@article {pmid41039221,
year = {2025},
author = {Ilmi, AFN and Kaewsapsak, P and Rotcheewaphan, S},
title = {Repression of mab_1999 impairs growth and alters cellular morphology of Mycobacterium abscessus.},
journal = {BMC microbiology},
volume = {25},
number = {1},
pages = {599},
pmid = {41039221},
issn = {1471-2180},
mesh = {*Mycobacterium abscessus/growth & development/genetics/drug effects/cytology/metabolism ; *Bacterial Proteins/genetics/metabolism ; Anti-Bacterial Agents/pharmacology ; Microbial Sensitivity Tests ; Cell Division/genetics ; Gene Expression Regulation, Bacterial ; Mycobacterium smegmatis/genetics ; Gene Knockdown Techniques ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Cell division is essential for bacterial survival and represents a promising target for the development of novel antibiotics, particularly in mycobacteria. The role of the division protein FtsL in Mycobacterium abscessus remains poorly understood. This study investigated the effects of MAB_1999, a predicted homolog of FtsL, on the growth and cell division of M. abscessus.

METHOD: To investigate the function of mab_1999, a knockdown mutant was generated via CRISPR interference (CRISPRi). The phenotypic impact of mab_1999 suppression was evaluated, with a focus on its effects on M. abscessus growth, cellular morphology, and antibiotic susceptibility.

RESULTS: The putative homolog of FtsL in M. abscessus (MAB_1999) shares 54% amino acid sequence identity with FtsL from M. smegmatis (MSMEG_4234). CRISPRi-mediated repression of mab_1999 expression resulted in cell elongation and growth defects, although complete growth arrest was not observed. Furthermore, reduced mab_1999 expression increased the susceptibility of M. abscessus to β-lactam antibiotics, including ceftriaxone and imipenem.

CONCLUSIONS: Our findings suggest that mab_1999 is involved in cell division and cell wall integrity in M. abscessus. However, further investigation is necessary to confirm its identity as FtsL and to fully elucidate its role in the cell division process and cell wall synthesis.},
}

*Mycobacterium abscessus/growth & development/genetics/drug effects/cytology/metabolism
*Bacterial Proteins/genetics/metabolism
Anti-Bacterial Agents/pharmacology
Microbial Sensitivity Tests
Cell Division/genetics
Gene Expression Regulation, Bacterial
Mycobacterium smegmatis/genetics
Gene Knockdown Techniques
CRISPR-Cas Systems

@article {pmid41039127,
year = {2025},
author = {Safenkova, IV and Kamionskaya, MV and Ivanov, AV and Zherdev, AV and Dzantiev, BB},
title = {A novel tripod probe and lateral flow test to improve CRISPR/Cas12a assay: benefits of branched probe based on trebler phosphoramidite modification.},
journal = {Mikrochimica acta},
volume = {192},
number = {11},
pages = {711},
pmid = {41039127},
issn = {1436-5073},
support = {23-46-10011//Russian Science Foundation/ ; },
mesh = {*CRISPR-Cas Systems ; *DNA Probes/chemistry ; *Biosensing Techniques/methods ; *Organophosphorus Compounds/chemistry ; Fluorescent Dyes/chemistry ; Limit of Detection ; Gold/chemistry ; *CRISPR-Associated Proteins/metabolism ; Metal Nanoparticles/chemistry ; *Bacterial Proteins/metabolism ; *Endodeoxyribonucleases/metabolism ; },
abstract = {CRISPR/Cas12a-based assays, when integrated with lateral flow tests (LFTs), provide highly specific nucleic acid detection in a simple, rapid, and equipment-free format. Nevertheless, traditional DNA probes utilized for cleavage by Cas12a have limitations as the cleaved probe only has one label. To overcome this challenge, we engineered a novel type of DNA probe with multiple fluorescein (FAM) labels and a biotin-labeled single-stranded DNA fragment (polyFAM probe). The cleaved polyFAM parts of the probes were detected using a specially designed sandwich LFT, where FAM-specific antibodies were immobilized in the test zone and conjugated with gold nanoparticles. The LFT ensured accurate recognition of the cleaved polyFAM fragments within 10 min. A comparison of five distinct polyFAM probes revealed that the highest signal-to-noise ratio was achieved with a tripod-branched probe synthesized via trebler phosphoramidite modification. Each arm of the tripod probe consists of a hexaethylene glycol spacer ending in a FAM label. Upon Cas12a cleavage, the tripod structure carrying three FAMs is released and detected by LFT. A rapid magnetic separation strategy was subsequently implemented, facilitating the efficient removal of uncleaved probes via biotin-streptavidin capture within 5 min. The CRISPR/Cas12a-tripod-LFT strategy demonstrated excellent sensitivity without preamplification, with a detection Limit of 1.4 pM for DNA target of Salmonella Typhimurium. The CRISPR/Cas12a-tripod-LFT with preliminary loop-mediated isothermal amplification enabled the detection of as few as 0.3 cells per reaction. This innovative tripod probe with corresponding LFT creates a universal, sensitive, rapid, and equipment-free biosensing platform for CRISPR/Cas12a-based diagnostics in point-of-care applications.},
}

*CRISPR-Cas Systems
*DNA Probes/chemistry
*Biosensing Techniques/methods
*Organophosphorus Compounds/chemistry
Fluorescent Dyes/chemistry
Limit of Detection
Gold/chemistry
*CRISPR-Associated Proteins/metabolism
Metal Nanoparticles/chemistry
*Bacterial Proteins/metabolism
*Endodeoxyribonucleases/metabolism

@article {pmid40847019,
year = {2025},
author = {Ramani, B and Rose, IVL and Teyssier, N and Pan, A and Danner-Bocks, S and Sanghal, T and Yadanar, L and Tian, R and Ma, K and Palop, JJ and Kampmann, M},
title = {CRISPR screening by AAV episome-sequencing (CrAAVe-seq): a scalable cell-type-specific in vivo platform uncovers neuronal essential genes.},
journal = {Nature neuroscience},
volume = {28},
number = {10},
pages = {2129-2140},
pmid = {40847019},
issn = {1546-1726},
support = {R01 AG082141/AG/NIA NIH HHS/United States ; K99 AG062776/AG/NIA NIH HHS/United States ; RF1 AG062234/AG/NIA NIH HHS/United States ; R25 NS070680/NS/NINDS NIH HHS/United States ; K08 NS133300/NS/NINDS NIH HHS/United States ; T32 NS115706/NS/NINDS NIH HHS/United States ; EDUC4-12812//California Institute for Regenerative Medicine (CIRM)/ ; UCSF Hillblom/BARI Graduate Fellowship Award//Larry L. Hillblom Foundation (Larry L. Hillblom Foundation, Inc.)/ ; R01 AG082141/AG/NIA NIH HHS/United States ; K99 AG062776/AG/NIA NIH HHS/United States ; RF1 AG062234/AG/NIA NIH HHS/United States ; R25 NS070680/NS/NINDS NIH HHS/United States ; K08 NS133300/NS/NINDS NIH HHS/United States ; T32 NS115706/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; *Dependovirus/genetics ; *Neurons/metabolism ; Mice ; *CRISPR-Cas Systems/genetics ; *Genes, Essential/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Mice, Inbred C57BL ; *Genetic Testing/methods ; Humans ; },
abstract = {There is a substantial need for scalable CRISPR-based genetic screening methods that can be applied in mammalian tissues in vivo while enabling cell-type-specific analysis. Here we developed an adeno-associated virus (AAV)-based CRISPR screening platform, CrAAVe-seq, that incorporates a Cre-sensitive sgRNA construct for pooled screening within targeted cell populations in mouse tissues. We used this approach to screen two large sgRNA libraries, which collectively target over 5,000 genes, in mouse brains and uncovered genes essential for neuronal survival, of which we validated Rabggta and Hspa5. We highlight the reproducibility and scalability of the platform and show that it is sufficiently sensitive for screening in a restricted subset of neurons. We systematically characterize the impact of sgRNA library size, mouse cohort size, the size of the targeted cell population, viral titer, and coinfection rate on screen performance to establish general guidelines for large-scale in vivo screens.},
}

Animals
*Dependovirus/genetics
*Neurons/metabolism
Mice
*CRISPR-Cas Systems/genetics
*Genes, Essential/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Mice, Inbred C57BL
*Genetic Testing/methods
Humans

@article {pmid41038958,
year = {2025},
author = {Chen, J and Huang, H and Chen, C and Xia, G and Huang, H and Xiong, Y and Luo, P and Chen, Y and Li, J and Wen, L and Li, L and Lin, J and Xu, G and Ji, C and Tian, W and Zhou, J and Wei, P and Shen, C and Wang, X},
title = {ABCC4 impairs the clearance of plasma LDL cholesterol through suppressing LDLR expression in the liver.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {1414},
pmid = {41038958},
issn = {2399-3642},
support = {8217051361//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Receptors, LDL/metabolism/genetics ; Animals ; *Liver/metabolism ; Mice ; *Cholesterol, LDL/blood/metabolism ; *Multidrug Resistance-Associated Proteins/metabolism/genetics ; Humans ; Male ; Proprotein Convertase 9/metabolism/genetics ; Hepatocytes/metabolism ; Mice, Inbred C57BL ; Hypercholesterolemia/metabolism/genetics ; CRISPR-Cas Systems ; Signal Transduction ; Hep G2 Cells ; },
abstract = {Low expression level of low-density lipoprotein receptor (LDLR) in hepatocytes leads to hypercholesterolemia and eventually contributes to atherosclerotic cardiovascular disease (ASCVD). Here, we report that inhibition of hepatocyte ABCC4, identified as a top hit from large-scale CRISPR/Cas9 screens, significantly increases hepatic LDLR abundance and enhances LDL cholesterol clearance. As a hepatic transporter for cAMP efflux, ABCC4 silencing alters its intracellular distribution and activates the downstream Epac2/Rap1a signaling pathway, which ultimately blocks PCSK9 protein expression, thereby preventing lysosomal degradation of LDLR. Furthermore, in both male mice and cell models, we demonstrate that liver-specific disruption and pharmacological inhibition of ABCC4 elevate hepatic plasma membrane LDLR levels and reduce plasma LDL cholesterol through ABCC4-cAMP-PCSK9 pathway. Collectively, our genome-wide CRISPR screening offers a valuable resource for identifying LDLR modifiers, providing potential insights for therapeutic strategies in hypercholesterolemia and atherosclerosis.},
}

*Receptors, LDL/metabolism/genetics
Animals
*Liver/metabolism
Mice
*Cholesterol, LDL/blood/metabolism
*Multidrug Resistance-Associated Proteins/metabolism/genetics
Humans
Male
Proprotein Convertase 9/metabolism/genetics
Hepatocytes/metabolism
Mice, Inbred C57BL
Hypercholesterolemia/metabolism/genetics
CRISPR-Cas Systems
Signal Transduction
Hep G2 Cells

@article {pmid41038616,
year = {2025},
author = {Aliciaslan, M and Erbasan, E and Erendor, F and Sanlioglu, S},
title = {Prime Editing: The Next Frontier in Precision Gene Therapy.},
journal = {The journal of gene medicine},
volume = {27},
number = {10},
pages = {e70040},
doi = {10.1002/jgm.70040},
pmid = {41038616},
issn = {1521-2254},
support = {//Akdeniz Üniversitesi/ ; },
mesh = {*Gene Editing/methods ; Humans ; *Genetic Therapy/methods ; *Precision Medicine/methods ; CRISPR-Cas Systems ; Animals ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Prime editing (PE) represents a significant advancement in genome editing, offering high precision for diverse genetic modifications without inducing double-strand breaks or requiring exogenous donor DNA templates. This "search-and-replace" technology employs a Cas9 nickase-reverse transcriptase fusion protein, guided by a PE guide RNA (pegRNA), to directly install specified edits including all 12 base-to-base conversions and targeted insertions/deletions with high fidelity. Since its introduction, PE systems have undergone rapid evolution (e.g., PE2-PE6, PEmax), markedly improving editing efficiency, product purity, and targeting scope. Although PE efficacy is context dependent, influenced by pegRNA design, cellular milieu, and DNA repair pathway engagement, ongoing research focuses on comprehensive system optimization. These efforts include engineering the Cas9 nickase and reverse transcriptase components for enhanced performance and processivity, alongside developing improved pegRNA architectures and chemical modifications to increase their stability and editing efficiency. Furthermore, strategies to modulate the cellular environment, such as transiently altering DNA repair pathway activities, particularly mismatch repair, are being explored to boost the accuracy and yield of precise edits. PE holds substantial promise for basic research, including precise disease modeling, and has demonstrated successful correction of pathogenic mutations in preclinical models of various genetic disorders like sickle cell disease, cystic fibrosis, and inherited retinal diseases. A significant milestone was the US Food and Drug Administration's granting of Investigational New Drug (IND) clearance for the first clinical trial of PM359, a therapeutic based on PE. This agent employs an ex vivo strategy, correcting the NCF1 gene in patient-derived hematopoietic stem cells for the treatment of chronic granulomatous disease. Despite considerable progress, unlocking the complete therapeutic promise of PE requires overcoming significant hurdles, particularly in developing effective in vivo delivery systems for its sizable components, with ongoing research actively investigating diverse viral and nonviral approaches. The translation of this versatile platform into transformative precision gene therapies is critically dependent upon its continued responsible advancement under robust ethical and regulatory oversight.},
}

*Gene Editing/methods
Humans
*Genetic Therapy/methods
*Precision Medicine/methods
CRISPR-Cas Systems
Animals
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid41037600,
year = {2025},
author = {de Alba, EL and Salguero, I and Giménez-Llorente, D and Montes-Torres, J and Fernández-Sanromán, Á and Casajús-Pelegay, E and Terrón-Bautista, J and Barroso-González, J and Bernal, JA and Macintyre, G and Fernández-Leiro, R and Losada, A and Cortés-Ledesma, F},
title = {A comprehensive genetic catalog of human double-strand break repair.},
journal = {Science (New York, N.Y.)},
volume = {390},
number = {6768},
pages = {eadr5048},
doi = {10.1126/science.adr5048},
pmid = {41037600},
issn = {1095-9203},
mesh = {Humans ; *DNA Breaks, Double-Stranded ; CRISPR-Cas Systems ; *DNA Repair/genetics ; Von Hippel-Lindau Tumor Suppressor Protein/genetics ; INDEL Mutation ; Gene Editing ; DNA End-Joining Repair/genetics ; Kidney Neoplasms/genetics ; DNA-Binding Proteins/genetics ; Transcription Factors/genetics ; CRISPR-Associated Protein 9 ; Genome, Human ; Gene Knockout Techniques ; Carcinoma, Renal Cell/genetics ; },
abstract = {The analysis of DNA sequence outcomes provides molecular insights into double-strand break (DSB) repair mechanisms. Using parallel in-pool profiling of Cas9-induced insertions and deletions (indels) within a genome-wide knockout library, we present a comprehensive catalog that assesses the influence of nearly every human gene on DSB repair outcomes. This REPAIRome resource uncovers uncharacterized mechanisms, pathways, and factors involved in DSB repair, including opposing roles for XLF and PAXX, a molecular explanation for Cas9-induced multinucleotide insertions, HLTF functions in Cas9-induced DSB repair, the involvement of the SAGA complex in microhomology-mediated end joining, and an indel mutational signature linked to VHL loss, renal carcinoma, and hypoxia. These results exemplify the potential of REPAIRome to drive future discoveries in DSB repair, CRISPR-Cas gene editing and the etiology of cancer mutational signatures.},
}

Humans
*DNA Breaks, Double-Stranded
CRISPR-Cas Systems
*DNA Repair/genetics
Von Hippel-Lindau Tumor Suppressor Protein/genetics
INDEL Mutation
Gene Editing
DNA End-Joining Repair/genetics
Kidney Neoplasms/genetics
DNA-Binding Proteins/genetics
Transcription Factors/genetics
CRISPR-Associated Protein 9
Genome, Human
Gene Knockout Techniques
Carcinoma, Renal Cell/genetics

