highlighting the necessity for personalized safety assessment in clinical genome editing applications. Tracking-seq2 provides a robust platform for sensitive off-target detection in primary cells, with sensitivity comparable to or exceeding current state-of-the-art methods.},
}

Humans
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Hematopoietic Stem Cells/metabolism
T-Lymphocytes/metabolism
DNA End-Joining Repair

@article {pmid42717063,
year = {2026},
author = {Ocampo, RF and Orosco, C and Huang, B and West, MS and Parker, B and Jain, PK and Taylor, DW},
title = {Architecture of a DNA-guided Cas12a.},
journal = {Nature structural & molecular biology},
volume = {},
number = {},
pages = {},
pmid = {42717063},
issn = {1545-9985},
abstract = {CRISPR-Cas systems have largely been restricted to RNA-guided nucleases. Here, we present the cryo-electron microscopy structure of Acidaminococcus sp. Cas12a bound to a pseudo-DNA (ΨDNA) guide and RNA target, revealing how Cas12a can accomplish DNA-guided RNA recognition. The ΨDNA hairpin bridges the recognition and nuclease lobes, mimicking a PAM-proximal duplex and positioning the spacer to allow formation of a canonical RNA-DNA heteroduplex along the recognition lobe. This provides a structural framework for its activity and provides a potential blueprint for future engineering.},
}

@article {pmid42717197,
year = {2026},
author = {Zhang, H and Luo, S and Wang, X and Lin, L and Liang, R and Zhong, C and Zhang, Y and Zhao, W and Chen, Z and Liu, X and Gao, Y and Chen, F and Sun, N and Huang, J and Fei, T},
title = {Towards efficient perturbation for the noncoding genome.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42717197},
issn = {2041-1723},
support = {32470673//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {CRISPR-Cas Systems/genetics ; *RNA, Untranslated/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Genome ; Humans ; Animals ; CRISPR-Associated Protein 9/metabolism/genetics ; *Gene Editing/methods ; },
abstract = {Deciphering the functionality of the noncoding genome, which includes important cis-regulatory elements (CREs) and transcribed noncoding RNA genes, remains technically challenging. Here, using massively parallel genetic screening, we systematically benchmark the performance of five representative loss-of-function perturbation tools, including single-guide RNA (gRNA) mediated SpCas9 cleavage or CRISPR interference, and paired gRNA (pgRNA) involved dual-SpCas9, Big Papi (paired SpCas9 and SaCas9) or dual-enAsCas12a fragment deletion methods, in decoding the roles of the noncoding genome. For targeting CREs such as enhancers, dual-SpCas9 outperforms other methods with superior efficiency in destroying functional genomic regions. For perturbing noncoding RNA genes, in addition to dual-SpCas9, other RNA-targeting methods such as RNA interference are recommended to discriminate transcript-dependent or -independent roles. A deep learning model, DeepDC, with an associated web server, is built to facilitate optimal dual-SpCas9 pgRNA design for efficiently deleting a genomic fragment. Together, our work provides practical guidance on selecting appropriate loss-of-function tools to resolve the functional complexity of the noncoding genome.},
}

CRISPR-Cas Systems/genetics
*RNA, Untranslated/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
*Genome
Humans
Animals
CRISPR-Associated Protein 9/metabolism/genetics
*Gene Editing/methods

@article {pmid42720682,
year = {2026},
author = {Ullah, I and Ahmad, N and Bhat, AH and Hussain, A and Wei, F and Hongzhi, W},
title = {Engineering cold stress resilience in capsicum annuum through functional genomics and precision breeding.},
journal = {Plant cell reports},
volume = {45},
number = {10},
pages = {},
pmid = {42720682},
issn = {1432-203X},
mesh = {*Capsicum/genetics/physiology ; *Genomics/methods ; *Plant Breeding/methods ; Gene Expression Regulation, Plant/genetics ; *Cold-Shock Response/genetics/physiology ; Cold Temperature ; },
abstract = {This review synthesizes the molecular mechanisms of cold tolerance in pepper, integrating multi-omics data,genome editing, and precision breeding strategies to accelerate the development of cold-resilient cultivars. Cold stress is a significant environmental factor that affects the growth, productivity, and fruit quality of Capsicum annuum by impairing membrane integrity photosynthesis and cellular redox homeostasis. Although pepper has several endogenous cold-responsive regulators such as CaNAC035 and CabHLH035, along with antioxidant defense systems, its cold tolerance remains limited due to low transcriptional activation of key regulators, functional redundancy among cold-responsive genes, and the polygenicity of cold tolerance. These complexities, combined with low genetic diversity and linkage drag, have hindered the improvement of cold-resistant cultivars through conventional breeding. This review brings together the recent progress in understanding the molecular mechanisms of cold stress perception, signal transduction, transcriptional regulation, metabolic reprogramming, and phytohormone interactions in pepper. Precision Breeding 2.0 is a new innovation that combines the integration of multi-omics-based target identification with next-generation genome-editing techniques, allowing precise and multiplex engineering of complex and interconnected regulatory networks instead of single genes. We cover new approaches such as engineering the DREB/CBF pathway, allele-specific editing and targeted disruption of negative regulators to enhance the pathway(s) involved in cold response. Moreover, we propose a roadmap for integration of transcriptomics, proteomics, metabolomics, high-throughput phenomics, and speed breeding to accelerate the identification, validation, and deployment of superior alleles to boost cold tolerance. This review provides a foundation for developing climate-resilient pepper cultivars by connecting functional genomics with precision genome engineering approaches to maintain productivity under variable environmental conditions.},
}

*Capsicum/genetics/physiology
*Genomics/methods
*Plant Breeding/methods
Gene Expression Regulation, Plant/genetics
*Cold-Shock Response/genetics/physiology
Cold Temperature

@article {pmid42720869,
year = {2026},
author = {Lu, D and Li, Z and Zhu, P and Zhang, Z and Liu, P and Meng, L and Sun, X and Yang, Q and Song, J and Li, Z},
title = {Development and evaluation of a single-tube RT-ERA-CRISPR/Cas12a assay for simultaneous detection of enterovirus G and recombinant strains with papain-like cysteine protease gene insertion.},
journal = {Veterinary research communications},
volume = {50},
number = {6},
pages = {},
pmid = {42720869},
issn = {1573-7446},
support = {202304BI090001//Project of Innovation Guide and S&T Enterprise Cultivation of Yunnan Province/ ; 202102AE090039//Major Science and Technology Project of Yunnan Province/ ; 202401AS070010//Applied Basic Research Foundation of Yunnan Province/ ; YNWR-QNBJ-2020-211//Ten Thousand Talent Plans for Youth Top-notch Talents of Yunnan Province/ ; },
mesh = {Animals ; *Swine Diseases/virology/diagnosis ; Swine ; *CRISPR-Cas Systems ; *Enterovirus Infections/veterinary/diagnosis/virology ; Nucleic Acid Amplification Techniques/veterinary/methods ; Sensitivity and Specificity ; *Cysteine Proteases/genetics ; *Enteroviruses, Porcine/genetics/isolation & purification ; Diarrhea/veterinary/virology/diagnosis ; Feces/virology ; Rapid Diagnostic Tests ; },
abstract = {Enterovirus G (EV-G) strains carrying the papain-like cysteine protease (PLCP) gene insertion have recently emerged as important pathogens associated with diarrhea in weaned piglets. Herein, a single-tube reverse transcription-enzymatic recombinase amplification coupled with CRISPR/Cas12a (RT-ERA-CRISPR/Cas12a) dual-readout assay was established to concurrently detect pan-EV-G via conserved RNA-dependent RNA polymerase 3D-encoding gene and screen pathogenic PLCP-carrying recombinant strains. After systematic optimization of primer-probe set and reaction parameters, the analytical and clinical performances of the established assay were evaluated. The assay exhibited no cross-reaction with other common porcine diarrhea-associated viruses or closely related members of the Picornaviridae family. Its limits of detection reached 5.19 copies·µL[-1] for the 3D-encoding gene and 5.51 copies·µL[-1] for the PLCP gene, demonstrating superior sensitivity compared with conventional RT-PCR, RT-qPCR, RPA, and LAMP assays. Testing 96 field fecal samples yielded almost perfect agreement with RT-qPCR (κ = 0.95, concordance = 97.92%) and moderate consistency with conventional RT-PCR (κ = 0.68, concordance = 87.88%), respectively. Unlike multiplex CRISPR-Cas12a platforms that suffer from reporters' interference from Cas12a non-specific cleavage and dual nfo-probe systems prone to oligonucleotide dimerization, this work implemented separated visual readouts: nfo-probe lateral flow strips for 3D-encoding gene amplicons and Cas12a collateral cleavage for PLCP gene amplification products. An 85 ℃ heating step neutralized endonuclease Ⅳ to remove potential CRISPR signal inhibition, realizing unified single-tube detection. This assay is rapid, cost-efficient, and features high specificity and sensitivity. It offers a practical on-farm surveillance tool for virulent PLCP-recombinant EV-G, facilitating prevention and control of swine diarrheal diseases and safeguarding pork production safety.},
}

Animals
*Swine Diseases/virology/diagnosis
Swine
*CRISPR-Cas Systems
*Enterovirus Infections/veterinary/diagnosis/virology
Nucleic Acid Amplification Techniques/veterinary/methods
Sensitivity and Specificity
*Cysteine Proteases/genetics
*Enteroviruses, Porcine/genetics/isolation & purification
Diarrhea/veterinary/virology/diagnosis
Feces/virology
Rapid Diagnostic Tests

@article {pmid42721033,
year = {2026},
author = {Park, HH},
title = {Diverse routes to Cas3 activation in type I CRISPR-Cas immunity.},
journal = {Cell reports},
volume = {45},
number = {9},
pages = {117899},
doi = {10.1016/j.celrep.2026.117899},
pmid = {42721033},
issn = {2211-1247},
abstract = {Jiang et al. show that the type I-A CRISPR system of Saccharolobus islandicus recruits Cas3 only after target recognition. Their study reveals different Cas3 activation mechanisms and a dual role of ATP in target degradation and collateral cleavage.},
}

@article {pmid42722935,
year = {2026},
author = {Demir, B and Aydin, EA and Sağlam, A},
title = {Bridging the bench-to-field gap: a quantitative multi-omics and CRISPR/Cas9 framework for climate-resilient agriculture.},
journal = {Transgenic research},
volume = {35},
number = {1},
pages = {},
pmid = {42722935},
issn = {1573-9368},
mesh = {Multiomics ; *CRISPR-Cas Systems/genetics ; *Crops, Agricultural/genetics/growth & development ; *Plants, Genetically Modified/genetics/growth & development ; Climate Change ; Gene Editing ; *Agriculture/methods ; Drought Resistance ; },
abstract = {Global climate change and shrinking freshwater supplies threaten agricultural sustainability. A major translational bottleneck exists: drought-tolerant genotypes developed in labs often fail in open-field conditions due to phenotypic mismatch. Traditional single-gene approaches and static lab tests overlook genotype-by-environment (G × E) interactions, hormonal tradeoffs (involving Abscisic Acid, Jasmonic Acid, Brassinosteroids, and Melatonin), and artificial effects of pots. This review presents an integrated quantitative selection framework to bridge lab and field results. In this review, we propose a conceptual three-stage Predictive Translational Workflow designed to bridge lab and field outcomes by integrating multi-omics envirotyping with advanced mixed models like (G × E)-BLUP and MegaLMM. These methods group complex transcriptomic and metabolomic data into functional networks, filtering out lab artifacts and pinpointing core regulatory nodes stable in real climates. Finally, we show how this pipeline enables precise CRISPR/Cas9 editing of native promoters, reducing agronomic yield penalties in normal conditions and offering a streamlined, non-GMO path to field-resilient crops.},
}

Multiomics
*CRISPR-Cas Systems/genetics
*Crops, Agricultural/genetics/growth & development
*Plants, Genetically Modified/genetics/growth & development
Climate Change
Gene Editing
*Agriculture/methods
Drought Resistance

@article {pmid42141726,
year = {2026},
author = {Dong, J and Zhang, K and Song, M and Xiao, M and Lu, X and Ge, S and Zheng, Z and Li, M and Zhu, JK and Lei, M},
title = {CRISPR-mediated intronic knock-in of pre-amiRNA enables targeted gene silencing.},
journal = {Plant communications},
volume = {7},
number = {9},
pages = {101916},
doi = {10.1016/j.xplc.2026.101916},
pmid = {42141726},
issn = {2590-3462},
mesh = {*Introns/genetics ; *Gene Silencing ; *MicroRNAs/genetics ; Plants, Genetically Modified ; *CRISPR-Cas Systems ; *Gene Knock-In Techniques/methods ; },
abstract = {This study introduces an intronic artificial microRNA (IamiRNA) strategy that combines CRISPR-Cas9-mediated knock-in with endogenous miRNA processing for targeted gene silencing in plants. By inserting amiRNA precursors into introns of endogenous genes, this approach enables effective, tissue-specific gene silencing without persistent transgene expression, offering a promising tool for functional genomics and crop improvement.},
}

*Introns/genetics
*Gene Silencing
*MicroRNAs/genetics
Plants, Genetically Modified
*CRISPR-Cas Systems
*Gene Knock-In Techniques/methods

@article {pmid42319116,
year = {2026},
author = {Qin, Z and Surnido, W and Hozumi, S and Hirano, T and Sawatsubashi, S and Mizuta, H and Uji, T},
title = {Expanding the scope of precision editing in seaweeds through the application of a novel CRISPR-associated nuclease 12a-aligned CRISPR system in Ulva prolifera.},
journal = {Journal of experimental botany},
volume = {77},
number = {17},
pages = {5560-5565},
doi = {10.1093/jxb/erag302},
pmid = {42319116},
issn = {1460-2431},
support = {//Open Innovation Platform with Enterprise, Research Institute and Academia/ ; //Japan Science and Technology Agency/ ; JPJS00420230001//Genome Editing Technology Research Grant, and the JSPS Program for Forming Japan's Peak Research Universities/ ; },
mesh = {*Ulva/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Seaweed/genetics ; Edible Seaweeds ; },
abstract = {Seaweeds, such as the fast-growing green alga Ulva prolifera, can be harnessed as valuable marine crops. The lack of scalable genome-editing tools hampers functional genomics to explore and elucidate algal molecular pathways with industrial importance. Here, we expanded precision genome modification in seaweeds by successfully demonstrating gene editing with a transgene-free AT-rich-targeting CRISPR-associated protein (Cas) system in U. prolifera. By evaluating various delivery buffers, comparing different Cas systems, and optimizing incubation temperatures, we determined suitable conditions for more widespread applicability of a novel Cas12a-aligned ST8 editor. We obtained >50 ST8-mediated knockout mutants of a toxin-based endogenous marker gene, UpAPT, at 28 °C post-delivery incubations. Our work diversified the applicable genome-editing tools in seaweeds, advancing algal functional genomics and providing more strategies to precisely target unexplored seaweed resources.},
}

*Ulva/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
*Seaweed/genetics
Edible Seaweeds

@article {pmid42323890,
year = {2026},
author = {Zhang, TL and Chen, MK and Li, YM and Yu, HW and Ye, LD},
title = {A CRISPR-Cas9 Toolkit Enabling Tunable Integration and Transient Homologous Recombination Enhancement in Yarrowia lipolytica.},
journal = {Biotechnology and bioengineering},
volume = {123},
number = {10},
pages = {2643-2654},
pmid = {42323890},
issn = {1097-0290},
support = {32471481//National Natural Science Foundation of China/ ; 226-2025-00043//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*Yarrowia/genetics ; *Homologous Recombination/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; },
abstract = {Although the oleaginous yeast Yarrowia lipolytica is a promising microbial cell factory, its application remains constrained by inefficient homology-directed repair (HDR) and a lack of precise genomic integration tools. To address these limitations, we developed a comprehensive genetic toolkit featuring three synergistic advancements. First, we systematically identified 55 neutral integration sites with tunable expression profiles, enabling stable, position-independent gene integration with predictable transcriptional output across a 12.88-fold dynamic range. Second, we established a dual-readout high-throughput screening platform combining colony morphology analysis with hrGFP fluorescence. This approach accurately measures locus-specific homologous recombination (HR) efficiency while eliminating false positives by dominant non-homologous end joining (NHEJ). Third, we engineered a transient HR enhancement system by fusing the Sae2 exonuclease to Cas9 via a flexible (GGGGS)3 linker. This fusion significantly boosts HR efficiency and surpasses the cleavage activity of unmodified Cas9 without introducing permanent genomic modifications or compromising cellular fitness. Finally, HR efficiency for single-gene integration was increased from 46.5% to 77.5% while the dual-locus editing efficiency reached 64.1% when using 500-bp homology arms, and the engineered strains demonstrated improved genetic stability compared to those with constitutive HR enhancement.},
}