@article {pmid41035653,
year = {2025},
author = {Naumovas, D and Rojas-Araya, B and Polanco, CM and Andrade, V and Čekauskienė, R and Valatkaitė-Rakštienė, B and Laurinaitytė, I and Jakubauskas, A and Stoškus, M and Griškevičius, L and Nalvarte, I and Inzunza, J and Baltriukienė, D and Arias, J},
title = {Identification of HLA-A, HLA-B, and HLA-C triple homozygous and double homozygous donors: a path toward synthetic superdonor advanced therapeutic medicinal products.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1626787},
pmid = {41035653},
issn = {1664-3224},
mesh = {Humans ; *Homozygote ; *HLA-B Antigens/genetics ; *HLA-C Antigens/genetics ; *HLA-A Antigens/genetics ; Alleles ; Gene Frequency ; Induced Pluripotent Stem Cells/immunology ; *Tissue Donors ; Genotype ; CRISPR-Cas Systems ; Female ; Male ; },
abstract = {Human-induced pluripotent stem cells with broad immune compatibility are highly desirable for regenerative medicine applications. Human leukocyte antigen (HLA) class I homozygous cell sources are ideal for immune compatibility modeling. Here, we profile HLA-A, HLA-B, and HLA-C alleles in 3,496 Lithuanian donors genotyped at three-field resolution. The five most frequent alleles constitute 74.6% of HLA-A, 43.2% of HLA-B, and 59.2% of HLA-C, with HLA-A*02:01:01, HLA-B*07:02:01, and HLA-C*07:02:01 being the most common. Lithuanian allele frequencies closely resemble those of European-American and British populations. We identified 153 double homozygotes and 51 triple homozygotes for HLA-A, HLA-B, and HLA-C. Compatibility modeling showed that triple homozygous profiles match 60.5% of Lithuanians, 13.4% of the British population, and 7.4% of European-Americans. CRISPR-Cas9 guide RNA design yielded 54 candidates predicted to disrupt HLA-A or HLA-B while preserving HLA-C, producing edited profiles matching over 97.9% of Lithuanians, 95.7% of European-Americans, and 95.5% of the British population. Finally, we established 15 fibroblast lines from triple homozygotes as a bioresource for the derivation of human-induced pluripotent stem cells and immune compatibility studies.},
}

Humans
*Homozygote
*HLA-B Antigens/genetics
*HLA-C Antigens/genetics
*HLA-A Antigens/genetics
Alleles
Gene Frequency
Induced Pluripotent Stem Cells/immunology
*Tissue Donors
Genotype
CRISPR-Cas Systems
Female
Male

@article {pmid41034738,
year = {2025},
author = {Devkar, V and Ghose, K and D'Agostino, L and Patil, GB},
title = {Exonuclease-fused CRISPR-cas system enhances targeted genome editing for functional genomics in soybean.},
journal = {BMC plant biology},
volume = {25},
number = {1},
pages = {1283},
pmid = {41034738},
issn = {1471-2229},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Glycine max/genetics ; *Genome, Plant ; *Exonucleases/genetics/metabolism ; Genomics/methods ; },
abstract = {CRISPR/Cas technologies have revolutionized plant genome editing, yet their inherent bias toward small insertions or deletions (indels) limits their utility for dissecting regulatory elements and generating impactful allelic variants. Here, we report the development and systematic evaluation of exonuclease-fused CRISPR/Cas systems in soybean to overcome this limitation. We engineered fusions of Cas9 and Cas12a with bacteriophage T5 exonuclease and human TREX2 and assessed their editing performance at the GmWOX5 locus using Agrobacterium rhizogenes-mediated transformation and deep amplicon sequencing. While native Cas9 and Cas12a predominantly generated micro-size deletions (1-10 bp), T5-Exo fusions shifted the mutation spectrum, producing a high frequency of moderate (26-50 bp) and large (> 50 bp) deletions. TREX2 fusions preferentially enhanced the generation of small (11-25 bp) to moderate deletions (26-50 bp). Fusion of exonucleases to Cas9 substantially reduced insertion frequencies and promoted more precise deletion patterns, as observed in T5-Exo-Cas9 and TREX2-Cas9. Deletions from both exonuclease fusions were biased toward the PAM-proximal region, reflecting altered repair outcomes likely driven by directional exonuclease activity and enhanced end resection. These results demonstrate that exonuclease fusions effectively expand the CRISPR toolkit by enabling efficient, targeted generation of larger deletions, which are often required for targeting cis-regulatory elements and microRNAs.},
}

*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Glycine max/genetics
*Genome, Plant
*Exonucleases/genetics/metabolism
Genomics/methods

@article {pmid40974621,
year = {2025},
author = {Zheng, X and Yao, S and Yin, C and Zhao, H and Wang, J and Su, T and Li, H and Wang, J and Zhao, C},
title = {CRISPR-integrated nanoconfined interparticle catalytic hairpin assembly for enhanced dual-mode SARS-CoV-2 detection in wastewater.},
journal = {Biosensors & bioelectronics},
volume = {290},
number = {},
pages = {118008},
doi = {10.1016/j.bios.2025.118008},
pmid = {40974621},
issn = {1873-4235},
mesh = {*SARS-CoV-2/isolation & purification/genetics ; *Wastewater/virology ; *Biosensing Techniques/methods/instrumentation ; *COVID-19/diagnosis/virology ; *CRISPR-Cas Systems ; Limit of Detection ; Humans ; *RNA, Viral/analysis/genetics/isolation & purification ; Colorimetry/methods ; Metal-Organic Frameworks/chemistry ; Nanoparticles/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Accurate monitoring of pathogenic viruses in wastewater is critical for early outbreak and risk assessment. This study presented a novel biosensing platform that combined an interparticle magnetic covalent organic framework (MCOF)-assisted mismatched catalytic hairpin assembly (iMMCHA) with CRISPR/Cas12a-activated colorimetric-photothermal dual-mode detection of SARS-CoV-2 RNA. The system strategically immobilized CHA reactants (H1 and mismatched H2) on separate MCOF nanoparticles, creating a spatially confined and collision-enhanced interparticle MCHA that achieved 270-fold higher local reactant concentration and 20-min faster kinetics than solution-phase CHA. Upon target recognition, the iMMCHA system generated dsDNA activators that triggered Cas12a-mediated cleavage of ssDNA linkers on magnetic bead-glucose oxidase conjugates. This cleavage event reduced the TMB-oxidizing activity of the magnetically isolated integrated enzyme system, producing inversely correlated colorimetric and photothermal signals. This iMMCHA-CRISPR dual-mode assay allowed for the rapid and sensitive detection of SARS-CoV-2 pseudovirus in sanitary wastewater samples, with detection limits of 100 and 120 copies/μL (colorimetric mode) and 100 and 140 copies/μL (photothermal mode) for S and N genes, respectively. This work established a powerful platform for aqueous environmental virus monitoring that combined the specificity of CRISPR with the signal enhancement and kinetics acceleration of nanoconfined interparticle CHA and the reliability of dual-mode detection.},
}

*SARS-CoV-2/isolation & purification/genetics
*Wastewater/virology
*Biosensing Techniques/methods/instrumentation
*COVID-19/diagnosis/virology
*CRISPR-Cas Systems
Limit of Detection
Humans
*RNA, Viral/analysis/genetics/isolation & purification
Colorimetry/methods
Metal-Organic Frameworks/chemistry
Nanoparticles/chemistry
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40961645,
year = {2025},
author = {He, Y and Zhang, Y and Xiang, H and Ren, K and Yin, Y and Gao, Y and Yang, Y and Zhang, W and Liu, L and Han, H and Wang, W},
title = {Magnetic bead-assisted one-pot RCA-activated CRISPR/Cas12a electrochemiluminescence biosensor for the detection of citrus Huanglongbing pathogen.},
journal = {Biosensors & bioelectronics},
volume = {290},
number = {},
pages = {117986},
doi = {10.1016/j.bios.2025.117986},
pmid = {40961645},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *Citrus/microbiology ; *Plant Diseases/microbiology ; Luminescent Measurements/methods ; CRISPR-Cas Systems/genetics ; Electrochemical Techniques/methods ; Limit of Detection ; Nucleic Acid Amplification Techniques/methods ; *Liberibacter/isolation & purification/genetics/pathogenicity ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Huanglongbing (HLB) poses a catastrophic threat to the global citrus industry, necessitating early detection of pathogen for disease control and minimize economic losses. Herein, we reported a one-pot electrochemiluminescence (ECL) biosensor for integrating rolling circle amplification (RCA)-activated CRISPR/Cas12a dual cleavage activity, and engineered magnetic beads-based quenched ECL emitter. Target-initiated RCA generated amplicons that activated Cas12a, simultaneously leveraging cis-cleavage for template recycling and trans-cleavage to degrade single stranded DNA attached on Ru(bpy)3[2+]-loaded magnetic beads. This dual-amplification strategy restored ECL signals, enabling ultrasensitive detection of Candidatus Liberibacter asiaticus (CLas) ribonucleotide-diphosphate reductase subunit beta gene fragments with high specificity. A linear range 10 fM-1 nM with the detection limit of 2 fM was obtained. The integrated platform eliminated multi-step incubations, and exhibited satisfactory performance in citrus leaf samples, offering a powerful tool for HLB diagnostics.},
}

*Biosensing Techniques/methods
*Citrus/microbiology
*Plant Diseases/microbiology
Luminescent Measurements/methods
CRISPR-Cas Systems/genetics
Electrochemical Techniques/methods
Limit of Detection
Nucleic Acid Amplification Techniques/methods
*Liberibacter/isolation & purification/genetics/pathogenicity
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40945113,
year = {2025},
author = {Che, R and Tang, D and Fu, B and Wen, T and Wang, Z and Feng, D and Huang, KJ and Xu, J},
title = {Smartphone-integrated tri-mode RCA-CRISPR/Cas12a biosensor with Fe3O4@Au nanozyme for on-site detection of sugarcane smut at attomolar level.},
journal = {Biosensors & bioelectronics},
volume = {290},
number = {},
pages = {117985},
doi = {10.1016/j.bios.2025.117985},
pmid = {40945113},
issn = {1873-4235},
mesh = {*Biosensing Techniques/instrumentation ; *Saccharum/microbiology ; Smartphone ; Gold/chemistry ; CRISPR-Cas Systems ; Nucleic Acid Amplification Techniques ; Limit of Detection ; *Plant Diseases/microbiology ; Colorimetry ; Electrochemical Techniques ; Metal Nanoparticles/chemistry ; },
abstract = {The devastating sugarcane smut causes up to 70 % sugar yield loss and secondary infections, but field-deployable diagnostics remain challenging due to the limitations of lab-dependent methods. Herein, we report a portable CRISPR/Cas12a-powered biosensor integrated with tri-functional Fe3O4@Au nanozymes and triple-modal signal readout for precise and on-site pathogen detection. By synergizing rolling circle amplification (RCA) with CRISPR/Cas12a trans-cleavage activity, the system achieves ultrasensitive target recognition (detection limit: 32.11 aM for electrochemical mode). The Fe3O4@Au@GOD bioconjugates simultaneously enables magnetic separation, optimizes GOD-mediated colorimetric signals (visual LOD: 49.28 fM), and enhances photothermal responses (LOD: 42.17 fM) via precise biocatalyst-catalyzed TMB oxidation. A smartphone-coupled 3D-printed device integrates electrochemical, colorimetric, and photothermal detection modes, providing cross-validated results that eliminate false positives in complex matrices (recovery: 98-104 %). This field-portable platform completes detection within 2.5-4.5 h (80 % cost reduction vs. qPCR) and demonstrates high specificity against non-target pathogens. The fusion of nanozyme engineering, CRISPR amplification, and multi-modal sensing offers a transformative tool for precision agriculture.},
}

*Biosensing Techniques/instrumentation
*Saccharum/microbiology
Smartphone
Gold/chemistry
CRISPR-Cas Systems
Nucleic Acid Amplification Techniques
Limit of Detection
*Plant Diseases/microbiology
Colorimetry
Electrochemical Techniques
Metal Nanoparticles/chemistry

@article {pmid40939269,
year = {2025},
author = {Zhou, C and Jiang, F and Chen, W and Nugen, SR and Huang, C},
title = {Synthetic biology meets diagnostics: Engineering biosensing platforms for rapid and accurate pathogen and viral detection.},
journal = {Biosensors & bioelectronics},
volume = {290},
number = {},
pages = {117946},
doi = {10.1016/j.bios.2025.117946},
pmid = {40939269},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods/instrumentation ; *Synthetic Biology/methods/instrumentation ; Humans ; *Viruses/isolation & purification/genetics/pathogenicity ; *Virus Diseases/diagnosis/virology ; Nanotechnology/methods ; CRISPR-Cas Systems ; },
abstract = {The integration of synthetic biology with biosensor technologies has catalyzed a paradigm shift in the development of programmable, field-deployable diagnostic systems for precision detection of pathogens and viral threats. This review provides a comprehensive overview of current synthetic biology toolkits, such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR-based) systems, argonaute proteins, and modular genetic circuits. These tools were integrated into biosensors and in vitro diagnostic devices. The applications of cell-free systems, modular genetic circuits, and nanomaterial-enhanced platforms have further expanded the versatility of these tools, which include infectious disease diagnostics, public health monitoring, and food safety. Recent studies integrate synthetic biology with artificial intelligence (AI) and nanotechnology, enabling the development of automated, low-cost, and high-throughput diagnostic systems. This review provides a comprehensive overview of current technologies, emerging trends, future directions, and challenges, which offers valuable insights for advancing pathogen detection and in vitro diagnostics through synthetic biology.},
}

*Biosensing Techniques/methods/instrumentation
*Synthetic Biology/methods/instrumentation
Humans
*Viruses/isolation & purification/genetics/pathogenicity
*Virus Diseases/diagnosis/virology
Nanotechnology/methods
CRISPR-Cas Systems

@article {pmid40914019,
year = {2025},
author = {Ding, Y and Zhang, J and Li, K and Wang, X and Shi, X and Zhao, C and Dai, J and Wang, Q and Yao, S and Wang, J},
title = {A cascade amplification platform integrating entropy-driven DNA nanomachine with CRISPR/Cas12a for microRNA-21 and Listeria monocytogenes detection.},
journal = {Biosensors & bioelectronics},
volume = {290},
number = {},
pages = {117947},
doi = {10.1016/j.bios.2025.117947},
pmid = {40914019},
issn = {1873-4235},
mesh = {*Listeria monocytogenes/isolation & purification/genetics ; CRISPR-Cas Systems/genetics ; *MicroRNAs/genetics/isolation & purification ; *Biosensing Techniques/methods ; Entropy ; Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; Metal Nanoparticles/chemistry ; Gold/chemistry ; Animals ; DNA/chemistry ; Swine ; Humans ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {As one of enzyme-free amplification strategies, entropy-driven catalytic (EDC) based on toehold-mediated strand displacement reaction could achieve efficient amplification without cumbersome temperature changing and expensive enzymes, which shows great potential in biological sensing. However, the limitations in reaction velocity and sensitivity need to be further improved. Herein, a cascade platform integrating entropy-driven DNA nanomachine with CRISPR/Cas12a was proposed. Benefiting from the increased local concentration of DNA on AuNPs, the reaction velocity was enhanced 2-fold compared to solution-based EDC efficiently and the signal was cascade amplified through specific recognition by the designed CRISPR/Cas12a with high sensitivity and selectivity. Impressively, utilizing the flexible design capabilities of DNA molecules, the proposed method achieved both nucleic acid and non-nucleic acid targets detection. The platform achieved a low limit of 6.1pM for microRNA-21 detection and 6 CFU/mL for Listeria monocytogenes detection. Moreover, it showed good performance in 10-fold diluted serum with 98.6-102.3 % recovery. And it has achieved good consistency with traditional plate culture methods in pork. Here, a rapid and sensitive platform based on entropy-driven DNA nanomachine coupled with CRISPR/Cas12a is proposed with great potential of application for early disease detection and food safety screening.},
}

*Listeria monocytogenes/isolation & purification/genetics
CRISPR-Cas Systems/genetics
*MicroRNAs/genetics/isolation & purification
*Biosensing Techniques/methods
Entropy
Nucleic Acid Amplification Techniques/methods
Limit of Detection
Metal Nanoparticles/chemistry
Gold/chemistry
Animals
DNA/chemistry
Swine
Humans
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40914018,
year = {2025},
author = {Deng, Z and Mao, X and Yang, Y and Wu, G and Meng, H and Hou, J and Yu, XF and Zhou, W and Mao, G and Ma, Y},
title = {Amplification-free CRISPR/Cas12a biosensor integrating AuNPs-mediated surface plasmon resonance for human papillomavirus detection and genotyping.},
journal = {Biosensors & bioelectronics},
volume = {290},
number = {},
pages = {117960},
doi = {10.1016/j.bios.2025.117960},
pmid = {40914018},
issn = {1873-4235},
mesh = {Humans ; Gold/chemistry ; *Surface Plasmon Resonance/methods ; CRISPR-Cas Systems/genetics ; *Papillomavirus Infections/virology/diagnosis ; Metal Nanoparticles/chemistry ; *Papillomaviridae/genetics/isolation & purification ; *DNA, Viral/genetics/isolation & purification ; *Biosensing Techniques ; Limit of Detection ; Genotyping Techniques ; Female ; Alkaline Phosphatase/chemistry ; Genotype ; Human Papillomavirus Viruses ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Screening for high-risk human papillomavirus (hrHPV) infection is essential for cervical cancer prevention. However, developing a simple, portable, and low-cost hrHPV genotyping method remains challenging, particularly in resource-limited settings. Herein, we present an innovative amplification-free, point-of-care hrHPV genotyping platform integrating CRISPR/Cas12a with alkaline phosphatase (ALP)-mediated surface plasmon effect. The platform detects HPV DNA through the Cas12a-crRNA complex recognition, activating the cleavage of ALP-labeled oligonucleotides within microwells and releasing ALP. The output signal is generated by changes in surface plasmon resonance of gold nanoparticles (AuNPs) induced by the ALP-mediated reaction of AuNPs with p-aminophenyl phosphate. This ALP-integrated CRISPR/Cas12a biosensing strategy enhances sensitivity by 10,000-fold compared to Cas12a-based detection integrating ALP-mediated p-nitrophenyl phosphate (p-NPP) hydrolysis. This approach allows the sensitive detection of HPV DNA with a detection limit of 300 aM. Moreover, integration with microplate separation allows specifically screen for the nine HPV subtypes targeted by the nine-valent HPV vaccine within 2.5 h. The platform's performance is validated using cervical swab samples, confirming its accuracy for HPV genotyping. Overall, this strategy provides a simple, portable, and cost-effective solution for multiplex nucleic acid targets detection without preamplification or instrumentation.},
}

Humans
Gold/chemistry
*Surface Plasmon Resonance/methods
CRISPR-Cas Systems/genetics
*Papillomavirus Infections/virology/diagnosis
Metal Nanoparticles/chemistry
*Papillomaviridae/genetics/isolation & purification
*DNA, Viral/genetics/isolation & purification
*Biosensing Techniques
Limit of Detection
Genotyping Techniques
Female
Alkaline Phosphatase/chemistry
Genotype
Human Papillomavirus Viruses
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40907452,
year = {2025},
author = {Hu, D and Lin, K and Xu, X and Chen, P and Wang, G and Parvin, R and Chen, X and Wang, D and Zhang, L and Ye, F},
title = {SEE-phAST: Spatially encapsulated emulsions for phenotypic antibiotic susceptibility testing via sequential digital RAA-CRISPR.},
journal = {Biosensors & bioelectronics},
volume = {290},
number = {},
pages = {117937},
doi = {10.1016/j.bios.2025.117937},
pmid = {40907452},
issn = {1873-4235},
mesh = {*Anti-Bacterial Agents/pharmacology ; *Biosensing Techniques ; Emulsions/chemistry ; Microbial Sensitivity Tests ; Humans ; DNA Copy Number Variations/genetics ; CRISPR-Cas Systems/genetics ; Escherichia coli/genetics/drug effects ; Pseudomonas aeruginosa/genetics/drug effects ; Nucleic Acid Amplification Techniques ; Recombinases/chemistry ; Klebsiella pneumoniae/genetics/drug effects ; DNA, Bacterial/genetics ; },
abstract = {The escalating threat of antimicrobial resistance is exacerbated by delayed diagnostics and improper antibiotics use, underscoring an urgent demand for rapid, versatile AST tools to support evidence-based prescribing. In this study, we present an innovative, generalizable phenotypic AST approach by quantifying bacterial gDNA copy number variations (CNVs) following 0.5-h-brief culturing with antibiotic exposure, termed spatially encapsulated emulsions (SEE)-phAST. It employed gelatin-PEG biomimetic phase separation and microfluidic technology to one-step fabricate spatial-confined microcarriers that enabled on-demand separation and mixing of recombinase-aided amplification (RAA) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 12a (CRISPR/Cas12a) reactions in a sequentially orchestrated manner. Importantly, it retained the superiority of droplet-based digitalization framework to identify 3-4 folds CNVs while synergistically enhancing signal-to-noise ratio and detection speed through RAA pre-amplification. By utilizing a phase diagram for precise separation, coupled with channel design and rate regulation, we controllably synthesized 180-210 μm microcarriers with 90-100 μm cores, achieving a 30-min RAA amplification that boosted sensitivity from 10[11] to 10[1] aM. The artificial urinary infection samples were prepared by introducing common bacteria, specifically Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa, into urinary samples obtained from healthy individuals. We demonstrated the system's capability to classify susceptibility and resistance to three specific drugs following a 30-min pre-culture, with epigenetic changes quantified as reduced to 0.5 for inhibition and maintained at 2 for unaffected growth. This strategy presents valuable potential for AST diagnosis and versatility in other sequence recognition scenarios.},
}

*Anti-Bacterial Agents/pharmacology
*Biosensing Techniques
Emulsions/chemistry
Microbial Sensitivity Tests
Humans
DNA Copy Number Variations/genetics
CRISPR-Cas Systems/genetics
Escherichia coli/genetics/drug effects
Pseudomonas aeruginosa/genetics/drug effects
Nucleic Acid Amplification Techniques
Recombinases/chemistry
Klebsiella pneumoniae/genetics/drug effects
DNA, Bacterial/genetics

@article {pmid40902593,
year = {2025},
author = {Chan, BKC and Zhang, C and Poon, CH and Lee, MHY and Chu, HY and Wang, B and Chen, SG and Yan, HHN and Leung, SY and Wong, ASL},
title = {A combined enteric neuron-gastric tumor organoid reveals metabolic vulnerabilities in gastric cancer.},
journal = {Cell stem cell},
volume = {32},
number = {10},
pages = {1595-1613.e10},
doi = {10.1016/j.stem.2025.08.006},
pmid = {40902593},
issn = {1875-9777},
mesh = {*Organoids/metabolism/pathology/drug effects ; *Stomach Neoplasms/metabolism/pathology ; Humans ; Animals ; *Neurons/metabolism/pathology/drug effects ; Mice ; Cell Line, Tumor ; Xenograft Model Antitumor Assays ; CRISPR-Cas Systems ; Lipid Metabolism/drug effects ; Acetyl-CoA Carboxylase/antagonists & inhibitors/metabolism ; },
abstract = {The discrepancy between organoid and immortalized cell line cultures for cancer target discovery remains unclear. Here, our multi-tiered clustered regularly interspaced short palindromic repeats (CRISPR) screens reveal in vivo-relevant metabolic dependencies and synthetic lethal pairs that can be uncovered with tumor organoids but not cell lines or even three-dimensional (3D) spheroids. These screens identify lanosterol synthase and acetyl-coenzyme A (CoA) carboxylase inhibitors as effective treatments that impede xenografted tumor growth in mice. These lipid metabolic inhibitors exhibit nanomolar half-maximal inhibitory concentration (IC50) values across diverse human gastric cancer organoids resistant to first-line treatments. Mechanistically, gastric cancer organoids and in vivo tumors exhibit lipid metabolic adaptations not seen in two-dimensional (2D) in vitro cultures. Additionally, enteric neurons modulate lipid metabolism in tumor organoids, altering drug sensitivity by up to two orders of magnitude. A neuron-cocultured CRISPR screen further reveals that acetyl-CoA carboxylase expression determines lanosterol synthase inhibitor efficacy. These findings highlight the critical roles of organoid environment and neuronal interaction in cancer lipid reliance.},
}

*Organoids/metabolism/pathology/drug effects
*Stomach Neoplasms/metabolism/pathology
Humans
Animals
*Neurons/metabolism/pathology/drug effects
Mice
Cell Line, Tumor
Xenograft Model Antitumor Assays
CRISPR-Cas Systems
Lipid Metabolism/drug effects
Acetyl-CoA Carboxylase/antagonists & inhibitors/metabolism

@article {pmid40674704,
year = {2025},
author = {Yamaguchi, K and Koya, J and Mizuno, K and Mizukami, Y and Yoshifuji, K and Saito, Y and Tabata, M and Shingaki, S and Yuasa, M and Ito, Y and Nakashima, K and Dreval, K and Morin, RD and Chiba, K and Okada, A and Shiraishi, Y and Murakami, K and Kogure, Y and Ohshima, K and Kataoka, K},
title = {In vivo CRISPR screening reveals cooperation of KMT2D and TP53 deficiencies in B-cell lymphomagenesis.},
journal = {Blood advances},
volume = {9},
number = {19},
pages = {5040-5055},
doi = {10.1182/bloodadvances.2024015519},
pmid = {40674704},
issn = {2473-9537},
mesh = {*Tumor Suppressor Protein p53/genetics/deficiency ; Humans ; Animals ; Mice ; *Lymphoma, Large B-Cell, Diffuse/genetics/pathology ; *DNA-Binding Proteins/genetics/deficiency ; *CRISPR-Cas Systems ; *Lymphoma, B-Cell/genetics ; *Neoplasm Proteins/genetics/deficiency ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Cell Line, Tumor ; },
abstract = {Although recent genetic studies have identified numerous genetic alterations in diffuse large B-cell lymphoma (DLBCL), their biological relevance remains elusive. Here, we performed in vivo CRISPR loss-of-function screening targeting 86 genes recurrently altered in DLBCL to examine oncogenicity of single-guide RNA (sgRNA)-targeted genes, association between genotype and lineage, occurrence of second-hit alterations, and cooperability among sgRNA-targeted genes and second-hit alterations. Transplantation of the CRISPR library-transduced hematopoietic stem/progenitor cells induces various hematologic malignancies, including B-cell lymphomas in mice. Enrichment analysis of sgRNA-targeted genes demonstrates significant overrepresentation of Kmt2d, Pax5, and Trp53 in B-cell lymphomas. Whole-exome sequencing identifies recurrent second-hit driver alterations, showing significant enrichment of Trp53 alterations in sgKmt2d-targeted B-cell lymphomas. Importantly, KMT2D and TP53 mutations are found to be the most prevalent co-occurring combination in human DLBCL, which is more prominent in relapsed/refractory DLBCL. Moreover, this combination confers significantly worse prognosis independent of clinical factors. Transcriptomic sequencing identifies overexpression of Yap1, the Hippo pathway component, in double sgKmt2d-targeted/Trp53-altered B-cell lymphomas. Furthermore, chromatin accessibility analysis demonstrates enrichment of transcriptional enhanced associate domain 1 binding motifs in regions that gained accessibility and increased expression of their nearest genes in these B-cell lymphomas. Most importantly, genetic and pharmacological inhibition of YAP1 suppresses in vitro cell proliferation and in vivo tumor growth of a human KMT2D/TP53-altered DLBCL cell line and prolongs survival of mice transplanted with double sgKmt2d-targeted/Trp53-altered B-cell lymphoma cells. Our findings demonstrate the utility of in vivo CRISPR screening to integrate human cancer genomics with mouse modeling and highlight the functional interplay between KMT2D and TP53 aberrations, providing insights into therapeutic strategies in DLBCL.},
}

*Tumor Suppressor Protein p53/genetics/deficiency
Humans
Animals
Mice
*Lymphoma, Large B-Cell, Diffuse/genetics/pathology
*DNA-Binding Proteins/genetics/deficiency
*CRISPR-Cas Systems
*Lymphoma, B-Cell/genetics
*Neoplasm Proteins/genetics/deficiency
*Clustered Regularly Interspaced Short Palindromic Repeats
Cell Line, Tumor

@article {pmid41033726,
year = {2025},
author = {Gupta, Y and Chosdol, K},
title = {Practical approaches to advanced molecular biology techniques.},
journal = {Methods in cell biology},
volume = {198},
number = {},
pages = {73-101},
doi = {10.1016/bs.mcb.2025.02.022},
pmid = {41033726},
issn = {0091-679X},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Molecular Biology/methods ; Single-Cell Analysis/methods ; Animals ; Chromatin Immunoprecipitation/methods ; Proteomics/methods ; },
abstract = {The field of molecular biology has undergone tremendous advancements in recent years, with the development of powerful techniques that allow for in-depth exploration of cellular processes at the molecular level. This chapter, "Advanced Molecular Biology Techniques," provides a detailed protocol of the molecular techniques. We begin with CRISPR-Cas9 genome editing, a transformative tool for precise and efficient gene manipulation, enabling targeted mutations and gene knockouts in various organisms. Gene amplification via Real-Time PCR is then discussed, highlighting its ability to quantify gene expression and detect rare genetic variants with high sensitivity. Flowcytometry follows, offering a robust platform for analyzing cellular populations based on specific markers, enabling the study of immune cells, cancer diagnostics, and cell cycle analysis. Chromatin Immunoprecipitation Sequencing (ChIP-Seq) is explored as a method for mapping protein-DNA interactions, providing insights into gene regulation and epigenetic modifications. The chapter also covers Single-cell RNA sequencing (scRNA-Seq), a groundbreaking technique for profiling gene expression at the single-cell level, allowing for the discovery of cell heterogeneity and complex biological processes. Next, we explore into proteomics through Mass Spectrometry-Based Analysis, which offers detailed proteome characterization and biomarker discovery by identifying and quantifying proteins in complex samples. Finally, Fluorescence In Situ Hybridization (FISH) is discussed as a method for visualizing the spatial localization of specific nucleic acid sequences within intact cells or tissues. Together, these advanced molecular biology techniques offer unparalleled precision and insight into the molecular mechanisms underlying health, disease, and cellular function.},
}

Humans
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Molecular Biology/methods
Single-Cell Analysis/methods
Animals
Chromatin Immunoprecipitation/methods
Proteomics/methods

@article {pmid41033723,
year = {2025},
author = {Hasan, N and Palungan, J and Ullah, M},
title = {Gene editing techniques in cancer research.},
journal = {Methods in cell biology},
volume = {198},
number = {},
pages = {287-312},
doi = {10.1016/bs.mcb.2025.03.002},
pmid = {41033723},
issn = {0091-679X},
mesh = {*Gene Editing/methods ; Humans ; *Neoplasms/genetics/therapy ; *CRISPR-Cas Systems/genetics ; Zinc Finger Nucleases/genetics ; Transcription Activator-Like Effector Nucleases/genetics ; Animals ; },
abstract = {The process of editing genes has emerged as a game-changing instrument in the field of cancer research. It has the potential to provide a whole new understanding of the biology of tumors and to facilitate the creation of tailored medicines. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system are the three basic methods of gene editing techniques that are discussed in this chapter. We investigate the protocol modifications that are specific to each approach, focusing on high-prevalence tumors, and we investigate the utility, efficiency, and application issues that are associated with each technique in oncology. In addition, we describe current developments in improving these methods to successfully target oncogenes and tumor suppressor genes, with the goal of driving forward advances in precision cancer therapy.},
}

*Gene Editing/methods
Humans
*Neoplasms/genetics/therapy
*CRISPR-Cas Systems/genetics
Zinc Finger Nucleases/genetics
Transcription Activator-Like Effector Nucleases/genetics
Animals

@article {pmid41033719,
year = {2025},
author = {Tiwari, PC and Chaudhary, MJ and Pal, R and Nath, R},
title = {In vivo cancer modeling using mouse models.},
journal = {Methods in cell biology},
volume = {198},
number = {},
pages = {221-250},
doi = {10.1016/bs.mcb.2025.02.013},
pmid = {41033719},
issn = {0091-679X},
mesh = {Animals ; Mice ; *Disease Models, Animal ; *Neoplasms/pathology/genetics ; Humans ; Tumor Microenvironment ; Xenograft Model Antitumor Assays/methods ; CRISPR-Cas Systems ; },
abstract = {Mouse models have contributed to a better understanding of cancer biology and the development of new treatments. This chapter elaborates on the various types of mouse models applied in cancer research, such as xenograft, syngeneic, and humanized models, together with the state-of-the-art techniques of genetic engineering involved in their generation. We described the methodologies of tumor induction and engraftment procedures and these model applications in drug development, efficacy testing, and studies on immuno-oncology. Further, the chapter covers ethical considerations and regulatory requirements on the use of animals in research, essentially aligned with international guidelines and those in India. The chapter illustrates that mouse models will not become outdated in preclinical testing any time soon but continue to be relevant for the study of tumor biology and the tumor microenvironment besides their use for investigating genetic and molecular pathways in cancer. Emerging technologies, such as CRISPR/Cas9 and organoid integration, are also highlighted for their work in improving the accuracy and translational potential of models. These developments combined with initiatives on collaborative and open science that enable the sharing of data and resources, hold great promise for the future of in vivo cancer modeling. The mouse models will continue to be one of the prime movers in advancing cancer research and formulating individual medication strategies that lead to improved patient outcomes through their integration of classical approaches with modern technologies.},
}

Animals
Mice
*Disease Models, Animal
*Neoplasms/pathology/genetics
Humans
Tumor Microenvironment
Xenograft Model Antitumor Assays/methods
CRISPR-Cas Systems