*Yarrowia/genetics
*Homologous Recombination/genetics
*CRISPR-Cas Systems/genetics
*Gene Editing/methods

@article {pmid42325108,
year = {2026},
author = {Guo, Q and Shen, Q and Zhao, L and Zhou, X and Yan, S and Xue, Y and Zheng, Y},
title = {One Plasmid Is All You Need: Genome Editing in Escherichia coli Using Endogenous TnpB and Endogenous Recombination System.},
journal = {Biotechnology and bioengineering},
volume = {123},
number = {10},
pages = {2690-2701},
pmid = {42325108},
issn = {1097-0290},
support = {2022YFC2105400//National Key Research and Development Program of China/ ; },
mesh = {*Escherichia coli/genetics ; *Plasmids/genetics ; *Gene Editing/methods ; *Recombination, Genetic ; *Escherichia coli Proteins/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Escherichia coli (E. coli) is a key workhorse of biotechnology. Commonly used CRISPR-Cas9 systems for E. coli genome editing are complex and impose metabolic stress on the host, creating demand for more streamlined strategies. Recent studies identified the IS605 transposon-associated TnpB as a programmable RNA-guided (ωRNA) DNA endonuclease, prompting us to explore whether endogenous TnpB in E. coli (EcoTnpB) could be harnessed for genome editing. Biochemical and cellular analyses demonstrated that EcoTnpB efficiently cleaves both chromosomal and plasmid DNA at custom-specified sites in a TAM-dependent manner. Interestingly, E. coli possesses an endogenous recombination machinery capable of repairing EcoTnpB-induced DNA double-strand breaks (DSBs), challenging the long-held view that bacteria lack efficient homologous recombination systems. Based on these findings, we established a single-plasmid editing system (SPEED) in which genome editing is achieved by simply providing ωRNA and a homologous recombination template. By utilizing endogenous EcoTnpB together with the host HR pathway, this system enabled inducible and seamless genome editing at multiple genomic loci in BL21 (DE3), with editing efficiencies ranging from approximately 29% to 56%. Our results demonstrate for the first time that endogenous TnpB can be harnessed for genome editing and may hold potential for broader applications, such as species-specific antimicrobial development.},
}

*Escherichia coli/genetics
*Plasmids/genetics
*Gene Editing/methods
*Recombination, Genetic
*Escherichia coli Proteins/genetics/metabolism
CRISPR-Cas Systems

@article {pmid42366734,
year = {2026},
author = {Köse, AM and Ranalli, M and Trivanovic, D and Maslovaric, I},
title = {Minimizing Off-Target Effects of CRISPR-Cas9 With Optimized sgRNA: Evaluation of Efficiency and Specificity in the Tumor Protein 53 (TP53) Region.},
journal = {Biotechnology and bioengineering},
volume = {123},
number = {10},
pages = {2677-2689},
pmid = {42366734},
issn = {1097-0290},
support = {//Ministry of Science, Technological Development, and Innovation of the Republic of Serbia/ ; },
mesh = {*CRISPR-Cas Systems ; *Tumor Suppressor Protein p53/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; *Gene Editing/methods ; Machine Learning ; },
abstract = {CRISPR-Cas9 is a widely used genetic tool with therapeutic potential in molecular biology. CRISPR-Cas9 enables precise genome editing by its ability to target specific DNA sequence. After off-target and on-target regions are identified, CRISPR-Cas9 is applied to these regions based on the match between the guide RNA (gRNA) and target DNA sequence. This study points to the off-target impact of mismatches between the gRNA and target DNA on exon regions of the TP53 gene, which are involved in regulating multiple genes and cellular functions. Off-target positions are typically evaluated using scoring methods. In this study, we have used latent class analysis to reveal subclasses of off-target positions. Thus, we have created the levels of off-target positions and evaluated the effects of mismatching positions within these classes using machine learning classifiers. The results revealed that mismatching positions could be categorized into three levels: low, middle, and high off-target positions. We have improved a computational framework to minimize off-target effects and to identify the PAM sequences in the gRNA design. Thus, carefully designed gRNAs will ensure that desired genetic edits are performed and target variants are achieved. This work will avail the future research aimed at optimizing genome editing by customizing CRISPR-Cas9 to target specific protospacer DNA through gRNA.},
}

*CRISPR-Cas Systems
*Tumor Suppressor Protein p53/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics
Humans
*Gene Editing/methods
Machine Learning

@article {pmid42496673,
year = {2026},
author = {Ning, Z and Wang, G},
title = {Context-Dependent Cancer Vulnerabilities: CRISPR Screening under Inflammatory Stress.},
journal = {Cancer research},
volume = {86},
number = {18},
pages = {4457-4459},
doi = {10.1158/0008-5472.CAN-26-3042},
pmid = {42496673},
issn = {1538-7445},
mesh = {Humans ; *Neoplasms/genetics/pathology/metabolism/immunology ; *Inflammation/genetics ; Tumor Microenvironment/genetics ; *CRISPR-Cas Systems ; Animals ; Cytokines/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Oxidative Stress ; Signal Transduction ; Stress, Physiological ; },
abstract = {Genome-wide CRISPR screens have systematically identified genes required for cancer cell survival, yet these studies are typically performed under standardized conditions that do not fully recapitulate the physiologic stresses encountered within the tumor microenvironment. In a recent issue of Nature Genetics, Cheruiyot and colleagues perform genome-wide loss-of-function screens under inflammatory conditions induced by interferon (IFN) β, IFNγ, and tumor necrosis factor (TNF), revealing that distinct cytokines impose different genetic requirements for tumor cell survival. The study shows that inflammatory signaling reshapes the genetic dependency landscape in a cytokine-specific manner. Mechanistic analyses identify the glycosylphosphatidylinositol (GPI) transamidase complex and Fitm2 as representative examples of genes that become selectively required under inflammatory stress by maintaining membrane protein maturation, endoplasmic reticulum homeostasis, and resistance to oxidative stress. These findings broaden our understanding of how inflammatory cytokines influence tumor cell biology beyond transcriptional regulation and immune recognition. More broadly, the study highlights the value of incorporating physiologically relevant conditions into functional genetic screens, suggesting that conventional dependency maps capture only part of the genetic requirements for tumor survival. Applying similar approaches to other microenvironmental stresses-including hypoxia, metabolic competition, extracellular matrix remodeling, and stromal signaling-may uncover additional therapeutic opportunities for cancer immunotherapy.},
}

Humans
*Neoplasms/genetics/pathology/metabolism/immunology
*Inflammation/genetics
Tumor Microenvironment/genetics
*CRISPR-Cas Systems
Animals
Cytokines/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats
Oxidative Stress
Signal Transduction
Stress, Physiological

@article {pmid42612243,
year = {2026},
author = {Xu, S and Neupane, S and Wang, L},
title = {CRISPR-Cas9-induced genetic mosaicism in three species of the microcrustacean Daphnia.},
journal = {Genome},
volume = {69},
number = {},
pages = {1-7},
doi = {10.1139/gen-2026-0049},
pmid = {42612243},
issn = {1480-3321},
mesh = {Animals ; *Mosaicism ; *CRISPR-Cas Systems ; *Daphnia/genetics/classification ; Female ; *Gene Editing ; *Daphnia magna/genetics ; Daphnia pulex/genetics ; Genotype ; },
abstract = {Genetic mosaicism can arise from in vivo CRISPR-Cas9 gene editing, especially in the embryos. This study evaluates the extent of genetic mosaicism resulted from CRISPR-Cas9-mediated knockout for 11 genes in the freshwater microcrustacean Daphnia magna, Daphnia pulex, and Daphnia sinensis. Based on extensive genotyping data of the asexually produced progenies of successfully edited females, we find strong evidence of mosaicism in 9 of these genes. The genotyping data also suggest that the gene editing activity can take place as early as the one-cell embryo stage and extends into the 32-cell and later stages. This study establishes genetic mosaicism as an important feature of Cas9-mediated gene editing in Daphnia.},
}

Animals
*Mosaicism
*CRISPR-Cas Systems
*Daphnia/genetics/classification
Female
*Gene Editing
*Daphnia magna/genetics
Daphnia pulex/genetics
Genotype