@article {pmid41032194,
year = {2025},
author = {Naderian, R and Alibabaei, F and Paraandavaji, E and Dehghan, P and Eslami, M},
title = {Phage-Microbiota Interactions in the Gut: Implications for Health and Therapeutic Strategies.},
journal = {Probiotics and antimicrobial proteins},
volume = {},
number = {},
pages = {},
pmid = {41032194},
issn = {1867-1314},
abstract = {The diversified ecology of microorganisms, including bacteria, archaea, fungi, protozoa, and viruses known collectively as the gut microbiota, which includes bacteriophages, is crucial to human health because it affects functions like immune system regulation, vitamin production, and pathogen protection. Bacteriophages are viruses that infect bacteria and are increasingly recognized as a viable treatment option for antibiotic-resistant strains, owing to their high host specificity, which enables precise targeting of drug-resistant bacteria while sparing commensal microbiota. The complex relationships between bacteriophages and gut microbiota are examined, with emphasis on their roles in maintaining health and contributing to disease. Gut microbiota homeostasis is influenced by a number of factors, including age, nutrition, and drugs. Bacteriophages, via lytic cycles and lysogenic conversion, influence the gut microbiota composition and microbial community structure. Gaining an understanding of these processes is crucial to appreciating their contribution to the stability and variety of microbes. Recent research highlights the gut phageome's potential for therapeutic interventions by demonstrating its substantial influence on immunological responses and metabolic problems. The study of phage-microbiota interactions has been transformed by cutting-edge technologies, including high-throughput sequencing, CRISPR-Cas systems, and viral metagenomics, which allow for thorough research and the creation of new therapeutics. Even though tailored medicine and pathogen management hold great potential, obstacles such as regulatory difficulties and bacterial resistance call for additional investigation. Phage-based therapeutic strategies are rapidly advancing, ranging from genetically engineered phages and phages with modified capsid proteins designed to enhance efficacy to phage cocktails that target multiple bacterial strains.},
}

@article {pmid40803956,
year = {2025},
author = {Murata, S and Kushiyama, N and Yabu, Y and Watanabe, K and Fujii, T and Yasui, R and Nojima, D and Maeda, Y and Yoshino, T and Matsuda, Y and Tanaka, T},
title = {Establishment of genome editing techniques in the marine oleaginous diatom Fistulifera solaris for improved oil accumulation.},
journal = {Journal of bioscience and bioengineering},
volume = {140},
number = {5},
pages = {271-276},
doi = {10.1016/j.jbiosc.2025.07.008},
pmid = {40803956},
issn = {1347-4421},
mesh = {*Diatoms/genetics/metabolism ; *Gene Editing/methods ; CRISPR-Cas Systems ; Biofuels ; Lipase/genetics/metabolism ; Gene Knockout Techniques ; *Oils/metabolism ; Microalgae/genetics/metabolism ; Triglycerides/metabolism ; *Lipid Metabolism/genetics ; },
abstract = {Biofuel production using microalgae has attracted considerable attention owing to high growth rate and lipid accumulation properties. However, further enhancement in lipid productivity is required to render this economically feasible. CRISPR/Cas9, which is one of the powerful genome editing tools, is an essential technique that may solve this problem. The marine diatom Fistulifera solaris JPCC DA0580 is a promising candidate of the biofuel production, since it accumulates significant amount of lipids. However, genome editing techniques have not yet been established for F. solaris, which prevent the construction of valuable strains. In this study, CRISPR/Cas9-mediated specific gene knockout technique was established in F. solaris, through targeting adenine phosphoribosyl transferase gene (apt) and triacylglycerol (TAG) lipase gene (tgl1). Mutations in the target sequence were detected in apt- and tgl1-edited mutants. Moreover, the mutants showed distinct phenotypes, such as suppression of TAG degradation and resistance to 2-fluoroadenine. These results indicate the successful demonstration of CRISPR/Cas9-mediated genome editing in the oleaginous marine diatom F. solaris. Furthermore, oil degradation was successfully suppressed by knocking-out tgl1. The CRISPR/Cas9-mediated genome editing established in this study provides key molecular tools for both the basic biology and the future biotechnological applications of F. solaris, such as biofuel production.},
}

*Diatoms/genetics/metabolism
*Gene Editing/methods
CRISPR-Cas Systems
Biofuels
Lipase/genetics/metabolism
Gene Knockout Techniques
*Oils/metabolism
Microalgae/genetics/metabolism
Triglycerides/metabolism
*Lipid Metabolism/genetics

@article {pmid40774862,
year = {2025},
author = {Karashima, T and Oda, K and Futagami, T and Hokazono, H and Takashita, H},
title = {Ribonucleoprotein-based CRISPR/Cas9 genome co-editing in Aspergillus luchuensis mut. kawachii.},
journal = {Journal of bioscience and bioengineering},
volume = {140},
number = {5},
pages = {298-305},
doi = {10.1016/j.jbiosc.2025.07.006},
pmid = {40774862},
issn = {1347-4421},
mesh = {*CRISPR-Cas Systems/genetics ; *Ribonucleoproteins/genetics/metabolism ; *Gene Editing/methods ; *Aspergillus/genetics/metabolism ; Gene Knockout Techniques ; *Genome, Fungal ; RNA, Guide, CRISPR-Cas Systems/genetics ; Sulfate Adenylyltransferase/genetics ; },
abstract = {In this study, we established a ribonucleoprotein-based clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome co-editing method for the white koji fungus, Aspergillus luchuensis mut. kawachii. To introduce the single guide RNA-Cas9 ribonucleoprotein complex into protoplast cells of A. luchuensis mut. kawachii, we investigated the conditions for protoplast preparation using Yatalase -Plus-. Subsequently, we employed the ribonucleoprotein-based method to knockout the ATP sulfurylase-encoding sC gene, which imparts selenate resistance in the model strain NBRC 4308 and the industrial strain No. 8046. Furthermore, we explored genome co-editing by simultaneously targeting sC along with either the orotidine 5'-phosphate decarboxylase-encoding pyrG gene or the transcriptional activator of protease genes-encoding prtR gene in NBRC 4308. The transformants were selected in medium containing selenate, resulting in the successful generation of pyrG- and prtR-knockout strains. Similarly, transformants were selected on medium containing selenate, resulting in the successful generation of prtR-knockout strain in No. 8046. These results demonstrate that the ribonucleoprotein-based genome co-editing method is applicable not only to the model strain but also to industrial strains, making it a promising approach for manipulating A. luchuensis mut. kawachii.},
}

*CRISPR-Cas Systems/genetics
*Ribonucleoproteins/genetics/metabolism
*Gene Editing/methods
*Aspergillus/genetics/metabolism
Gene Knockout Techniques
*Genome, Fungal
RNA, Guide, CRISPR-Cas Systems/genetics
Sulfate Adenylyltransferase/genetics

@article {pmid40634141,
year = {2025},
author = {Shan, L and Verstrepen, KJ and Wang, Q and Dai, Z},
title = {A homologous recombination-proficient Yarrowia lipolytica chassis for multiplex genome manipulation.},
journal = {Trends in biotechnology},
volume = {43},
number = {10},
pages = {2627-2645},
doi = {10.1016/j.tibtech.2025.06.009},
pmid = {40634141},
issn = {1879-3096},
mesh = {*Yarrowia/genetics ; *Homologous Recombination/genetics ; *Gene Editing/methods ; DNA End-Joining Repair/genetics ; *Genome, Fungal/genetics ; CRISPR-Cas Systems ; },
abstract = {Homologous recombination (HR) greatly facilitates precise genome editing. However, most organisms prefer error-prone non-homologous end joining (NHEJ) for DNA double-strand break (DSB) repair. Here, the NHEJ-proficient Yarrowia lipolytica was transformed into a HR-proficient strain by systematic engineering of recombination machinery, regulating the multiinvasion-induced rearrangement (MIR) process, and expressing cognate single-stranded DNA-annealing protein (SSAP)-single-stranded DNA-binding protein (SSB) pairs. These strategies improved HR efficiency by 38.9, 1.6, and 1.2-fold compared with the NHEJ-deficient strain for multifragment multisite integration, and multi- and single-fragment single-site integration, respectively. Moreover, HR efficiency remained high at 58% even with 50-base pair (bp) homology arms (HAs) and reached 11% for simultaneously integrating two mega-DNA fragments (18.0 kb and 13.5 kb) at two genome sites. This strain also enabled simultaneous editing, repression, and activation of multiple genes, while cellular robustness parameters showed marked increases over the NHEJ-deficient strain. Our work provides a HR-proficient Y. lipolytica chassis allowing efficient and precise genome editing of this increasingly important microbe.},
}

*Yarrowia/genetics
*Homologous Recombination/genetics
*Gene Editing/methods
DNA End-Joining Repair/genetics
*Genome, Fungal/genetics
CRISPR-Cas Systems

@article {pmid40605271,
year = {2025},
author = {Stigzelius, V and Cavallo, AL and Chandode, RK and Nitsch, R},
title = {Peeling back the layers of immunogenicity in Cas9-based genomic medicine.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {10},
pages = {4714-4730},
doi = {10.1016/j.ymthe.2025.06.045},
pmid = {40605271},
issn = {1525-0024},
mesh = {*CRISPR-Cas Systems/immunology ; Humans ; *Gene Editing/methods ; *Genetic Therapy/methods ; Animals ; *CRISPR-Associated Protein 9/immunology/genetics ; *Genomics/methods ; },
abstract = {The CRISPR-Cas9 genome editing system is rewriting the treatment of genetic disorders, offering unprecedented potential for detrimental and previously untreatable diseases. As this technology advances toward wider utilization in clinical applications, the immunogenicity of Cas9 nuclease has emerged as a potential challenge for in vivo therapies. Immune recognition of CRISPR-Cas9 components can trigger both innate and adaptive responses. The complex interactions between Cas9, delivery vectors, and host immune reactivity play a crucial role in determining the safety and efficacy of CRISPR-based treatments. Recent advances in mitigating Cas9 immunogenicity include epitope engineering, optimized delivery systems, and nucleic acid modifications. These strategies, explored across various tissue contexts and delivery methods, show promise in enhancing the tolerability of CRISPR-based therapies. However, pre-existing immunity to Cas9 and the potential for long-term adaptive immune responses remain important considerations. Addressing these immunological challenges requires an integrated approach, combining insights from immunology with innovative engineering solutions. As the field progresses, overcoming Cas9 immunogenicity will be crucial for realizing the full therapeutic potential of the CRISPR-Cas9 system in diverse clinical applications.},
}

*CRISPR-Cas Systems/immunology
Humans
*Gene Editing/methods
*Genetic Therapy/methods
Animals
*CRISPR-Associated Protein 9/immunology/genetics
*Genomics/methods

@article {pmid40368676,
year = {2025},
author = {Zhang, S and Xu, D and Li, F and Wang, J},
title = {CRISPR-based non-nucleic acid detection.},
journal = {Trends in biotechnology},
volume = {43},
number = {10},
pages = {2494-2508},
doi = {10.1016/j.tibtech.2025.04.012},
pmid = {40368676},
issn = {1879-3096},
mesh = {*CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Nucleic Acids/genetics/analysis ; Gene Editing/methods ; Humans ; },
abstract = {Characterization of clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) trans-cleavage activities has initiated the era of next-generation CRISPR diagnostics. By using the trans-cleavage reaction for signal output, CRISPR systems have been engineered to detect non-nucleic acids (NNAs), including ions, inorganic small molecules, organic compounds, proteins, and bacteria. Diverse strategies are being used to specifically recognize NNAs and regulate Cas trans-cleavage activities, via generation or depletion of output signals. In this review, we introduce the principles and advantages of CRISPR-based NNA detection. We then classify CRISPR-based NNA detection strategies into three classes: the generation or depletion of free activators, synthesis of crRNAs, and reconstruction of active Cas effectors. Finally, we discuss the challenges and potential strategies to advance both clinical and nonclinical applications of CRISPR-based NNA detection.},
}

*CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*Nucleic Acids/genetics/analysis
Gene Editing/methods
Humans

@article {pmid41032193,
year = {2025},
author = {Yetiman, AE and Horzum, M and Kanbur, E and Çadir, M and Bahar, D and Gürbüz, Ş and Karaman, MZ and Fidan, Ö and Kaya, M and Yetiman, S and Doğan, M and Akbulut, M},
title = {Pangenome Analysis and Genome-Guided Probiotic Evaluation of Cyclic Dipeptides Producing Levilactobacillus brevis DY55bre Strain from a Lactic Acid Fermented Shalgam to Assess Its Metabolic, Probiotic Potentials, and Cytotoxic Effects on Colorectal Cancer Cells.},
journal = {Probiotics and antimicrobial proteins},
volume = {},
number = {},
pages = {},
pmid = {41032193},
issn = {1867-1314},
support = {FBA-2025-14779//Bilimsel Araştırma Projeleri, Erciyes Üniversitesi/ ; },
abstract = {This study investigates the genetic, metabolic, and probiotic characteristics of Levilactobacillus brevis DY55bre, a strain isolated from the traditional Turkish fermented beverage, shalgam. Whole-genome sequencing revealed a circular genome of 2.485 Mb with a GC content of 45.72%, predicted 2791 genes, and multiple CRISPR-Cas systems. Pangenome analysis demonstrated an open structure, with 18.9% core genes and 103 strain-specific genes, highlighting its genetic diversity. The DY55bre exhibits heterofermentative carbohydrate metabolism due to the presence of the araBAD operon and the lack of 1-phosphofructokinase (pfK) and fructose-1,6-bisphosphate aldolase enzymes. Probiotic evaluation revealed firm survival under simulated gastrointestinal conditions, including resistance to acidic pH (as low as 3.0) and bile salts (up to 1%), along with significant adhesion to intestinal epithelial cell lines (HT29;59.3%, Caco-2;87%, and DLD-1;60.8%). The strain exhibited high auto-aggregation (84.55%) and cell surface hydrophobicity (56.69%), essential for gut colonization. Safety assessments confirmed its non-hemolytic nature and absence of horizontally acquired antibiotic resistance genes. Notably, GC-MS analysis identified bioactive cyclic dipeptides, Cyclo(D-Phe-L-Pro) and Cyclo(L-Leu-L-Pro), which demonstrated cytotoxic effects against colorectal cancer cell lines, with IC50 values of 7.71 mg/mL for HT29 and 3.19 mg/mL for DLD-1. The cell-free supernatant exhibited antimicrobial activity against pathogens, likely due to the synergistic effects of cyclic dipeptides, organic acids, and other metabolites. Antioxidant assays revealed significant ABTS[+] (76.63%) and DPPH (34.25%) radical scavenging activities, while cholesterol assimilation tests showed a 27.29% reduction. These findings position the DY55bre as a promising candidate for functional foods, nutraceuticals, and therapeutic applications, warranting further in vivo validation.},
}

@article {pmid41032156,
year = {2025},
author = {Meshram, V and Jadhav, SK and Chandrawanshi, NK},
title = {Strain improvement of Cordyceps militaris for optimized bioactive metabolite biosynthesis: current progress and prospective approaches.},
journal = {Antonie van Leeuwenhoek},
volume = {118},
number = {11},
pages = {162},
pmid = {41032156},
issn = {1572-9699},
support = {201610136180//University Grants Commission/ ; },
mesh = {*Cordyceps/metabolism/genetics ; *Metabolic Engineering/methods ; Gene Editing ; CRISPR-Cas Systems ; Biosynthetic Pathways ; },
abstract = {Cordyceps militaris is a rare and highly valued medicinal fungus that has attracted considerable attention due to its production of diverse bioactive compounds, including nucleosides such as cordycepin, polysaccharides, lovastatin, carotenoids, etc., all of which exhibit significant nutritional and therapeutic potential. However, the large-scale utilization of C. militaris is constrained by several critical challenges. A major limitation is the progressive degeneration of strains over successive subcultures, which adversely affects fruiting body formation and metabolite biosynthesis. Moreover, genetic instability during long-term culture, contamination risks in large-scale production, and the lack of standardized cultivation and extraction protocols often result in variable product quality. The absence of efficient genetic transformation systems and the low success rate of genome editing approaches further complicate efforts in molecular strain improvement. This review provides a comprehensive overview of the principal bioactive compounds produced by C. militaris and critically evaluates the current challenges and limitations associated with both conventional and advanced strain improvement strategies. These include conventional approaches such as mutagenesis and protoplast fusion, as well as genome-editing technologies like CRISPR/Cas9, which are employed to enhance the biosynthesis of target metabolites. Moreover, the integration of metabolic engineering frameworks offers significant potential for rational strain design, optimization of bioprocesses, and the discovery of novel therapeutic agents.},
}

*Cordyceps/metabolism/genetics
*Metabolic Engineering/methods
Gene Editing
CRISPR-Cas Systems
Biosynthetic Pathways

@article {pmid41032013,
year = {2025},
author = {Huang, X and Li, H and Du, J and Xie, W and Liu, Y and Lin, Y and Xing, H},
title = {HUH endonuclease-mediated DNA-protein conjugates: sequence-specific tools and cellular applications.},
journal = {Chemical communications (Cambridge, England)},
volume = {},
number = {},
pages = {},
doi = {10.1039/d5cc04628a},
pmid = {41032013},
issn = {1364-548X},
abstract = {This highlight review article summarizes recent advances in employing HUH endonucleases as self-labeling protein tags for the sequence-specific covalent conjugation of unmodified ssDNA and examines their applications in cellular studies via engineered DNA-protein conjugates. We outline the structural basis and catalytic mechanism of the conserved HUH and Y motifs, which enable high selectivity, bioorthogonality, and robust conjugation under physiological conditions. Recent applications demonstrate the versatility of HUH-based DNA-protein conjugates in programmable cellular interface engineering, targeted therapeutic delivery, and enhancement of genome editing systems such as CRISPR-Cas. In the perspective section, we further highlight two emerging directions: computational tools such as the HUHgle platform for predictive substrate design, and directed evolution strategies extending HUH reactivity toward RNA substrates. Together, these advancements establish HUH endonucleases as powerful, programmable tools for generating DNA-protein conjugates that enable innovations in chemical biology, synthetic biology, and therapeutics.},
}

@article {pmid41030550,
year = {2025},
author = {Yang, ZQ and Li, MJ and Ahmad, F and Jin, CZ and Li, T and Jin, FJ and Shin, KS and Jin, L},
title = {Application of the transposon-associated TnpB system of CRISPR-Cas in bacteria: Deinococcus.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1604032},
pmid = {41030550},
issn = {1664-302X},
abstract = {Deinococcus radiodurans is one of the most radioresistant organisms found on Earth to date, showing extreme resistance to damage factors such as UV, drought, and mutagens, and is of great interest to scientists around the world. It was determined that the TnpB protein from D. radiodurans ISDra2 functions as an RNA-guided endonuclease, serving as a functional ancestor for the widely used CRISPR-Cas endonucleases. The CRISPR-Cas system is an "acquired immune system" found in most Bacteria and Archaea, and used in a wide range of biological and medical research fields. Cas12f is the smallest RNA-directed nuclease that is currently known and possesses unique characteristics. There has been extensive research conducted on the origin, classification, and mechanism of action of CRISPR-Cas12f, as well as its application in the field of gene editing. TnpB, as the protein closest to Cas12f in the evolutionary tree, has the potential to be used as a new micro-editing tool. Systematic studies have been conducted on it to develop smaller volumes of precision gene editing and treatment tools. In this review, the research progress, mechanism, and application of TnpB protein in D. radiodurans were reviewed. In addition, the classification of CRISPR-Cas system and the application and function of CRISPR-Cas12f in gene editing are also introduced and summarized.},
}

@article {pmid41028589,
year = {2026},
author = {Bhargava, CN and Karuppannasamy, A and Ramasamy, A},
title = {CRISPR/Cas9-Mediated Genome Editing in the Management of Oriental Fruit Fly, Bactrocera dorsalis (Hendel) (Tephritidae: Diptera).},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2966},
number = {},
pages = {259-270},
pmid = {41028589},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Tephritidae/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genome, Insect ; Pest Control, Biological/methods ; },
abstract = {The oriental fruit fly, Bactrocera dorsalis (Hendel), is a highly invasive polyphagous pest that causes significant damage to horticultural crops of global importance. Traditional management practices have not been effective in controlling this pest, and therefore, there is a need for alternative management strategies. CRISPR/Cas9-driven genome editing has been successfully used in a wide range of insects to induce site-specific, off-target minimized mutations that result in loss of function. This technique can be used to develop precision-guided sterile insect technique (pgSIT) and gene drive programs, which can be used for area-wide suppression of the pest. This chapter provides a brief overview of the workflow for RNP-based genome editing, which can be used to validate and establish gene function for large-scale gene drive programs aimed at combating this pest. The RNP, or ribonucleoprotein complex, comprises the sgRNA and Cas9 protein, which are microinjected into the G0 stage embryos for heritable editing of the target gene(s).},
}

Animals
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Tephritidae/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Genome, Insect
Pest Control, Biological/methods

@article {pmid41028581,
year = {2026},
author = {Ashokkumar, S and Ponnurangan, V and Krish, KK and Loganathan, A and Eswaran, K and Vaikuntavasan, P and Duraialagaraja, S and Shanmugam, V},
title = {CRISPR-Mediated Gene Editing for Inducing Thermosensitive Genic Male Sterility and Sheath Blight Resistance in Rice.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2966},
number = {},
pages = {127-140},
pmid = {41028581},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; *Oryza/genetics/microbiology ; *CRISPR-Cas Systems ; *Disease Resistance/genetics ; *Plant Infertility/genetics ; *Plant Diseases/genetics/microbiology ; Plants, Genetically Modified/genetics ; },
abstract = {Recent advances in genome editing enable the researchers to focus more and more on the ability to manipulate genomes at specific sites. Efficient methods for genome editing further promote gene discovery and functional gene analyses in model plants as well as the introduction of novel desired agricultural traits in important species. CRISPR/Cas9 technology enables precise genetic modification through the creation of double-strand breaks in a target region and the generation of desired alterations during the repair process. In this chapter, we describe the cloning strategy, transformation protocols, triparental mating procedure, and characterization of genome-edited genetic male sterile mutants and sheath blight disease-resistant mutant plants.},
}

*Gene Editing/methods
*Oryza/genetics/microbiology
*CRISPR-Cas Systems
*Disease Resistance/genetics
*Plant Infertility/genetics
*Plant Diseases/genetics/microbiology
Plants, Genetically Modified/genetics

@article {pmid41028476,
year = {2026},
author = {Raschmanová, H and Weninger, A and Kovar, K},
title = {Engineering Pichia pastoris Strains Using CRISPR/Cas9 Technologies: The Basic Protocol.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2697},
number = {},
pages = {361-371},
pmid = {41028476},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Saccharomycetales/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Metabolic Engineering/methods ; *Pichia/genetics ; Genome, Fungal ; },
abstract = {The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats, CRISPR-associated protein 9) system has become a commonly used tool for genome editing and metabolic engineering. For Komagataella phaffii, commercialized as Pichia pastoris, the CRISPR/Cas9 protocol for genome editing was established in 2016 and since then has been employed to facilitate genetic modifications such as markerless gene disruptions and deletions as well as to enhance the efficiency of homologous recombination.In this chapter, we describe a robust basic protocol for CRISPR-based genome editing, demonstrating near 100% targeting efficiency for gene inactivation via a frameshift mutation. As described in other chapters of this volume, CRISPR/Cas9 technologies for use in P. pastoris have been further optimized for various specific purposes.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Saccharomycetales/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
*Metabolic Engineering/methods
*Pichia/genetics
Genome, Fungal

@article {pmid41028475,
year = {2026},
author = {Smirnov, K and Rieder, L and Glieder, A},
title = {High-Throughput Generation of Pichia pastoris Knock-Out Strains by Using CRISPR/Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2697},
number = {},
pages = {345-360},
pmid = {41028475},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Plasmids/genetics ; *Gene Knockout Techniques/methods ; *Saccharomycetales/genetics ; Genetic Vectors/genetics ; },
abstract = {The CRISPR/Cas9 (CRISPR is an acronym for clustered regularly interspaced short palindromic repeats) system is a powerful molecular biological tool simplifying the process of genome engineering. Originally demonstrated to function in human and mouse cells, the portfolio of organisms that can be engineered by the new and groundbreaking technology was soon expanded. In the past years, CRISPR/Cas9 tools for use in Komagataella phaffii were reported to allow the generation of K. phaffii mutant strains in less than 2 weeks. In addition, the K. phaffii tailored system uses episomal vectors for the expression of the CRISPR/Cas9 elements, which allows the recycling of the plasmid after the CRISPR editing to obtain empty mutant strains. This means that the engineered strains do not carry the expression cassette of the resistance marker and CRISPR/Cas9 plasmid in their genome and are therefore a superb starting point for further investigations.In this chapter, we describe a pipeline for the high-throughput generation of K. phaffii mutant strains with interrupted open reading frames of genes, by using the CRISPR/Cas9 system in combination with error-prone repair of the double-strand break by NHEJ. The pipeline we developed consists of four steps: (a) CRISPR/Cas9 plasmids assembly, (b) transformation of K. phaffii, (c) screening for mutant strains, and (d) plasmid elimination and is due to the detailed description of every step being easily reproducible. To intensify and simplify the research work, most of the described procedures can be performed in a 96-well format.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Plasmids/genetics
*Gene Knockout Techniques/methods
*Saccharomycetales/genetics
Genetic Vectors/genetics

@article {pmid41028463,
year = {2026},
author = {Pichler, C and Weiss, F and Glieder, A},
title = {Autonomously Replicating Sequence-Bearing Plasmids Utilized in Pichia pastoris.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2697},
number = {},
pages = {191-203},
pmid = {41028463},
issn = {1940-6029},
mesh = {*Plasmids/genetics ; *Saccharomycetales/genetics ; *DNA Replication ; Transformation, Genetic ; Saccharomyces cerevisiae/genetics ; },
abstract = {Plasmids are a common tool in biotechnology to deliver recombinant DNA into microbial cells for the production of enzymes, pharmaceutical proteins, chemicals, or metabolites. Therefore, a stable plasmid system that provides reliable gene expression over generations is essential for the successful utilization of single-cell organisms in research and production applications. Most Komagataella phaffii expression clones are generated by the integration of linear plasmids into the genome, as circular episomal plasmids are not stable under non-selective conditions. The low rate of homology-directed specific integration and the large variation among transformants of random integration limit the organism's application in enzyme engineering approaches or comparative studies where high transformation rates and uniform expression levels are desired. In the yeast Saccharomyces cerevisiae, the problem of circular plasmid stability and partition to the daughter cells during mitosis has been solved by combining centromeric sequences or elements of the 2-micron plasmid with an autonomously replicating sequence (ARS) that serves as an origin of replication. Similar attempts have not yet been successful or widely adapted in K. phaffii; hence, permanent selection pressure is required to maintain episomal plasmids in K. phaffii. There are no reports so far about functional 2-micron plasmids for P. pastoris, and CEN/ARS plasmids for P. pastoris are usually rather large and do not provide the high transformation rates as known for episomal plasmids of S. cerevisiae expression systems. However, the availability of a broad set of resistance, auxotrophic, and carbon source utilization markers facilitates reliable plasmid selection in small-scale screening applications and recently also proved to be successful for bioreactor-scale expression. This allows the combined advantages of high transformation rates and low clonal variability of ARS plasmids to be exploited. This article describes the successful utilization of ARS1-containing plasmids in K. phaffii, including antibiotic-free selection, complementation of knockout strains, or even for the application of CRISPR/Cas by transient gRNA and CAS9 gene expression in K. phaffii.},
}

*Plasmids/genetics
*Saccharomycetales/genetics
*DNA Replication
Transformation, Genetic
Saccharomyces cerevisiae/genetics

@article {pmid41028441,
year = {2025},
author = {Kannan, S and Tennyson, J},
title = {Regeneration of Transgenic Nicotiana benthamiana Raised from the Genome-Edited Protoplast.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2973},
number = {},
pages = {261-274},
pmid = {41028441},
issn = {1940-6029},
mesh = {*Nicotiana/genetics/growth & development ; *Protoplasts/metabolism ; *Gene Editing/methods ; *Plants, Genetically Modified/genetics ; CRISPR-Cas Systems ; *Genome, Plant ; Transfection/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Regeneration/genetics ; },
abstract = {Plant genome editing is an emerging technique that has revolutionized plant genome engineering which helps to edit the plant genome precisely for the development of traits in many crops. Specifically, with clustered regularly interspaced short palindromic sequence (CRISPR)-CRISPR-associated protein (Cas) system, a progressive improvement in genome editing has been achieved with protoplast. Though protoplast isolation, transfection, and regeneration are available for many plants, regeneration of protoplast for many plants remains major challenge. In this methodology chapter, we outlined the construction of sgRNA for genome editing, transfection, and regeneration of transgenic N. benthamiana from the genome-edited protoplast and assay for gene targeting.},
}

*Nicotiana/genetics/growth & development
*Protoplasts/metabolism
*Gene Editing/methods
*Plants, Genetically Modified/genetics
CRISPR-Cas Systems
*Genome, Plant
Transfection/methods
RNA, Guide, CRISPR-Cas Systems/genetics
*Regeneration/genetics

@article {pmid41028420,
year = {2025},
author = {Andrew-Peter-Leon, MT and Pillai, MA and Kumar, KK and Sumithra, V},
title = {Agrobacterium-Mediated Genetic Transformation and Genome Editing Using CRISPR-Cas9 Constructs in Rice.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2973},
number = {},
pages = {27-49},
pmid = {41028420},
issn = {1940-6029},
mesh = {*Oryza/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Transformation, Genetic ; *Agrobacterium/genetics ; Plants, Genetically Modified/genetics ; *Genome, Plant ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas)9 has become an essential tool in every molecular plant breeding laboratory. CRISPR-Cas9 causes highly precise knock-out mutations in plants that can be exploited in crop improvement programmes. However, poor response to Agrobacterium-mediated genetic transformation in recalcitrant rice genotypes is a major limiting factor. This protocol describes a detailed procedure for genome editing with CRISPR-Cas9 in recalcitrant rice genotypes that otherwise show a poor response to tissue culture. With this method, high transformation efficiency can be achieved in relatively a short period.},
}

*Oryza/genetics
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Transformation, Genetic
*Agrobacterium/genetics
Plants, Genetically Modified/genetics
*Genome, Plant

@article {pmid41028383,
year = {2026},
author = {Yang, H and Bao, A and Tran, LP and Cao, D},
title = {CRISPR/Cas9-Based Gene Editing in Soybean.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2977},
number = {},
pages = {251-267},
pmid = {41028383},
issn = {1940-6029},
mesh = {*Glycine max/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Plasmids/genetics ; Agrobacterium/genetics ; Transformation, Genetic ; },
abstract = {CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated Cas9)-based gene editing is a robust tool for functional genomics research and breeding programs in various crops. In soybean (Glycine max), a number of laboratories have obtained mutants by the CRISPR/Cas9 system; however, there has not yet been a detailed method for the CRISPR/Cas9-based gene editing in soybean. Here, we describe the procedures for constructing the CRISPR/Cas9 plasmid suitable for soybean gene editing and the modified protocols for Agrobacterium-mediated soybean transformation and regeneration from cotyledonary node explants containing the Cas9/sgRNA (single-guide RNA) transgenes.},
}

*Glycine max/genetics
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Plants, Genetically Modified/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Plasmids/genetics
Agrobacterium/genetics
Transformation, Genetic

@article {pmid41028112,
year = {2025},
author = {Najar, IN and Sharma, P and Das, R and Mondal, K and Singh, AK and Radha, A and Sharma, V and Sharma, S and Thakur, N and Gandhi, SG and Kumar, V},
title = {Unveiling the probiotic potential of the genus Geobacillus through comparative genomics and in silico analysis.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {33748},
pmid = {41028112},
issn = {2045-2322},
mesh = {*Probiotics ; *Geobacillus/genetics ; *Genome, Bacterial ; *Genomics/methods ; Computer Simulation ; CRISPR-Cas Systems ; Humans ; Prophages/genetics ; },
abstract = {Pursuing new probiotic targets has surged, driven by next-generation sequencing, facilitating a thorough exploration of bacterial traits. The genus Geobacillus stands out as a promising candidate for probiotics. The study explored the genetic attributes of the genus Geobacillus for their resilience to gastrointestinal conditions, nutrient production, and immunomodulatory compound creation, revealing potential probiotic traits. Predictive analyses of genomic elements like prophages, CRISPR-Cas systems, insertion sequences, genomic islands, antibiotic resistance genes, and CAZymes were conducted to assess safety. Comparative genomic analysis was performed using 18 published Geobacillus genomes and a few Lactobacillus and Bifidobacterium genomes as controls. Genes associated with probiotic traits, such as adhesion, stress tolerance (acid/bile, osmotic, oxidative), immune modulation, and molecular chaperones, were uniformly detected in Geobacillus. Mobile genetic elements (such as plasmids, prophages, and insertion sequences), virulence factors, toxins, and antibiotic resistance genes were absent, while CRISPR-Cas systems and CAZymes were present. The pan-genome comprised 25,284 protein-coding genes. Comparative genomic analysis revealed an open pan-genome for Geobacillus. Pan-genome exhibited variability, particularly in genes linked to environmental interaction and secondary metabolite synthesis. Geobacillus appears potentially safe and well-suited for the gut habitat. However, further in vitro studies are essential to confirm its probiotic potential.},
}

*Probiotics
*Geobacillus/genetics
*Genome, Bacterial
*Genomics/methods
Computer Simulation
CRISPR-Cas Systems
Humans
Prophages/genetics