@article {pmid42626870,
year = {2026},
author = {Ding, G and Li, Y and Xiao, Y and Li, Y and Yang, J and Zhao, R and Tang, Y and Li, B},
title = {A lock-and-key aptamer probe gates CRISPR/Cas12a trans-cleavage for antibiotic detection.},
journal = {Chemical communications (Cambridge, England)},
volume = {62},
number = {72},
pages = {17979-17982},
doi = {10.1039/d6cc04487e},
pmid = {42626870},
issn = {1364-548X},
mesh = {*Aptamers, Nucleotide/chemistry/metabolism ; *Anti-Bacterial Agents/analysis ; *CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism ; *Biosensing Techniques/methods ; *Endodeoxyribonucleases/metabolism ; Bacterial Proteins ; },
abstract = {A lock-and-key aptamer-CRISPR/Cas12a strategy was developed to convert antibiotic recognition into signal-off fluorescence. Antibiotic binding disrupted the locking domain-maintained stem-loop structure, suppressed T4 DNA polymerase-mediated activator generation and inhibited Cas12a trans-cleavage, enabling selective antibiotic detection and providing a programmable strategy for small-molecule sensing.},
}

*Aptamers, Nucleotide/chemistry/metabolism
*Anti-Bacterial Agents/analysis
*CRISPR-Cas Systems
*CRISPR-Associated Proteins/metabolism
*Biosensing Techniques/methods
*Endodeoxyribonucleases/metabolism
Bacterial Proteins

@article {pmid42707868,
year = {2026},
author = {Lin, L and Luo, F and Liu, S and Tong, X and Xu, X and Yi, W and Deng, X and Luo, H},
title = {CRISPR-based point-of-care diagnostics for viral hepatitis: from molecular design to clinical implementation.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {14},
number = {},
pages = {1892898},
pmid = {42707868},
issn = {2296-4185},
abstract = {Timely etiological diagnosis of viral hepatitis is essential for surveillance, treatment initiation, linkage to care, and elimination efforts. In resource-limited settings, however, diagnostic pathways are frequently disrupted by centralized nucleic acid testing, specimen-transport requirements, delayed reporting, and loss to follow-up. CRISPR-Cas-based point-of-care testing may help shorten these pathways, but its clinical value cannot be judged by analytical sensitivity alone. This review evaluates CRISPR-Cas strategies for detecting hepatitis A, B, C, D, and E viruses by linking molecular design to virus-specific clinical and public-health decisions. We compare target selection, Cas effectors, amplification strategies, readout formats, specimen processing, genotype coverage, clinical validation, and implementation feasibility. For each virus, the evidence is evaluated in relation to the principal diagnostic need, the potential contribution of CRISPR-Cas, the maturity of the available evidence, and the remaining requirements for deployment. Current evidence is most advanced for HBV and HCV, whereas HAV, HDV, and HEV applications remain less developed or more context dependent. Future platforms should prioritize clinically meaningful detection thresholds, closed-tube sample-to-answer workflows, internal controls, genotype-diverse validation, reagent stability, non-expert usability, and transparent pathway-level cost evaluation.},
}

@article {pmid42708411,
year = {2026},
author = {Feussner, M and Birkholz, N and Lee, SY and Beisel, CL and Park, HH and Brown, CM and Fineran, PC and Weinberg, Z},
title = {An expanded realm of anti-CRISPR-associated proteins and regulatory mechanisms.},
journal = {Nucleic acids research},
volume = {54},
number = {17},
pages = {},
pmid = {42708411},
issn = {1362-4962},
support = {01IM19001//German Academic Exchange Service (DAAD)/ ; 468749960//German Research Foundation/ ; //Health Sciences Career Development Postdoctoral Fellowship/ ; //University of Otago/ ; //Bioprotection Aotearoa/ ; //Centre of Research Excellence/ ; //Tertiary Education Commission, NZ/ ; //James Cook Research Fellowship/ ; //Royal Society of New Zealand, Te Apārangi/ ; //Leipzig University/ ; },
mesh = {5' Untranslated Regions ; Operon ; *Viral Proteins/metabolism/genetics ; *Bacteriophages/genetics ; *Repressor Proteins/metabolism/genetics ; *CRISPR-Cas Systems ; Helix-Turn-Helix Motifs ; RNA, Messenger/metabolism ; Nucleotide Motifs ; Gene Expression Regulation, Bacterial ; },
abstract = {Many bacteriophages encode anti-CRISPR (Acr) proteins that inhibit bacterial CRISPR-Cas immune systems. Rapid acr gene expression upon phage entry enables CRISPR-Cas neutralization but can impact phage fitness if unregulated. Therefore, Acr production is often controlled by distinct families of co-encoded anti-CRISPR-associated (Aca) proteins, which are usually helix-turn-helix (HTH) regulators that bind DNA within acr-aca operon promoters. Previously, we demonstrated that the Aca2 family additionally represses Acr production translationally by binding structured RNA motifs within the 5' untranslated region (UTR) of the acr-aca mRNA. Here, through systematic bioinformatic analyses, we provide evidence of structured RNA motifs in the 5' UTRs of operons encoding members of other Aca families and show that Aca1 also specifically binds its cognate RNA motif. Additionally, many Aca proteins are predicted to regulate not only their own but also adjacent operons with potential anti-defence genes. Indeed, we show that Aca14, newly identified in this study, represses two predicted anti-defence operons. Aca14 is a ribbon-helix-helix domain protein, revealing regulatory diversity beyond the canonical HTH Aca family members. Collectively, our findings expand our understanding of acr regulation in mobile genetic elements and reveal novel mechanisms by which phages fine-tune anti-defence gene expression.},
}

5' Untranslated Regions
Operon
*Viral Proteins/metabolism/genetics
*Bacteriophages/genetics
*Repressor Proteins/metabolism/genetics
*CRISPR-Cas Systems
Helix-Turn-Helix Motifs
RNA, Messenger/metabolism
Nucleotide Motifs
Gene Expression Regulation, Bacterial

@article {pmid42709801,
year = {2026},
author = {Kumar, A and Nahar, N and Chouhan, D and McLean, JS and Bor, B and Dong, PT and He, X},
title = {A host-encoded prophage targets a Candidate Phyla Radiation bacterium and shapes episymbiotic interactions.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {37},
pages = {e2615890123},
doi = {10.1073/pnas.2615890123},
pmid = {42709801},
issn = {1091-6490},
support = {R01DE023810//HHS | NIH | National Institute of Dental and Craniofacial Research (NIDR)/ ; R01DE031274//HHS | NIH | National Institute of Dental and Craniofacial Research (NIDR)/ ; T90DE026110//HHS | NIH | National Institute of Dental and Craniofacial Research (NIDR)/ ; },
mesh = {*Prophages/genetics/physiology ; *Symbiosis ; Lysogeny ; Humans ; Bacteriophages ; },
abstract = {The Patescibacteriota, also known as the Candidate Phyla Radiation (CPR), represent a large lineage of ultrasmall bacteria with highly reduced genomes and obligate dependence on bacterial hosts. Although genomic analyses have revealed CRISPR-Cas and restriction-modification systems in many CPR genomes, no cognate bacteriophages (phages) have been isolated, leaving CPR-phage interactions unexplored. Nanosynbacter lyticus TM7x, the first cultivated CPR bacterium, grows episymbiotically on its host, Schaalia odontolytica XH001, in the human oral microbiome. Here, we identify Xhp1, an inducible prophage of XH001 that is preferentially activated during episymbiosis with TM7x. Released Xhp1 particles infect prophage-free XH001 via distinct strategies determined by host growth mode, establishing lysogeny under planktonic conditions but driving lytic infection during surface-associated growth. Xhp1 also binds efficiently to TM7x and exhibits limited infection under the conditions tested, indicating direct phage-CPR interactions. Importantly, TM7x modulates Xhp1 availability in a spatially dependent manner. In planktonic culture, free-floating TM7x reduces lysogenic conversion of XH001ΔXhp1, consistent with TM7x acting as a phage sink that lowers effective phage concentration. In contrast, during surface-associated growth, TM7x increases XH001ΔXhp1 susceptibility to lytic infection, likely by locally concentrating phage particles within a constrained niche. These results demonstrate that CPR bacteria can regulate viral encounter rates through spatial organization. In spatially structured environments such as oral biofilms, such modulation may shape infection dynamics and community structure. Together, this work characterizes the first CPR-targeting phage and reveals a an important role for phages in CPR-host bacteria interactions.},
}