@article {pmid41027434,
year = {2025},
author = {Jin, S and Zhu, Z and Li, Y and Zhang, S and Liu, Y and Li, D and Li, Y and Luo, Y and Cheng, Z and Zhao, KT and Gao, Q and Yang, G and Li, H and Liang, R and Zhang, R and Qiu, JL and Zhang, YE and Liu, JG and Gao, C},
title = {Functional RNA splitting drove the evolutionary emergence of type V CRISPR-Cas systems from transposons.},
journal = {Cell},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.cell.2025.09.004},
pmid = {41027434},
issn = {1097-4172},
abstract = {Transposon-encoded TnpB nucleases gave rise to type V CRISPR-Cas12 effectors through multiple independent domestication events. These systems use different RNA molecules as guides for DNA targeting: transposon-derived right-end RNAs (reRNAs or omega RNAs) for TnpB and CRISPR RNAs for type V CRISPR-Cas systems. However, the molecular mechanisms bridging transposon activity and CRISPR immunity remain unclear. We identify TranCs (transposon-CRISPR intermediates) derived from distinct IS605- or IS607-TnpB lineages. TranCs utilize both CRISPR RNAs and reRNAs to direct DNA cleavage. The cryoelectron microscopy (cryo-EM) structure of LaTranC from Lawsonibacter sp. closely resembles that of the ISDra2 TnpB complex; however, unlike a single-molecule reRNA, the LaTranC guide RNA is functionally split into a tracrRNA and crRNA. An engineered RNA split of ISDra2 TnpB enabled activity with a CRISPR array. These findings indicate that functional RNA splitting was the primary molecular event driving the emergence of diverse type V CRISPR-Cas systems from transposons.},
}

@article {pmid41026506,
year = {2025},
author = {Demissie, HA and Das, S and Thompson, JR and Lucks, JB},
title = {An Integrated Nucleic Acid Sequence-Based Amplification (NASBA) and CRISPR-Cas13a-Based Platform for Accurate and Sensitive Detection of Cucumber Mosaic Virus.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.5c00406},
pmid = {41026506},
issn = {2161-5063},
abstract = {Cucumber mosaic virus (CMV) is a highly prevalent ssRNA viral crop pathogen that contributes to substantial losses in agricultural productivity worldwide. The first step in managing the impact of this pathogen is an accurate and timely diagnosis. However, current sensing strategies are hampered by several limitations, including insufficient sensitivity, off-target effects, and the need for complex instrumentation. To address these challenges, we refined a highly specific and sensitive system that pairs nucleic acid sequence-based amplification (NASBA) with clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13a to selectively amplify and detect crop pathogens. To configure this system for CMV biosensing, we first screened guide RNAs and successfully validated designs that detect attomolar concentrations of purified CMV fragments. We then developed a simplified reaction assembly workflow toward optimizing the system for downstream point-of-use utility. Using this workflow, we demonstrated minimal matrix effects when detecting purified CMV fragments in a range of plant lysate backgrounds and showed high test specificity to CMV in the presence of common nontarget viral crop pathogens. We also showed that the NASBA-Cas13a system effectively detects the viral target in infected plant samples, as validated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Finally, we optimized the system for lyophilization and long-term storage, toward preparing it for point-of-use settings. This work expands the suite of CMV diagnostic tools, offering a sensitive, specific, and user-friendly biosensing strategy. Through modular design, this assay has the potential to be reconfigured for the detection of a range of crop viruses, enhancing viral surveillance and improving infection management.},
}

@article {pmid41026406,
year = {2025},
author = {Rahman, MU and Shah, JA and Khan, MN and Bilal, H and Zhu, D and Du, Z and Mu, DS},
title = {Innovative Approaches to Combat Antimicrobial Resistance: A Review of Emerging Therapies and Technologies.},
journal = {Probiotics and antimicrobial proteins},
volume = {},
number = {},
pages = {},
pmid = {41026406},
issn = {1867-1314},
abstract = {The threat of antimicrobial resistance (AMR) presents a challenge in infectious diseases, leading to higher illness and deaths worldwide. No new antibiotic has been introduced, leaving healthcare systems vulnerable to resistant pathogens. Researchers are exploring innovative approaches to overcome this growing resistance crisis. One promising strategy is synergistic therapy using combined drugs to enhance efficacy and reduce resistance. Other approaches focus on targeting the specific enzymes or proteins responsible for resistance mechanisms, thereby neutralizing the defense strategies of microorganisms. Advances in drug delivery systems have also shown promise in improving the effectiveness of existing antimicrobial agents. Biotechnological breakthroughs, such as bacteriophages and antibodies, have seen partial clinical implementation, while newer approaches like antimicrobial peptides (AMPs), lysins, and probiotics are still under development. Emerging technologies such as CRISPR-Cas and engineered phages demonstrate significant potential in preclinical studies, offering precision targeting of resistance genes and pathogen-specific lysis, respectively. However, their translational success hinges on overcoming delivery challenges, scalability, and regulatory hurdles. Additionally, physicochemical methods that disrupt microbial activity are being explored as alternative treatments. While innovative therapies like phage-derived lysins and CRISPR-Cas systems show promise in preclinical models, their clinical impact remains to be validated through large-scale trials. Their integration into mainstream medicine will depend on addressing practical challenges such as manufacturing consistency, cost considerations, and real-world efficacy assessments. These efforts are crucial for addressing the growing threat of AMR and advancing more effective, sustainable infection control strategies in clinical settings.},
}

@article {pmid41025805,
year = {2025},
author = {Nadar, S and Brown, JC and Coe, LSY and Koukoulidis, NM and Czyż, EM and Czyż, DM},
title = {Antimicrobial resistance and One Health in the high school biology curriculum.},
journal = {Journal of microbiology & biology education},
volume = {},
number = {},
pages = {e0014525},
doi = {10.1128/jmbe.00145-25},
pmid = {41025805},
issn = {1935-7877},
abstract = {Antimicrobial resistance (AMR) is the ability of a microbial organism to resist treatment designed to kill it. It poses a significant global threat to public health, affecting humans, animals, and the environment, in a concept collectively referred to as One Health. While one of the major mitigators of this pressing issue is education, the high school curriculum in the United States does not cover any aspects of AMR. As such, to address this challenge, we developed and delivered a one-week-long unit on AMR within a One Health framework into a high school biology curriculum. The unit aimed to enhance students' understanding of AMR and its implications across the One Health sectors. A survey was designed and administered to measure current knowledge, awareness, interest, and motivation. Through a combination of lectures developed using Universal Design of Learning principles, interactive discussions using team-based learning (TBL) with the help of content experts, hands-on laboratory exercise, and poster presentations, biology students explored the mechanisms of resistance and novel mitigation strategies. Pre- and post-assessments revealed a marked improvement in students' knowledge and comprehension of AMR and therapeutic strategies, such as silver nanoparticles, bacteriocins, bacteriophages, CRISPR-Cas, and immunotherapy. This research study provides a detailed overview of the curriculum design, instructional strategies, and assessment outcomes, offering a replicable model for broadly integrating AMR education into high school curricula. We found that the AMR mitigation strategies lesson, delivered through TBL, significantly enhanced students' understanding of novel therapeutic strategies and fostered high levels of engagement throughout the AMR and One Health unit.},
}

@article {pmid41025245,
year = {2025},
author = {Shin, K and Kim, ET},
title = {Efficient CRISPR-based genome editing for inducible degron systems to enable temporal control of protein function in large double-stranded DNA virus genomes.},
journal = {Journal of microbiology (Seoul, Korea)},
volume = {63},
number = {9},
pages = {e2504008},
doi = {10.71150/jm.2504008},
pmid = {41025245},
issn = {1976-3794},
support = {RS-2024-00352590//Ministry of Science and ICT/ ; RS-2023-00270936//Ministry of Education/ ; //Korea Health Industry Development Institute/ ; RS-2022-KH129726//Ministry of Health and Welfare/ ; RS-2024-00438990//Ministry of Health and Welfare/ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Humans ; *Cytomegalovirus/genetics ; *Genome, Viral ; *Viral Proteins/genetics/metabolism ; Indoleacetic Acids/metabolism/pharmacology ; Green Fluorescent Proteins/genetics ; Recombinational DNA Repair ; Degrons ; },
abstract = {CRISPR-Cas9-based gene editing enables precise genetic modifications. However, its application to human cytomegalovirus (HCMV) remains challenging due to the large size of the viral genome and the essential roles of key regulatory genes. Here, we establish an optimized CRISPR-Cas9 system for precise labeling and functional analysis of HCMV immediate early (IE) genes. By integrating a multifunctional cassette encoding an auxin-inducible degron (AID), a self-cleaving peptide (P2A), and GFP into the viral genome via homology-directed repair (HDR), we achieved efficient knock-ins without reliance on bacterial artificial chromosome (BAC) cloning, a labor-intensive and time-consuming approach. We optimized delivery strategies, donor template designs, and component ratios to enhance HDR efficiency, significantly improving knock-in success rates. This system enables real-time fluorescent tracking and inducible protein degradation, allowing temporal control of essential viral proteins through auxin-mediated depletion. Our approach provides a powerful tool for dissecting the dynamic roles of viral proteins throughout the HCMV life cycle, facilitating a deeper understanding of viral pathogenesis and potential therapeutic targets.},
}

*CRISPR-Cas Systems
*Gene Editing/methods
Humans
*Cytomegalovirus/genetics
*Genome, Viral
*Viral Proteins/genetics/metabolism
Indoleacetic Acids/metabolism/pharmacology
Green Fluorescent Proteins/genetics
Recombinational DNA Repair
Degrons

@article {pmid41025215,
year = {2025},
author = {Park, J and Sipe, GO and Tang, X and Ojha, P and Fernandes, G and Leow, YN and Zhang, C and Osako, Y and Natesan, A and Drummond, GT and Jaenisch, R and Sur, M},
title = {Astrocytic modulation of population encoding in mouse visual cortex via GABA transporter 3 revealed by multiplexed CRISPR/Cas9 gene editing.},
journal = {eLife},
volume = {14},
number = {},
pages = {},
pmid = {41025215},
issn = {2050-084X},
support = {R01DA049005/NH/NIH HHS/United States ; R01MH126351/NH/NIH HHS/United States ; R01NS130361/NH/NIH HHS/United States ; R01MH133066/NH/NIH HHS/United States ; Multidisciplinary University Research Initiative W911NF2110328//United States Department of Defense/ ; F32EY022264/NH/NIH HHS/United States ; Autism Research Initiative Bridge to Independence award//Simons Foundation/ ; Picower Postdoctoral Fellowship//JPB Foundation/ ; },
mesh = {Animals ; *Visual Cortex/physiology ; *Astrocytes/physiology/metabolism ; *GABA Plasma Membrane Transport Proteins/metabolism/genetics ; Mice ; *CRISPR-Cas Systems ; *Gene Editing ; Mice, Knockout ; Neurons/physiology ; },
abstract = {Astrocytes, which are increasingly recognized as pivotal constituents of brain circuits governing a wide range of functions, express GABA transporter 3 (Gat3), an astrocyte-specific GABA transporter responsible for maintenance of extra-synaptic GABA levels. Here, we examined the functional role of Gat3 in astrocyte-mediated modulation of neuronal activity and information encoding. First, we developed a multiplexed CRISPR construct applicable for effective genetic ablation of Gat3 in the visual cortex of adult mice. Using in vivo two-photon calcium imaging of visual cortex neurons in Gat3 knockout mice, we observed changes in spontaneous and visually driven single neuronal response properties such as response magnitudes and trial-to-trial variability. Gat3 knockout exerted a pronounced influence on population-level neuronal activity, altering the response dynamics of neuronal populations and impairing their ability to accurately represent stimulus information. These findings demonstrate that Gat3 in astrocytes profoundly shapes the sensory information encoding capacity of neurons and networks within the visual cortex.},
}

Animals
*Visual Cortex/physiology
*Astrocytes/physiology/metabolism
*GABA Plasma Membrane Transport Proteins/metabolism/genetics
Mice
*CRISPR-Cas Systems
*Gene Editing
Mice, Knockout
Neurons/physiology

@article {pmid41024337,
year = {2025},
author = {Zhang, L and Fu, J and Long, T and Zhang, C and Fan, F and Lang, Z and Zhu, JK},
title = {A Modular and Customizable CRISPR/Cas Toolkit for Epigenome Editing of Cis-regulatory Modules.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {},
number = {},
pages = {e03917},
doi = {10.1002/advs.202503917},
pmid = {41024337},
issn = {2198-3844},
support = {KQTD20240729102038044//Shenzhen Science and Technology Program/ ; 32188102//National Natural Science Foundation of China/ ; },
abstract = {Epigenome and cis-regulome, comprising cis-regulatory elements (CREs) and modules (CRMs), jointly define the architecture of gene regulation. However, the causal mechanisms by which epigenetic marks influence CRM function remain elusive. To address this, modular epigenome editing frameworks, exemplified by dead Cas9-coupled DNA demethylation (dCd) and DNA methylation (dCm) platforms, are developed for programmable dissection and engineering of CRM activity. The dCd system modulates methylation levels and transcriptional output at CRMs in situ or ex situ, in accordance with CRM-specific methylation responsiveness, and alters co-transcriptional RNA processing to yield predictable phenotypic outcomes in plants. These findings underscore the reliability of targeted DNA demethylation. In parallel, the dCm system reconstitutes methylation-dependent and -sensitive CRMs of diverse origins in Saccharomyces cerevisiae, a species devoid of native DNA methylation, enabling causal dissection of epigenetic regulation and revealing cross-species portability. This system further uncovers crosstalk between DNA methylation and chromatin modifications, and enables logic-gated control of endogenous genes through CRM engineering. Incorporation of optogenetic and temperature-sensitive anti-CRISPR inhibitors confers tunable, reversible regulation, proposing dCm as a foundation for input-responsive synthetic epigenome editors. Together, these frameworks provide a versatile platform to decode and reprogram cis-regulatory epigenetic logic, with broad applications in trait design and synthetic biology.},
}

@article {pmid41023727,
year = {2025},
author = {Zhao, XY and Gao, C and Zhao, WW and Zhou, ZH and Zhuang, TC and Guo, C and Ji, MH},
title = {Development of a single-tube, dual-target CRISPR Cas12a/Cas13a system for rapid screening of coinfection with respiratory syncytial virus and rhinovirus.},
journal = {Virology journal},
volume = {22},
number = {1},
pages = {311},
pmid = {41023727},
issn = {1743-422X},
support = {Jiangsu Education Department (2023) No.11//"Nursing Science" Funded by the 4th Priority Discipline Development Program of Jiangsu Higher Education Institutions/ ; Jiangsu Education Department (2023) No.11//"Nursing Science" Funded by the 4th Priority Discipline Development Program of Jiangsu Higher Education Institutions/ ; Jiangsu Education Department (2023) No.11//"Nursing Science" Funded by the 4th Priority Discipline Development Program of Jiangsu Higher Education Institutions/ ; Jiangsu Education Department (2023) No.11//"Nursing Science" Funded by the 4th Priority Discipline Development Program of Jiangsu Higher Education Institutions/ ; Jiangsu Education Department (2023) No.11//"Nursing Science" Funded by the 4th Priority Discipline Development Program of Jiangsu Higher Education Institutions/ ; Jiangsu Education Department (2023) No.11//"Nursing Science" Funded by the 4th Priority Discipline Development Program of Jiangsu Higher Education Institutions/ ; Ym2023064//Jiangsu Provincial Health Commission Public Health Research Project/ ; Ym2023064//Jiangsu Provincial Health Commission Public Health Research Project/ ; Ym2023064//Jiangsu Provincial Health Commission Public Health Research Project/ ; Ym2023064//Jiangsu Provincial Health Commission Public Health Research Project/ ; Ym2023064//Jiangsu Provincial Health Commission Public Health Research Project/ ; Ym2023064//Jiangsu Provincial Health Commission Public Health Research Project/ ; NJYFKT202409//Nanjing Preventive Medicine Research Project/ ; NJYFKT202409//Nanjing Preventive Medicine Research Project/ ; NJYFKT202409//Nanjing Preventive Medicine Research Project/ ; NJYFKT202409//Nanjing Preventive Medicine Research Project/ ; NJYFKT202409//Nanjing Preventive Medicine Research Project/ ; NJYFKT202409//Nanjing Preventive Medicine Research Project/ ; },
mesh = {Humans ; *Coinfection/diagnosis/virology ; *Respiratory Syncytial Virus Infections/diagnosis/virology ; *Rhinovirus/genetics/isolation & purification ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; *Respiratory Syncytial Virus, Human/genetics/isolation & purification ; *Picornaviridae Infections/diagnosis/virology ; *Molecular Diagnostic Techniques/methods ; *Respiratory Tract Infections/diagnosis/virology ; CRISPR-Associated Proteins/genetics ; Infant ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {BACKGROUND: Respiratory syncytial virus (RSV) and human rhinovirus (HRV) are leading causes of respiratory infections in children, with increasing reports of coinfections leading to severe complications. Current CRISPR-based detection systems, such as Cas12a and Cas13a, are limited in multiplex detection due to the lack of specific reporter cleavage mechanisms. This study aims to develop a rapid, sensitive, and single-tube dual-gene detection method for RSV and HRV using the orthogonal trans-cleavage activities of CRISPR-Cas12a/13a combined with reverse transcription-recombinase polymerase amplification (RT-RPA).

METHODS: We designed a novel detection system leveraging RT-RPA for amplification and the distinct cleavage activities of Cas12a and Cas13a for simultaneous dual-gene detection.

RESULTS: The reaction components were optimized to complete detection within 30 min, achieving sensitivities of 10 copies/µL for RSV and 10[2] copies/µL for HRV. Clinical validation was performed on 543 respiratory infection samples, confirming high accuracy and specificity.

CONCLUSIONS: The RT-RPA-CRISPR-Cas12a/13a system provides a rapid, sensitive, and efficient solution for RSV and HRV coinfection detection. This method supports early diagnosis and improved clinical management, offering significant potential for public health applications in preventing severe respiratory complications in children.},
}

Humans
*Coinfection/diagnosis/virology
*Respiratory Syncytial Virus Infections/diagnosis/virology
*Rhinovirus/genetics/isolation & purification
*CRISPR-Cas Systems
Sensitivity and Specificity
*Respiratory Syncytial Virus, Human/genetics/isolation & purification
*Picornaviridae Infections/diagnosis/virology
*Molecular Diagnostic Techniques/methods
*Respiratory Tract Infections/diagnosis/virology
CRISPR-Associated Proteins/genetics
Infant
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid41023259,
year = {2025},
author = {Ravenel, K and Poirier, W and Razafimandimby, B and Bouchara, JP and Gastebois, A and Giraud, S},
title = {Optimization of the Genome Editing CRISPR-Cas9 Technology in Scedosporium apiospermum.},
journal = {Mycopathologia},
volume = {190},
number = {6},
pages = {94},
pmid = {41023259},
issn = {1573-0832},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Scedosporium/genetics ; },
abstract = {Scedosporium species are opportunistic pathogens causing a large variety of human infections. To date, there is limited information on the pathogenic mechanisms of these fungi, partly because of the limited number of genetic tools available. Here, the CRISPR-Cas9 technology, which provided promising results for functional genomic studies in filamentous fungi, was optimized for Scedosporium species using in vitro assembled Cas9 ribonucleoprotein (RNP) complexes. In these fungi, functional genomic studies are particularly complex in a wild-type strain, because of the high frequency of non-homologous recombination. Prior disruption of the KU70 gene encoding one of the components of the non-homologous end joining system is required, which necessitates the use of a first selection marker. The cleavage of the target gene at each end using a dual RNA-guided Cas9 complex, followed by recombination with a repair template containing the hygromycin resistance gene, allowed disruption of the target gene in the ΔKU70 mutant. Four genes encoding dioxygenases, catalyzing the critical ring-opening step in aromatic hydrocarbons, were successfully disrupted, and the optimum efficiency was observed using 5 μg of the HygR repair cassette. Alternatively, in the wild-type strain, the exclusive use of two Cas9 RNP complexes was enough to achieve an efficient deletion method; one dioxygenase gene was successfully deleted in up to 20% of the obtained colonies. These last experimental conditions path the way to multiple gene deletions and complementation experiments, which cannot be reached using our first procedure since only two selection markers are available for Scedosporium species.},
}

*CRISPR-Cas Systems
*Gene Editing/methods
*Scedosporium/genetics

@article {pmid41022734,
year = {2025},
author = {Fu, C and Saddawi-Konefka, R and Chinai, JM and Kim, SY and Kammula, AV and Perera, JJ and Jiang, A and Tiwari, P and Kistler, EN and Tang, S and Luna, SM and Colvin, KJ and Dubrot, J and Anderson, S and Fetterman, RA and Chuong, CL and Lane-Reticker, SK and Cheruiyot, CK and Muscato, AJ and Alipour, Z and Adkins, DR and Griffin, GK and Bernstein, BE and Egloff, AM and Yates, KB and Chernock, RD and Gutkind, JS and Uppaluri, R and Manguso, RT},
title = {In vivo CRISPR screening in head and neck cancer reveals Uchl5 as an immunotherapy target.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {8572},
pmid = {41022734},
issn = {2041-1723},
support = {U01DE029188//U.S. Department of Health & Human Services | NIH | National Institute of Dental and Craniofacial Research (NIDCR)/ ; },
mesh = {Animals ; Humans ; *Ubiquitin Thiolesterase/genetics/metabolism/immunology ; Mice ; *Head and Neck Neoplasms/genetics/immunology/therapy/pathology ; *Immunotherapy/methods ; *Squamous Cell Carcinoma of Head and Neck/genetics/immunology/therapy/pathology ; Epithelial-Mesenchymal Transition/genetics/immunology ; Cell Line, Tumor ; CD8-Positive T-Lymphocytes/immunology ; CRISPR-Cas Systems ; Extracellular Matrix/metabolism ; Immune Checkpoint Inhibitors/pharmacology/therapeutic use ; Tumor Escape/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Expression Regulation, Neoplastic ; Female ; },
abstract = {Recurrent/metastatic head and neck squamous cell carcinoma (HNSCC) is an aggressive malignancy with a significant unmet need for enhancing immunotherapy response given current modest efficacy. Here, we perform an in vivo CRISPR screen in an HNSCC mouse model to identify immune evasion genes. We identify several regulators of immune checkpoint blockade (ICB) response, including the ubiquitin C-terminal hydrolase 5 (UCHL5). Loss of Uchl5 in tumors increases CD8[+] T cell infiltration and improved ICB responses. Uchl5 deficiency attenuates extracellular matrix (ECM) production and epithelial-mesenchymal-transition (EMT) transcriptional programs, which contribute to stromal desmoplasia, a histologic finding we describe as associated with reduced anti-PD1 response in human HNSCCs. COL17A1, a collagen highly and specifically expressed in HNSCC, mediates in part Uchl5-mediated immune evasion. Our findings suggest an unappreciated role for UCHL5 in promoting EMT in HNSCC and highlight ECM modulation as a strategy to improve immunotherapy responses.},
}

Animals
Humans
*Ubiquitin Thiolesterase/genetics/metabolism/immunology
Mice
*Head and Neck Neoplasms/genetics/immunology/therapy/pathology
*Immunotherapy/methods
*Squamous Cell Carcinoma of Head and Neck/genetics/immunology/therapy/pathology
Epithelial-Mesenchymal Transition/genetics/immunology
Cell Line, Tumor
CD8-Positive T-Lymphocytes/immunology
CRISPR-Cas Systems
Extracellular Matrix/metabolism
Immune Checkpoint Inhibitors/pharmacology/therapeutic use
Tumor Escape/genetics
Clustered Regularly Interspaced Short Palindromic Repeats
Gene Expression Regulation, Neoplastic
Female

@article {pmid41022133,
year = {2025},
author = {Jin, X and Ouyang, C and Sun, T and Li, C and Gu, J and An, B and Wang, Z},
title = {A CRISPR/Cas9 mutant resource for OsSm RNA-binding genes in rice.},
journal = {The Plant journal : for cell and molecular biology},
volume = {124},
number = {1},
pages = {e70475},
doi = {10.1111/tpj.70475},
pmid = {41022133},
issn = {1365-313X},
support = {2021CX02N173//Guangdong Pearl River Talents Program/ ; 2025B03J0025//Guangzhou Science and Technology Plan Project/ ; 2024KJ31//the Provincial Rural Revitalization Strategy Special Project of Guangdong in 2024/ ; ZDYF2024XDNY179//Hainan Province Science and Technology Special Fund/ ; 32100252//National Natural Science Foundation of China/ ; 32100294//National Natural Science Foundation of China/ ; 32171292//National Natural Science Foundation of China/ ; 211207157080997//Zhanjiang plan for navigation/ ; QT2024-017//Young Talent Support Project of Guangzhou Association for Science and Technology/ ; 2023A1515010428//Guangdong Basic and Applied Basic Research Foundation/ ; },
mesh = {*Oryza/genetics/growth & development/metabolism ; *CRISPR-Cas Systems/genetics ; *Plant Proteins/genetics/metabolism ; Mutation ; Gene Expression Regulation, Plant ; *RNA-Binding Proteins/genetics/metabolism ; Genes, Plant ; RNA Splicing ; },
abstract = {Pre-mRNA, produced by eukaryotic DNA transcription, undergoes splicing by the spliceosome, which removes introns and joins exons to form mRNA. The spliceosome is a large and highly dynamic molecular machine. Its core components include five small nuclear ribonucleoproteins (snRNPs) and the various spliceosome-related proteins. The conserved Smith (Sm) complex and the Sm-like proteins (LSm) serve as primary components of the snRNPs. Sm proteins are involved in processes such as pre-mRNA splicing and mRNA degradation, which can regulate gene expression, thereby influencing plant growth, development, and stress responses. While 25 Sm proteins have been identified in rice, their specific roles in regulating rice growth and development remain unclear. In this study, we employed the CRISPR/Cas9 system to edit 15 OsSm genes, and 13 mutants were obtained, with mutation rates ranging from 20.83 to 83.87%. In comparison to the wild type (WT), the mutants exhibited dwarfism, reduced tiller numbers, lower seed-setting rates or sterility, and increased susceptibility to diseases. One Sm mutant, ossmf-2, exhibited dwarfism, delayed flowering, and small grains. Through transcriptome analysis, three target genes, OsMRG702, OsRGG2, and OsLA1, were identified. Mutations of the OsSmF protein may lead to the abnormal splicing of these genes and finally lead to the inhibition of growth and development. Our study first edited the OsSm genes and generated a mutant library in rice. Most of the mutants exhibited abnormal growth and development, underscoring the essential roles of OsSm proteins in rice physiology. Furthermore, this work addresses a critical gap in the functional characterization of Sm proteins in rice. The resulting mutant collection offers valuable germplasm resources and lays a theoretical foundation for elucidating the molecular regulatory networks involving spliceosomal components and their target genes in the control of crop growth, development, and reproduction.},
}

*Oryza/genetics/growth & development/metabolism
*CRISPR-Cas Systems/genetics
*Plant Proteins/genetics/metabolism
Mutation
Gene Expression Regulation, Plant
*RNA-Binding Proteins/genetics/metabolism
Genes, Plant
RNA Splicing

@article {pmid41021290,
year = {2025},
author = {Johnson, KA and Cooper, C and Philippe, C and Catchpole, RJ and Mitchell, S and Terns, MP},
title = {A Phage Variable Region Encodes Anti-CRISPR Proteins Inhibiting All Streptococcus thermophilus CRISPR Immune Systems.},
journal = {The CRISPR journal},
volume = {},
number = {},
pages = {},
doi = {10.1177/25731599251369720},
pmid = {41021290},
issn = {2573-1602},
abstract = {Bacteria and archaea utilize CRISPR-Cas systems to defend against invading mobile genetic elements (MGEs) such as phages and plasmids. In turn, MGEs have evolved anti-CRISPR (Acr) proteins to counteract these defenses. While several type II-A Acrs have been identified in Streptococcus thermophilus (Sth) phages, a more comprehensive understanding of Acr diversity in Sth phages has yet to be explored. Guided by the genomic context of known Acrs, we systematically screened uncharacterized phage proteins and identified several novel Acrs that inhibit type I-E, type II-A or type III-A Sth CRISPR-Cas systems. These acr genes are clustered within a variable phage genomic region, indicating a hotspot for anti-defense activity. We also identified neighboring proteins with predicted enzymatic or structural domains that may modulate phage-host interactions through Acr-independent mechanisms. Together, our findings expand the known repertoire of Sth Acrs and highlight the phage variable region as a key reservoir of immune-modulating factors.},
}

@article {pmid41021273,
year = {2025},
author = {Sgodda, M and Gebel, E and Dignas, L and Alfken, S and Eggenschwiler, R and Stalke, A and Dröge, C and Pfister, ED and Baumann, U and Luedde, T and Esposito, I and Keitel, V and Cantz, T},
title = {iPSC-based hepatic organoids reveal a heterozygous MYO5B variant as driver of intrahepatic cholestasis.},
journal = {Hepatology communications},
volume = {9},
number = {10},
pages = {},
pmid = {41021273},
issn = {2471-254X},
mesh = {Humans ; *Cholestasis, Intrahepatic/genetics/metabolism ; *Organoids/metabolism ; ATP Binding Cassette Transporter, Subfamily B, Member 11/genetics/metabolism ; *Induced Pluripotent Stem Cells ; Heterozygote ; *Myosin Type V/genetics ; *Myosin Heavy Chains/genetics ; ATP Binding Cassette Transporter, Subfamily B/genetics/metabolism ; Liver/metabolism ; Multidrug Resistance-Associated Protein 2 ; },
abstract = {BACKGROUND: Hereditary intrahepatic cholestasis is caused by variants of various genes involved in enterohepatic bile circulation, metabolization, and conjugation. Originally classified into 3 groups, the number of contributing genes is still increasing, underlining the need for a deeper understanding of the molecular interaction during intrahepatic cholestasis.

METHODS: In the present study, we investigate the interplay of heterozygous variants in 3 cholestasis-associated genes (ABCB11, ABCB4, and MYO5B) by exploiting iPSC-based hepatic organoids from a patient suffering from recurrent intrahepatic cholestasis.

RESULTS: Functional characterization of MRP2-mediated cholyl-lysyl-fluorescein (CLF) and BSEP-mediated Tauro-nor-THCA-24-DBD transport demonstrated a marked reduction of transport in MYO5B-deficient organoids, in comparison to unaffected control organoids. Moreover, iPSC-based organoids derived from the patient carrying 3 heterozygous variants in ABCB11, ABCB4, and MYO5B also exhibited absence of BSEP-mediated Tauro-nor-THCA-24-DBD transport, but functional MRP2-mediated CLF-transport. Interestingly, CRISPR/Cas9-mediated correction of the mutated ABCB11 allele could not restore the impaired BSEP function, suggesting the heterozygous MYO5B variant as the main driver of the transport deficiency. In fact, CRISPR/Cas-mediated correction of the MYO5B variant finally resulted in a restoration of the BSEP-mediated Tauro-nor-THCA-24-DBD transport.