*Prophages/genetics/physiology
*Symbiosis
Lysogeny
Humans
Bacteriophages

@article {pmid42709802,
year = {2026},
author = {Yang, X and Ouyang, H and Xu, H and Peng, L and Cui, J and Zhang, B and Huang, B and Wang, C and Li, G and Cao, X},
title = {In vivo genome-wide CRISPR screens identify FOXR1 as a suppressor of CD8[+] T cell antitumor immunity.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {37},
pages = {e2534362123},
doi = {10.1073/pnas.2534362123},
pmid = {42709802},
issn = {1091-6490},
support = {82388201//National Natural Science Foundation of China/ ; 32525028//National Natural Science Foundation of China/ ; 2025-I2M-XHZY-019//Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences/ ; 2024-I2M-ZD-005//Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences/ ; SZS2023005//Suzhou1114 Key Laboratory Program/ ; BK20250003//Basic Research Program of Jiangsu/ ; },
mesh = {Animals ; *Forkhead Transcription Factors/genetics/metabolism/immunology ; Humans ; *CD8-Positive T-Lymphocytes/immunology ; Mice ; Lymphocyte Activation/immunology ; CRISPR-Cas Systems ; *Neoplasms/immunology/therapy/genetics ; Cell Line, Tumor ; },
abstract = {T cell dysfunction critically limits the efficacy of T cell-based immunotherapies in solid tumors, yet the intrinsic regulators of T cell dysfunction remain incompletely understood. Through an in vivo genome-wide CRISPR screen in tumor-infiltrating CD8[+] T cells, we identified Forkhead Box R1 (FOXR1) as a potent transcriptional suppressor of CD8[+] T cell effector functions. Genetic ablation of FOXR1 significantly enhanced cytokine production and cytotoxic capacity in both murine and human CD8[+] T cells, whereas its overexpression impaired T cell activation and effector molecule expression. Mechanistically, multiomics integration of RNA-seq, CUT&Tag-seq, and ATAC-seq revealed that FOXR1 binds directly to promoter regions of key effector genes, including IL2, GZMB, and PRF1, and represses their expression. Importantly, FOXR1 deletion in human anti-CD19 CAR T cells improved their efficacy against solid tumors, demonstrating that FOXR1 is a checkpoint of T cell effector function and targeting FOXR1 is a promising strategy to enhance CAR T cell efficacy against solid tumors.},
}

Animals
*Forkhead Transcription Factors/genetics/metabolism/immunology
Humans
*CD8-Positive T-Lymphocytes/immunology
Mice
Lymphocyte Activation/immunology
CRISPR-Cas Systems
*Neoplasms/immunology/therapy/genetics
Cell Line, Tumor

@article {pmid42710586,
year = {2026},
author = {Suzuki, S and Aung, MS and Matsubara, K and Kimura, Y and Ito, M and Urushibara, N and Kawaguchiya, M and Ohashi, N and Higuchi, T and Takahashi, S and Kobayashi, N},
title = {Clonal diversity and genetic characteristics of extraintestinal pathogenic Escherichia coli in northern Japan: identification of the potentially prevalent clones phylogroup B2-ST357 and ST1193.},
journal = {Journal of global antimicrobial resistance},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jgar.2026.09.001},
pmid = {42710586},
issn = {2213-7173},
abstract = {OBJECTIVES: Extraintestinal pathogenic Escherichia coli (ExPEC) is a leading cause of bloodstream and urinary tract infections, often showing multiple drug resistance associated with production of extended-spectrum beta-lactamases (ESBLs). The present study aimed at revealing the clonal structure and diversity of ExPEC in northern Japan and its relation to antimicrobial resistance (AMR) profiles and determinants, and genetic features.

METHODS: Phylogenetic group and sequence type (ST) based on MLST scheme were determined for clinical isolates of ExPEC. PCR and sequencing were used to identify AMR determinants, virulence factors and CRISPR-Cas systems, along with antimicrobial susceptibility test by the broth microdilution method.

RESULTS: A total of 454 ExPEC isolates (150 from blood and 304 from urine/other specimens) were collected for 6 months. The major phylogenetic group was B2 (79.5%), and the remaining were D (9.9%), B1 (5.7%), F (3.1%), and other groups (A, C, G; 1.8% in all). Among the 68 STs identified, ST131 was the most common (33.7%), followed by ST95 (15.6%), ST357 (9.9%), ST1193 (7%), and ST73 (4%), all of which belonged to phylogroup B2. Five types of ESBL genes including the dominant blaCTX-M-15 or blaCTX-M-27 were detected in 17% of isolates, which belonged mostly to the ST131 complex. CRISPR-Cas type I-E was present mostly in phylogroup B1 and D, while I-F in 3 STs of phylogroup B2.

CONCLUSION: The potentially increased prevalence of ST357, ST1193, and animal-related clones was revealed to be novel clonal features of ExPEC in northern Japan.},
}

@article {pmid42712777,
year = {2026},
author = {Pattapulavar, V and Ramanujam, S and Yellampalli, A and Badiginchala, HS and Christopher, JG},
title = {Molecular biology and integrated strategies for activating cryptic biosynthetic gene clusters toward next-generation antibiotic discovery.},
journal = {Frontiers in bioinformatics},
volume = {6},
number = {},
pages = {1893206},
pmid = {42712777},
issn = {2673-7647},
abstract = {Antimicrobial resistance (AMR) has been identified as one of the 21st century's severest global public health crises. AMR led to an estimated 4.95 million deaths in 2019 and will claim 10 million lives a year by 2050 in the absence of targeted interventions. During the same period, the number of novel antibiotics discovered has decreased drastically as many researchers are rediscovering known antibiotics, non-model microorganisms are poorly understood or difficult to culture and antibiotic research and development investment has declined drastically. However, high-throughput whole genome sequencing and the subsequent application of bioinformatics in bacterial and fungal genomes have shown that a numerous of cryptic or silent biosynthetic gene clusters (BGCs) remain latent at ambient laboratory conditions since their genes are transcriptionally inactive. Cryptic BGCs represent a vast source of unique secondary metabolites, many of which may yield novel antibacterial, antifungal, anti-cancer and other potentially valuable natural products. This review discusses the biological relevance of cryptic BGCs, the major limiting factors that restricts their activation and novel strategies that have been employed to activate them and exploit their potential to produce novel natural products. The review focuses on biological approaches including CRISPR-Cas mediation for the activation of cryptic BGCs, promoter engineering, pathway refactoring, and heterologous expression; biochemical strategies such as Osman, OsMAC, Precursor Feeding, Chemical Elicitation, Epigenetic Regulation and Co-cultivation and technology-based strategies such as Genome mining, Microfluidic Cultivation systems, High-Throughput Screening, Metabolomics, Molecular Networking and Artificial Intelligence and Machine Learning based prediction of BGCs and their metabolites. The use of multi-omics technologies combined with synthetic biology to achieve better discovery, characterization and large-scale production of novel natural products is also discussed herein. Finally, we will talk about the ecological significance and evolutionary advantage of cryptic BGCs' role in interactions between microorganisms, such as competition, communication, symbiosis and environmental adaptability, so as to provide a useful background for accelerating next-generation antibiotics.},
}

@article {pmid42713177,
year = {2026},
author = {Nwagwu, CS and Nwachukwu, OL and Emencheta, SC and Onugwu, AL and Ezeibe, EN and Ugwu, CN and Nnamani, PO and Tarirai, C and Ofokansi, K and Attama, A},
title = {Role of nanoclusters in the design and development of novel strategies for diagnosis and treatment of infectious diseases.},
journal = {Nanoscale advances},
volume = {},
number = {},
pages = {},
pmid = {42713177},
issn = {2516-0230},
abstract = {Despite major advances in sanitation, vaccination, and antimicrobial chemotherapy, infectious diseases continue to pose a huge threat to global health. The ever-increasing occurrences of antimicrobial resistance, emerging pathogens, and persistent diagnostic delays further exacerbate this situation. Over the years, nanotechnology has generated a lot of material platforms to address these failures, and in this study, we critically examine nanoclusters-ultrasmall, atomically precise assemblies (<2 nm cores) with discrete electronic states, as an under-recognized but potentially transformative class of materials for infectious disease diagnostics and therapeutics. Their quantized optical properties, precision ligand chemistry, and unusually high surface-to-volume ratios enable behaviours fundamentally inaccessible to larger nanomaterials, including ratiometric fluorescence reporting, rapid redox-driven antimicrobial activity, and programmable bio-recognition at near-molecular resolution. First, we disentangle NCs from larger nanomaterials by focusing on the consequences of discrete electronic states, ultrasmall hydrodynamic radii, and well-defined ligand shells for transport, clearance, and interactions with pathogens and host-derived biomolecules. Furthermore, the review synthesizes advances in NC-enabled diagnostics, including DNA and aptamer templated Au, Ag, and Cu NC probes, CRISPR/Cas integrated ratiometric sensors, and nanozyme-based readouts that routinely achieve single colony-forming unit or sub-femtomolar detection of bacterial, viral, fungal, and protozoan nucleic acids and antigens. On the therapeutic side, we discuss how the same structural characteristics can be exploited to engineer NCs that mediate controlled ROS generation, membrane and biofilm disruption, quorum sensing interference, photothermal and NIR II photodynamic therapy, and ligand-directed drug delivery, with case studies spanning multidrug-resistant bacteria, opportunistic fungi, and selected parasitic infections. At the same time, we looked at some of the major barriers to clinical translation that remain unaddressed. We show with this study that nanoclusters are not merely "smaller nanoparticles" but a distinct chemical system, capable of enabling modular, multiplexed, and clinically relevant infectious disease interventions. To realize this potential, future work must couple atomic-level design with rigorous pharmacokinetic, toxicodynamic, and translational engineering frameworks.},
}

@article {pmid42713468,
year = {2026},
author = {Riaz Ahmed, S and Khan, J and Ijaz, I and Ali, Z and Rasul, I and Aslam, M and Ud-Din, N and Liu, Y and Tariq, MS and Skalicky, M and Tan, DKY},
title = {CRISPR-Cas technologies for precision genome editing in plants: advances, applications, and future perspectives.},
journal = {Frontiers in plant science},
volume = {17},
number = {},
pages = {1902159},
pmid = {42713468},
issn = {1664-462X},
abstract = {Developing climate-smart crops with enhanced crop productivity, nutritional quality, resistance to biological and environmental stressors is vital for global food security. While hybrid breeding forms the cornerstone of modern crop improvement, conventional breeding approaches are limited by genetic barriers and prolonged breeding cycles. CRISPR-Cas based genome editing has revolutionized plant biology by allowing precise, efficient, and multiplex genetic modifications. This review provides a comprehensive synthesis of a recent advances in CRISPR-Cas technologies and their strategic applications in crop genetics and hybrid breeding. We summarize major genome-editing strategies, including gene knock-out, base editing (BE), knock-in, gene replacement, epigenome editing, and transcriptional regulation. Furthermore, we contrast stable, transient, and DNA-free delivery systems, highlighting ribonucleoprotein (RNP)-mediated delivery for minimizing off-target effects and avoiding transgene integration. We showcase how these technologies accelerate hybrid breeding by engineering male sterility systems, fixing heterosis, and generating high-throughput mutant libraries for trait discovery. Finally, we synthesize major bottlenecks in tissue culture-independent transformation and delivery systems, while outlining how emerging paradigms like de novo domestication and synthetic biology will shape the future of climate-resilient agriculture.},
}

@article {pmid42714094,
year = {2026},
author = {Zheng, S and Li, X and Li, F and Ren, X and Chen, J and Zou, Y and Yang, FH and Xia, Y and Tan, W},
title = {Generation and Phenotypic Characterization of a CRISPR/Cas9-Engineered Cracd-Deficient Mouse Model for Post-Myocardial Infarction Remodeling Studies.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {235},
pages = {},
doi = {10.3791/71451},
pmid = {42714094},
issn = {1940-087X},
mesh = {Animals ; Mice ; *Myocardial Infarction/genetics/physiopathology/pathology ; *Disease Models, Animal ; Mice, Inbred C57BL ; *CRISPR-Cas Systems ; *Ventricular Remodeling ; Phenotype ; Male ; },
abstract = {Myocardial infarction (MI) remains a major cause of morbidity and mortality worldwide. This protocol describes a method for generating and characterizing a Cracd-deficient mouse line on the C57BL/6N background using CRISPR/Cas9 technology. Zygotes were co-injected with Cas9 mRNA, a gRNA construct, and a donor template designed to generate a 3153-bp genomic deletion. The edited allele was confirmed by PCR genotyping and Sanger sequencing. To assess the functional role of CRACD in post-MI remodeling, Cracd-deficient and wild-type (WT, C57BL/6N) mice underwent MI induced by permanent ligation of the left anterior descending coronary artery. On postoperative day 7, cardiac function and left ventricular wall motion were assessed using transthoracic echocardiography and speckle-tracking strain imaging, followed by histopathological evaluation with H&E and Masson's trichrome staining. Representative results showed that Cracd-deficient mice exhibited reduced ventricular dilation and preserved systolic function compared to WT controls. This protocol provides a reliable experimental platform for mechanistic studies of CRACD in cardiac pathophysiology.},
}

Animals
Mice
*Myocardial Infarction/genetics/physiopathology/pathology
*Disease Models, Animal
Mice, Inbred C57BL
*CRISPR-Cas Systems
*Ventricular Remodeling
Phenotype
Male

@article {pmid42714611,
year = {2026},
author = {Yaoyu, T and Xiang, X and Peizhi, Y and Xiaoxia, S},
title = {Genetic transformation of forage crops: comparative barriers, evidence, and emerging strategies.},
journal = {Plant cell reports},
volume = {45},
number = {10},
pages = {},
pmid = {42714611},
issn = {1432-203X},
support = {32302478//National Natural Science Foundation of China/ ; 2452024004//Northwest A&F University/ ; },
mesh = {*Crops, Agricultural/genetics ; *Transformation, Genetic ; Plants, Genetically Modified/genetics ; *Fabaceae/genetics ; *Poaceae/genetics ; Gene Transfer Techniques ; CRISPR-Cas Systems/genetics ; },
abstract = {Forage crops include phylogenetically and biologically distinct legumes and grasses, and their genetic transformation is constrained by different combinations of host response, DNA-delivery efficiency, regeneration competence, genotype dependence, and genome stability. This review critically compares evidence from forage legumes and forage grasses rather than treating these groups as a single transformation category. We evaluate Agrobacterium-mediated transformation, protoplast-based delivery, particle bombardment, CRISPR/Cas-enabled applications, developmental regulators (DRs), viral vectors, and nanomaterial-mediated delivery according to four practical outcomes: reproducibility across genotypes, recovery of regenerated plants, heritable transmission, and genetic stability. Direct evidence in forage crops shows that protocol performance is strongly species-, genotype-, explant-, and endpoint-dependent; efficiencies based on transient reporters or resistant callus therefore cannot be directly equated with stable, fertile events. DR-assisted regeneration has direct proof of concept in recalcitrant forage grasses, whereas stable nanomaterial-mediated transformation and virus-induced heritable editing remain unvalidated in forage crops. We conclude that current progress is best interpreted as the engineering of interacting delivery and regeneration constraints, not as a universal transition to genotype-independent transformation. Priority should be given to standardized outcome reporting, multi-genotype and inter-laboratory validation, controlled DR expression, and rigorous molecular and phenotypic assessment of regenerated plants.},
}

*Crops, Agricultural/genetics
*Transformation, Genetic
Plants, Genetically Modified/genetics
*Fabaceae/genetics
*Poaceae/genetics
Gene Transfer Techniques
CRISPR-Cas Systems/genetics

@article {pmid42715083,
year = {2026},
author = {Jiang, Q and Xu, R and Lin, Z and Sun, C and Ye, F and Ye, Y and Lin, D and Li, Z and Mai, J and Lin, Z and Qu, J and Deng, X and Ge, C and Wang, D},
title = {Harmonizing amplification and cleavage kinetics through crRNA scaffold mutation enables robust one-pot CRISPR-Cas12 diagnostics.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {37},
pages = {e2613134123},
doi = {10.1073/pnas.2613134123},
pmid = {42715083},
issn = {1091-6490},
support = {2024YFC2310203//the Key National R&D Program of China/ ; SYSPG20241211173949065//the Shenzhen Key Laboratory of Biochip/ ; JCYJ20240813113233044//the Shenzhen Science and Technology Program/ ; S202510570075//College Students' Innovative Entrepreneurial Training Plan Program/ ; S202510570072//College Students' Innovative Entrepreneurial Training Plan Program/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Kinetics ; Humans ; *Nucleic Acid Amplification Techniques/methods ; *SARS-CoV-2/genetics/isolation & purification ; CRISPR-Associated Proteins/genetics/metabolism ; Rapid Diagnostic Tests ; Mutation ; Endodeoxyribonucleases/genetics/metabolism ; Bacterial Proteins ; },
abstract = {CRISPR-based diagnostics (CRISPR-Dx) integrated with isothermal nucleic acid amplification have emerged as a promising strategy for point-of-care molecular testing. Their practical deployment, however, remains constrained by workflow-related limitations. Conventional two-step formats are laborious and highly susceptible to aerosol contamination during amplicon transfer, whereas simplified one-pot formats often suffer from reduced sensitivity because amplification and CRISPR-mediated cleavage compete kinetically within the same reaction vessel. In this work, we present a broadly applicable one-pot detection strategy termed mutant scaffold-mediated RPA-CRISPR/Cas12a (MS-CRISPR). This approach introduces rationally designed point mutations into the crRNA scaffold to moderately attenuate Cas12a activation and cleavage kinetics. Such kinetic tuning prevents the enzyme from prematurely depleting nascent RPA amplicons, a common source of signal loss in wild-type systems. By delaying cleavage until sufficient amplification products have accumulated, MS-CRISPR markedly enhances endpoint signal output. Using this kinetically balanced system, we achieved a limit of detection of 1 copy/μL for both SARS-CoV-2 and influenza A virus within 15 min, while preserving a fully closed-tube workflow that minimizes aerosol contamination and simplifies operation. We coupled the assay with an instrument-free lateral-flow readout and an ultraportable, multiplex, low-cost microfluidic platform (portable wireless isothermal nucleic acid detection). Clinical validation with patient samples showed 100% sensitivity and specificity for both viral targets. Collectively, MS-CRISPR establishes a sensitive, versatile, and field-deployable platform for rapid pathogen screening.},
}

*CRISPR-Cas Systems/genetics
Kinetics
Humans
*Nucleic Acid Amplification Techniques/methods
*SARS-CoV-2/genetics/isolation & purification
CRISPR-Associated Proteins/genetics/metabolism
Rapid Diagnostic Tests
Mutation
Endodeoxyribonucleases/genetics/metabolism
Bacterial Proteins

@article {pmid42716374,
year = {2026},
author = {Lau, CH and Xia, Q and Liang, Q and Guo, R and Zhu, H},
title = {Opto-CRISPR technologies in translational neuropharmacology.},
journal = {Neuropharmacology},
volume = {301},
number = {},
pages = {111185},
doi = {10.1016/j.neuropharm.2026.111185},
pmid = {42716374},
issn = {1873-7064},
abstract = {Opto-CRISPR (optically controlled CRISPR/Cas) represents a major advancement in precision neurology by enabling localized gene editing within the nervous system at precise times and locations. While preclinical studies still rely largely on classical CRISPR/Cas systems, opto-CRISPR offers unprecedented opportunities to safely treat neurological diseases at their genetic roots. It achieves this by spatiotemporally correcting pathogenic mutations, halting toxic protein accumulation, repairing neurons, rebuilding neural circuitry, and accelerating drug discovery through highly realistic disease models. This review discusses this emerging field and its transformative potential for translational neuropharmacology, focusing on therapies for neurodevelopmental and neurodegenerative disorders. Opto-CRISPR is classified into four generations: Generation 1 (light-controlled Cas protein systems), Generation 2 (light-activated guide RNA systems), Generation 3 (light-controlled inhibitor systems), and Generation 4 (NIR upconversion and epigenetic multiplexing). Their working mechanisms, applications, advantages, and limitations are comprehensively described. We also critically discuss how opto-CRISPR offers distinct advantages over traditional biologics and remote modalities (e.g., small-molecule drugs, monoclonal antibodies, antisense oligonucleotides, viral-mediated gene therapy, magnetogenetics, and sonogenetics) in neuropharmacology. Finally, we address current challenges and the therapeutic outlook for the clinical translation of opto-CRISPR.},
}

@article {pmid42705625,
year = {2026},
author = {Sharma, S and Subramanyam, R},
title = {Carotenoids in Stress Tolerance: Current Understanding and Future Prospects.},
journal = {Physiologia plantarum},
volume = {178},
number = {5},
pages = {e71095},
pmid = {42705625},
issn = {1399-3054},
support = {BT/PR55080/BSA/33/289/2024//Department of Biotechnology, Ministry of Science and Technology, India/ ; 6-8/2018 (IC)//Joint UGC-ISF Research/ ; CRG/2020/000489//Science & Engineering Research Board/ ; no. 38 (1504)/21/EMR-ll//Council of Scientific and Industrial Research, India/ ; UoH/IoE/RC1/RC1-20-019//Institute of Eminence/ ; UoH/IoE/IIRC-23-004//Institute of Eminence/ ; },
mesh = {*Carotenoids/metabolism ; *Stress, Physiological ; Photosynthesis/physiology ; Signal Transduction ; Plants/metabolism ; Adaptation, Physiological ; },
abstract = {Carotenoids are ubiquitous isoprenoid metabolites whose biological significance extends far beyond their classical roles as antioxidants and light-harvesting pigments. Across the tree of life, these molecules exhibit remarkable structural and functional diversity, enabling organisms to fine-tune photoprotection, redox homeostasis, membrane organization, and stress-responsive signaling. In this review, we provide a conceptual synthesis of carotenoid diversity and function across organisms, with emphasis on how carotenoid composition and accumulation are dynamically regulated under environmental and biotic stresses. We highlight stress-induced carotenoid plasticity, manifested as selective accumulation, rerouting of pathways, and the production of signaling apocarotenoids as a central adaptive strategy across photosynthetic organisms. Finally, we discuss emerging evidence and competing hypotheses regarding the mechanistic diversity of non-photochemical quenching and the evolving yet debated role of carotenoids in energy-dependent quenching. Together, this review emphasizes carotenoids as dynamic metabolic regulators that integrate many facets of photosynthetic performance, stress responses and signaling.},
}

*Carotenoids/metabolism
*Stress, Physiological
Photosynthesis/physiology
Signal Transduction
Plants/metabolism
Adaptation, Physiological

@article {pmid42706676,
year = {2026},
author = {Camargo, JA and Lee, MC and Souza, MM and Silva, LPD and Boas, GCV and Silva, KSD and Mioshi, CM and Pimenta, RCA and Candido, P and Silva, IAD and Trarbach, EB and Leite, KRM and Nahas, WC and Reis, STD},
title = {MMP9 Knockout by CRISPR-Cas9 Reduces Bladder Cancer Malignancy But Does Not Synergize With BCG and Anti-PD-L1 in an Orthotopic Mouse Model.},
journal = {IUBMB life},
volume = {78},
number = {9},
pages = {e70130},
pmid = {42706676},
issn = {1521-6551},
support = {2022/08864-3//Fundação de Amparo à Pesquisa do Estado de São Paulo/ ; 2022/02288-0//Fundação de Amparo à Pesquisa do Estado de São Paulo/ ; //Coordenação de Aperfeiçoamento de Pessoal de Nível Superior/ ; },
mesh = {Animals ; Mice ; *Matrix Metalloproteinase 9/genetics ; Female ; *CRISPR-Cas Systems ; *Urinary Bladder Neoplasms/pathology/genetics/therapy/drug therapy ; *B7-H1 Antigen/antagonists & inhibitors ; *BCG Vaccine/pharmacology ; Mice, Inbred C57BL ; Cell Line, Tumor ; *Immune Checkpoint Inhibitors/pharmacology ; Disease Models, Animal ; Gene Expression Regulation, Neoplastic ; Mice, Knockout ; Humans ; },
abstract = {Bladder cancer (BC) is the 10th most common cancer worldwide, accounting for approximately 5% of new cases. Several factors contribute to tumor progression, including increased MMP9. Recently, CRISPR-Cas9 and immunotherapy have offered high specificity for treatments; therefore, we edited MMP9 using CRISPR-Cas9 methodology to inhibit metastatic mechanisms and evaluated potential synergy with BCG and anti-PD-L1 therapy. We performed CRISPR-Cas9 gene editing using an RNP complex in the MB49 murine BC cell line. Gene expression of MMPs, integrins, and BAX was analyzed, along with protein expression. Cells were divided into a Scramble control group and CRISPR-Cas9 for the MMP9 edited group (MMP9-/-). Female C57BL/6 mice received orthotopic BC cells (scramble and MMP9-/- groups) treated with BCG and anti-PD-L1. Statistical analyses were performed using t test or ANOVA. MMP9 gene and protein expression were reduced in the MMP9-/- group compared with the scramble group. No differences were observed in other MMPs. Decreased ITGB3, increased BAX gene expression, as well as reduced migration and adhesion to ECM were observed in the MMP9-/- group. Animals in the scramble group showed greater weight loss and lower survival than treated groups. Overall, MMP9 modulated key cellular mechanisms, but did not show synergistic effects with BCG and anti-PD-L1 in vivo.},
}

Animals
Mice
*Matrix Metalloproteinase 9/genetics
Female
*CRISPR-Cas Systems
*Urinary Bladder Neoplasms/pathology/genetics/therapy/drug therapy
*B7-H1 Antigen/antagonists & inhibitors
*BCG Vaccine/pharmacology
Mice, Inbred C57BL
Cell Line, Tumor
*Immune Checkpoint Inhibitors/pharmacology
Disease Models, Animal
Gene Expression Regulation, Neoplastic
Mice, Knockout
Humans

@article {pmid42707485,
year = {2026},
author = {Fabrizi, A and Valenti, M and Zacchino, S and Russo, K and Baschetti, E and De Rosa, L and De Luca, M},
title = {Gene therapy for genodermatoses at the crossroads of innovation and clinical translation.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {14},
number = {},
pages = {1901459},
pmid = {42707485},
issn = {2296-4185},
abstract = {Inherited genodermatoses are a heterogeneous group of rare monogenic disorders. Among these, epidermolysis bullosa (EB) and ichthyoses represent paradigmatic disorders characterized by severe skin fragility and hyperkeratosis, respectively, and impaired barrier function, often with profound effects on quality of life and systemic health. Current management remains largely palliative, underscoring the urgent need for disease-modifying therapies. Over the past 2 decades, advances in epithelial stem cell biology, vector engineering and genome editing technologies have transformed the therapeutic landscape for genodermatoses. Ex vivo gene therapy has provided the first proof that genetically corrected epidermal stem cells can achieve long-term tissue regeneration in EB skin patients, establishing a new paradigm for regenerative medicine. In parallel, the emergence of programmable genome engineering platforms, including CRISPR/Cas nucleases, base editors and prime editors, have enabled increasingly precise strategies for mutation-specific correction in both recessive and dominant disorders. Furthermore, the development of in vivo topical approaches is expanding the possibility of directly targeting the skin. Despite these advances, substantial translational barriers continue to limit broad clinical implementation. Efficient and durable targeting of epidermal stem cells within a highly regenerative tissue, together with safe delivery across the skin barrier, stringent control of off-target activity, scalable manufacturing and demonstration of long-term safety, remain major challenges for the clinical translation of these approaches. In this Review, we discuss the current state of gene therapy for genodermatoses, highlighting key clinical milestones, emerging genome editing technologies and next-generation delivery systems. We further examine the biological and regulatory challenges that need to be overcome to bridge the gap between experimental innovation and clinically accessible therapies for patients with inherited skin diseases.},
}

@article {pmid42707723,
year = {2026},
author = {Tao, J and Zeng, B and Bi, C and Xu, M and Gu, X and Jing, D and Sun, J},
title = {An RCA-Coupled CRISPR/Cas12a Cascade-Enhanced SERS Platform for Ultrasensitive Detection of Exosomal miRNA-21 in Colorectal Cancer Serum.},
journal = {International journal of nanomedicine},
volume = {21},
number = {},
pages = {622664},
pmid = {42707723},
issn = {1178-2013},
mesh = {*MicroRNAs/blood/genetics ; *Spectrum Analysis, Raman/methods ; Humans ; *Colorectal Neoplasms/blood/genetics/diagnosis ; *CRISPR-Cas Systems/genetics ; *Exosomes/chemistry/genetics ; Gold/chemistry ; Metal Nanoparticles/chemistry ; Limit of Detection ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {PURPOSE: Ultra-sensitive detection of exosomal miR-21 is crucial for the early diagnosis of colorectal cancer. This study aims to develop a cascade amplification platform based on surface-enhanced Raman scattering (SERS), combining rolling circle amplification (RCA) and CRISPR/Cas12a, to enable ultra-sensitive quantitative detection of exosomal miR-21.

METHODS: A SERS substrate was constructed using streptavidin-modified concave gold nanocubes (CGNs), and gold double-stranded probes loaded with Raman reporter molecules were used as SERS probes. The two components are controllably linked via a single-stranded DNA molecule with a thiol group at one end and biotin at the other, forming a stable detection system. Target-triggered RCA generates a repeat sequence, which, under the mediation of crRNA, activates Cas12a cleavage, causing the probe to be released from the substrate surface and resulting in a significant attenuation of the SERS signal.

RESULTS: This platform achieves three-stage cascading signal amplification via RCA, CRISPR/Cas12a, and SERS, with a detection limit at the femto-mole (fM) level and a total detection time of approximately 80 minutes. The results were consistent with those of qRT-PCR, demonstrating high sensitivity, high specificity, and reliability.

CONCLUSION: This study proposes an RCA-CRISPR-SERS cascade amplification platform that provides a highly sensitive and specific method for detecting miR-21 in exosomes, offering potential clinical value for the early diagnosis and dynamic monitoring of colorectal cancer.},
}

*MicroRNAs/blood/genetics
*Spectrum Analysis, Raman/methods
Humans
*Colorectal Neoplasms/blood/genetics/diagnosis
*CRISPR-Cas Systems/genetics
*Exosomes/chemistry/genetics
Gold/chemistry
Metal Nanoparticles/chemistry
Limit of Detection
Nucleic Acid Amplification Techniques/methods

@article {pmid42628215,
year = {2026},
author = {Eom, KH and Gim, GM and Lee, MG and Choi, BY and Kim, DY and Jung, D and Heo, S and Yum, SY and Jang, G},
title = {Evaluation of germline transmission of electroporation-mediated double gene-edited cattle lines.},
journal = {Theriogenology},
volume = {266},
number = {},
pages = {118142},
doi = {10.1016/j.theriogenology.2026.118142},
pmid = {42628215},
issn = {1879-3231},
mesh = {Animals ; Cattle/genetics ; Male ; *Electroporation/veterinary ; Female ; *Gene Editing/veterinary/methods ; CRISPR-Cas Systems ; Gene Knockout Techniques/veterinary ; Germ Cells ; Myostatin/genetics ; Animals, Genetically Modified ; Spermatozoa ; Galactosyltransferases/genetics ; },
abstract = {Gene editing in livestock using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) offers a promising approach for genetic improvement in cattle. This study evaluated germline transmission and mutation stability of double-knockout cattle generated by zygote electroporation. Previously reported myostatin/beta-lactoglobulin (MSTN/BLG) and newly generated α-1,3-galactosyltransferase (GGTA1/BLG) double-knockout cattle were produced using CRISPR/Cas9-mediated genome editing. Targeted deep sequencing demonstrated extensive somatic mosaicism across multiple tissues. Computer-assisted sperm analysis (CASA) demonstrated normal sperm motility in MSTN/BLG double-knockout males. Fertilization of wild-type oocytes produced heterozygous embryos, with mutation frequencies of 37.76 ± 10.74% at the MSTN locus and 54.80 ± 7.73% at the BLG locus, as assessed by T7 endonuclease I (T7E1) assay. MSTN/BLG double-knockout sperm were subsequently used for embryo production and for artificial insemination of GGTA1/BLG double-knockout females. Healthy offspring were successfully obtained (n = 3), alongside one stillborn calf. Targeted deep sequencing of all four progenies revealed highly variable allele frequencies that deviated substantially from the approximately 50% expected for heterozygous germline transmission. In contrast, whole-genome sequencing (WGS) results were consistent with Mendelian expectations, underscoring the limitations of PCR-based targeted sequencing for assessing germline transmission in mosaic founders. These results show that CRISPR/Cas9-edited embryos generated by electroporation can develop into healthy, sexually mature cattle capable of germline transmission. While variable transmission rates were observed owing to founder mosaicism, non-mosaic F1 offspring were successfully generated. However, direct, embryo-mediated gene-editing strategies remain technically and economically challenging for large-scale commercial calf production, and reports in cattle are limited. This study provides a reference for future applications of gene-edited embryos and their germline propagation.},
}

Animals
Cattle/genetics
Male
*Electroporation/veterinary
Female
*Gene Editing/veterinary/methods
CRISPR-Cas Systems
Gene Knockout Techniques/veterinary
Germ Cells
Myostatin/genetics
Animals, Genetically Modified
Spermatozoa
Galactosyltransferases/genetics