CONCLUSIONS: iPSC-based organoids serve as an authentic model for functional assessment of the hepatobiliary transport with fluorescent substrates. This allows the characterization of variants of uncertain significance and other variants in cholestasis-associated genes and revealed that a heterozygous MYO5B variant increases the susceptibility to defective hepatobiliary BSEP-mediated transport.},
}

Humans
*Cholestasis, Intrahepatic/genetics/metabolism
*Organoids/metabolism
ATP Binding Cassette Transporter, Subfamily B, Member 11/genetics/metabolism
*Induced Pluripotent Stem Cells
Heterozygote
*Myosin Type V/genetics
*Myosin Heavy Chains/genetics
ATP Binding Cassette Transporter, Subfamily B/genetics/metabolism
Liver/metabolism
Multidrug Resistance-Associated Protein 2

@article {pmid41021067,
year = {2025},
author = {Zou, S and Chen, W and Cao, Y and Liu, X and Wang, J and Wang, Y and Zhou, S},
title = {Lethal endotoxin (ccdB) based counterselection improved the efficiency of sequential gene editing in Escherichia coli.},
journal = {Biotechnology letters},
volume = {47},
number = {5},
pages = {118},
pmid = {41021067},
issn = {1573-6776},
support = {Guike AA24206048//Science and Technology Major Project of Guangxi/ ; AA24206050//Science and Technology Major Project of Guangxi/ ; 4301/00960//Hubei University of Technology High-Level Talent Research Startup Fund Program/ ; },
mesh = {*Gene Editing/methods ; *Escherichia coli/genetics ; Plasmids/genetics ; CRISPR-Cas Systems ; *Endotoxins/genetics ; *Escherichia coli Proteins/genetics ; },
abstract = {The CRISPR/Cas9-based technology has been used for sequential gene editing in E. coli. The plasmids carrying the sgRNA and/or Cas9 genes need to be cured after each round of editing. Curing of these plasmids, particularly the sgRNA plasmid, limits the efficiency of sequential gene editing. In this study, a lethal endotoxin (ccdB) based counterselection was established for improving the overall efficiency of sequential gene editing in E. coli. This approach was validated for sequential editing (deletion) of cstA and ppsA genes in HBUT-P2 strain (W derivative). The experimental results showed that the transformation efficiency of sgRNA plasmid (pTargetF-tcr-PL-ccdB-N20) reached 10[8]-10[9] cfu/μg-DNA, resulting in a 100% and 93.75% recombination rate for cstA and ppsA gene, respectively. Upon completion of cstA gene editing, the sgRNA plasmid (pTargetF-tcr-PL-ccdB-N20 (cstA)) was effectively cured through ccdB based counterselection at 42 °C, with a 43.75% efficiency. At the end of sequential editing of ppsA gene, both Cas9 (25A) and sgRNA (pTargetF-tcr-PL-ccdB-N20 (ppsA)) plasmids were cured simultaneously through the sacB and ccdB based counterselections by incubating the cells on LB-sucrose (5%) plate at 42 °C, achieving a curing rate of 100% for Cas9 plasmid (25A), 37.5% for sgRNA plasmid (pTargetF-tcr-PL-ccdB-N20 (ppsA)), and 37.5% for both Cas9 and sgRNA plasmids. Moreover, this approach was further validated through efficient site-specific insertion of the csc operon into the slmA gene in DH5α (K12 derivative) and S322 (C derivative) strains. These results demonstrated that the endotoxin (ccdB) based counterselection improved the transformation efficiency of sgRNA plasmid, the recombination rate of the editing target gene, the curing rate of sgRNA plasmid, and the overall efficiency of sequential gene editing.},
}

*Gene Editing/methods
*Escherichia coli/genetics
Plasmids/genetics
CRISPR-Cas Systems
*Endotoxins/genetics
*Escherichia coli Proteins/genetics

@article {pmid41020501,
year = {2025},
author = {Yu, L and Jin, Y and Chen, J and Zhu, Z and Su, S and Wilkerson, EM and Gongora, J and Cloer, EW and Major, MB and Liu, P},
title = {Stable Cas9 expression regulates cell growth by facilitating mTORC2 activation.},
journal = {Nucleic acids research},
volume = {53},
number = {18},
pages = {},
pmid = {41020501},
issn = {1362-4962},
support = {//Mary Kay Ash Foundation/ ; //University of North Carolina/ ; //Chapel Hill University Cancer Research/ ; },
mesh = {Humans ; Ribosomal Proteins/metabolism/genetics ; *Mechanistic Target of Rapamycin Complex 2/metabolism/genetics ; *CRISPR-Associated Protein 9/genetics/metabolism ; Cell Line, Tumor ; *Cell Proliferation/genetics ; Signal Transduction ; *CRISPR-Cas Systems ; Adaptor Proteins, Signal Transducing/metabolism/genetics ; Repressor Proteins/metabolism/genetics ; Gene Editing ; Phosphatidylinositol 3-Kinases/metabolism ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR), widely used for gene editing, relies on bacterial endonucleases like Cas9 to study gene functions and develop therapies. However, its potential effects on mammalian cellular behavior remain unclear. Here, we systematically profiled effects of stable Cas9 expression on growth of 32 cell lines spanning 9 cancer types and non-cancerous cells, finding growth alterations in a subset. To investigate mechanisms, we established the SpCas9 interactome in DU145 and MDA-MB-231 cells, both showing Cas9-enhanced growth, and identified ribosomal proteins as the top shared interactors. RNA-seq analysis revealed that Cas9 expression in DU145 cells activated PI3K signaling. Mechanistic studies showed that ribosomal proteins, including RPL26 and RPL23a, bind to Sin1, a core mTORC2 component, leading to mTORC2 activation. Notably, SpCas9 interacts with both RPL26/RPL23a and Sin1, acting as a scaffold to stabilize their association and enhance mTORC2 activation, even in the absence of growth factors. Our study systematically characterizes Cas9's effects on cell growth regulation and uncovers a novel Cas9-ribosome-mTORC2 signaling axis that promotes cell growth. These findings underscore the need to consider unintended cellular effects in CRISPR applications and highlight the importance of engineering safer Cas9 variants for biomedical research and clinical therapies.},
}

Humans
Ribosomal Proteins/metabolism/genetics
*Mechanistic Target of Rapamycin Complex 2/metabolism/genetics
*CRISPR-Associated Protein 9/genetics/metabolism
Cell Line, Tumor
*Cell Proliferation/genetics
Signal Transduction
*CRISPR-Cas Systems
Adaptor Proteins, Signal Transducing/metabolism/genetics
Repressor Proteins/metabolism/genetics
Gene Editing
Phosphatidylinositol 3-Kinases/metabolism

@article {pmid41020266,
year = {2025},
author = {Back, F and Sandoval, A and Vu, LM and Hong, VM and Bhaskara, A and Rodriguez, SR and O'Brien, JT and Kolber, BJ and Kroener, S and Ploski, JE},
title = {Adeno-associated viral vector resource for the RNA-targeting Cas13d: A comparison of high-fidelity variants, DjCas13d and hfCas13d.},
journal = {Molecular therapy. Methods & clinical development},
volume = {33},
number = {4},
pages = {101565},
pmid = {41020266},
issn = {2329-0501},
abstract = {RNA-targeting CRISPR-Cas systems have emerged as alternatives to RNA-interference technology to knock down specific RNA transcripts. In particular, Cas13d derived from Ruminococcus flavefaciens (CasRx, RfxCas13d) has generated interest due to its superior knockdown efficiencies; however, accumulating evidence indicates that CasRx is prone to inducing transcriptome alterations due to its tendency to cleave bystander RNAs. High-fidelity Cas13d (hfCas13d) derived from CasRx and DjCas13d, an ortholog of Cas13d derived from Ruminococcus sp. UBA7013 (gut metagenome), are two recently identified variants that are superior to CasRx, as they both show a reduced tendency to cleave bystander RNAs. In this study, we created a resource of adeno-associated viral (AAV) vectors designed to deliver Cas13d, including hfCas13d and DjCas13d. We directly compared hfCas13d and DjCas13d for their on- and off-target potential in 293FT and neuro 2A cells. Specifically, we examined their ability to knockdown several endogenous and ectopically expressed transcripts using several different guide RNAs (gRNAs), and we examined knockdown specificity using a combination of reporter assays, RNA integrity analysis, and RNA sequencing (RNA-seq). We report that while both of these enzymes exhibit generally similar levels of knockdown potential, with DjCas13d sometimes outperforming hfCas13d, hfCas13d consistently caused significantly fewer transcriptome alterations when targeting highly expressed genes compared to DjCas13d.},
}

@article {pmid40974028,
year = {2025},
author = {Teng, J and Chen, Y and Zhang, W and Xu, H and Ke, L and Xu, H and Wang, J},
title = {An RCA-CRISPR-Enhanced SERS Platform for Ultrasensitive and Single-Nucleotide-Resolved Detection of Exosomal miRNA-21 in Early Lung Cancer.},
journal = {Analytical chemistry},
volume = {97},
number = {38},
pages = {21098-21105},
doi = {10.1021/acs.analchem.5c04448},
pmid = {40974028},
issn = {1520-6882},
mesh = {*Lung Neoplasms/diagnosis/genetics ; Humans ; *MicroRNAs/genetics/analysis/blood ; *Exosomes/genetics/chemistry ; *Spectrum Analysis, Raman/methods ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; CRISPR-Cas Systems ; Biomarkers, Tumor/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Exosomal miRNA-21 has emerged as a promising biomarker for early-stage lung cancer due to its close association with tumor progression and its stability in circulation. However, its low abundance, short sequence length, and high-sequence similarity present significant detection challenges. To address this, we developed an ultrasensitive surface-enhanced Raman scattering (SERS) platform that integrates rolling circle amplification (RCA) with clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 12a (Cas12a) for the detection of exosomal miRNA-21. RCA provides target-dependent amplification with stringent sequence discrimination via padlock probe ligation, while the CRISPR/Cas12a system facilitates robust signal generation through trans-cleavage activity. The final SERS readout enables molecular-level sensitivity by detecting nanotag-labeled cleavage events. The assay achieved a limit of detection as low as 0.62 aM and effectively discriminated miRNA-21 from multiple single- and multinucleotide variants. As a proof of concept, we applied this method to the detection of exosomal miRNA-21 extracted from the serum of 20 early-stage lung cancer patients and 20 healthy controls, achieving 100% sensitivity and 100% specificity (AUC = 1.0) in this preliminary cohort. These findings demonstrate the strong potential of the RCA-CRISPR-SERS platform for noninvasive early-stage lung cancer diagnosis based on exosomal miRNA-21 detection.},
}

*Lung Neoplasms/diagnosis/genetics
Humans
*MicroRNAs/genetics/analysis/blood
*Exosomes/genetics/chemistry
*Spectrum Analysis, Raman/methods
*Nucleic Acid Amplification Techniques/methods
Limit of Detection
CRISPR-Cas Systems
Biomarkers, Tumor/genetics
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40961329,
year = {2025},
author = {Joseph, RA and Haley, RM and Padilla, MS and Ricciardi, AS and Yamagata, HM and Mitchell, MJ},
title = {Cas9 Protein Outperforms mRNA in Lipid Nanoparticle-Mediated CFTR Repair.},
journal = {Nano letters},
volume = {25},
number = {39},
pages = {14348-14355},
doi = {10.1021/acs.nanolett.5c03548},
pmid = {40961329},
issn = {1530-6992},
mesh = {*Cystic Fibrosis Transmembrane Conductance Regulator/genetics/metabolism ; *Cystic Fibrosis/genetics/therapy ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *CRISPR-Associated Protein 9/genetics ; Humans ; *Nanoparticles/chemistry ; Animals ; *RNA, Messenger/genetics/chemistry/administration & dosage ; RNA, Guide, CRISPR-Cas Systems/genetics ; Mice ; *Lipids/chemistry ; Liposomes ; },
abstract = {Lipid nanoparticles (LNPs) are currently one of the most clinically advanced delivery systems for nucleic acid cargo and hold great potential for clinical applications in gene editing and the treatment of genetic diseases. LNP-mediated delivery of Cas9 with single guide RNA (sgRNA) and homology-directed repair DNA template (ssDNA) enables efficient and precise editing in vitro and in vivo. Comparative analysis of LNP delivery of Cas9 as protein or mRNA for relevant clinical targets, such as cystic fibrosis (CF), which is caused by mutations in the CFTR gene, is imperative in the design of corrective therapeutics for genetic diseases. Here, we show that delivery of Cas9 protein LNPs outperforms Cas9 mRNA LNPs when evaluated for in vivo lung editing as well as corrective CRISPR/Cas9 editing and functional recovery of the CFTR protein. These results demonstrate the ability to optimize the use of CRISPR/Cas9 LNPs for cystic fibrosis applications.},
}

*Cystic Fibrosis Transmembrane Conductance Regulator/genetics/metabolism
*Cystic Fibrosis/genetics/therapy
*CRISPR-Cas Systems/genetics
Gene Editing/methods
*CRISPR-Associated Protein 9/genetics
Humans
*Nanoparticles/chemistry
Animals
*RNA, Messenger/genetics/chemistry/administration & dosage
RNA, Guide, CRISPR-Cas Systems/genetics
Mice
*Lipids/chemistry
Liposomes

@article {pmid40955638,
year = {2025},
author = {Sun, F and Mao, R and Li, J and Wang, X and Hou, P and Zhou, H},
title = {Organic Photoelectrochemical Transistor/Visual Sensing Platform Based on CS/MCS Schottky Heterojunction and CRISPR/Cas12a-Driven Triple-Modal Synergistic Signal Amplification.},
journal = {Analytical chemistry},
volume = {97},
number = {38},
pages = {21079-21088},
doi = {10.1021/acs.analchem.5c04321},
pmid = {40955638},
issn = {1520-6882},
mesh = {*MicroRNAs/analysis/genetics ; *Transistors, Electronic ; *Electrochemical Techniques/methods ; Humans ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; DNA, Catalytic/chemistry/metabolism ; Photochemical Processes ; G-Quadruplexes ; Colorimetry ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Developing novel signal amplification and transduction technologies is the key to overcoming the bottlenecks of high-sensitivity and on-site detection in nucleic acid analysis. In this study, a dual-mode sensing platform based on organic electrochemical transistors (OPECT) and colorimetry was established to achieve ultrasensitive detection of miRNA-21. 1D/3D Co9S8/Mn0.3Cd0.7S Schottky heterojunction was synthesized as the photoactive material, which significantly enhanced the photoelectric conversion efficiency. The sensing and detection system cleverly integrated a quadruple signal amplification mechanism. The target triggered the catalytic hairpin assembly (CHA) reaction, generating H1 and H2 long chains. These chains activated the CRISPR/Cas12a system, which carried out nondiscriminatory cleavage to block the tandem strand displacement reaction (TSDR). This triggered the hybrid chain reaction (HCR) and formation of G-quadruplex/hemin DNAzyme (GQH DNAzyme), realizing cascade signal amplification. Under the catalysis of GQH DNAzyme, the detection had dual-signal outputs. It catalyzed the oxidation of 4-CN to form a deposition layer, inhibiting electron transport and achieving cascade signal amplification for OPECT. It catalyzed the H2O2-mediated TMB colorimetric reaction to complete the visual colorimetric analysis. Through triple-modal synergistic signal amplification of biological, chemical, and electronic modalities, this biosensing platform reduced the detection limits to as low as 36.5 aM and 3.8 fM, respectively. It provided a new solution for the accurate analysis of miRNA markers in the early diagnosis of cancer.},
}

*MicroRNAs/analysis/genetics
*Transistors, Electronic
*Electrochemical Techniques/methods
Humans
*CRISPR-Cas Systems
*Biosensing Techniques/methods
DNA, Catalytic/chemistry/metabolism
Photochemical Processes
G-Quadruplexes
Colorimetry
Limit of Detection
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40938323,
year = {2025},
author = {Martin-Vicente, A and Nywening, AV and Xie, J and Thorn, HI and Guruceaga, X and Fortwendel, JR},
title = {Genetic analysis of common triazole resistance mechanisms in a collection of Aspergillus lentulus clinical isolates from the United States.},
journal = {Antimicrobial agents and chemotherapy},
volume = {69},
number = {10},
pages = {e0069025},
doi = {10.1128/aac.00690-25},
pmid = {40938323},
issn = {1098-6596},
support = {R01AI143197/NH/NIH HHS/United States ; R01AI158442/NH/NIH HHS/United States ; R21AI178048/NH/NIH HHS/United States ; },
mesh = {*Triazoles/pharmacology ; *Antifungal Agents/pharmacology ; *Aspergillus/drug effects/genetics/isolation & purification ; *Drug Resistance, Fungal/genetics ; Microbial Sensitivity Tests ; United States ; Fungal Proteins/genetics/metabolism ; Aspergillosis/microbiology/drug therapy ; Humans ; Cytochrome P-450 Enzyme System/genetics/metabolism ; Aspergillus fumigatus/drug effects/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {Aspergillus fumigatus continues to be the leading cause of invasive aspergillosis. However, the number of cases by drug-resistant cryptic species has increased in recent years. Aspergillus lentulus is a sibling species of Aspergillus section Fumigati that can only be distinguished from A. fumigatus by molecular methods. The clinical importance of this species resides in its low susceptibility to triazoles and intrinsic resistance to amphotericin B, making invasive aspergillosis treatments extremely challenging and producing high mortality rates. In this study, we investigate known molecular mechanisms important for triazole resistance in A. fumigatus in a collection of 25 clinical A. lentulus isolates from the United States. Using CRISPR-Cas9 gene editing technology, we performed cyp51A and hmg1 allele replacements between susceptible and resistant isolates. Phenotypic characterization of the resulting mutants, together with mRNA expression analyzes of cyp51A, cyp51B, and the putative ABC efflux pump, abcC, suggests that triazole resistance in our A. lentulus isolates is independent of the mechanisms studied.},
}

*Triazoles/pharmacology
*Antifungal Agents/pharmacology
*Aspergillus/drug effects/genetics/isolation & purification
*Drug Resistance, Fungal/genetics
Microbial Sensitivity Tests
United States
Fungal Proteins/genetics/metabolism
Aspergillosis/microbiology/drug therapy
Humans
Cytochrome P-450 Enzyme System/genetics/metabolism
Aspergillus fumigatus/drug effects/genetics
CRISPR-Cas Systems/genetics

@article {pmid40916664,
year = {2025},
author = {Barker, EN and Ashiri, M and Saville, JT and Hemming, R and Furletti, N and Dhume, SH and Yu, S and Anjos, E and Wu, X and Fresnoza, A and Merz, DC and Jackson, M and Del Bigio, MR and Siddiqui, TJ and Fuller, M and Mark, BL and Triggs-Raine, B},
title = {Generation of mice with combined Hexa Gly269Ser KI or KO and Neu3 KO alleles to create new models of GM2 gangliosidoses.},
journal = {Biology open},
volume = {14},
number = {9},
pages = {},
doi = {10.1242/bio.062045},
pmid = {40916664},
issn = {2046-6390},
support = {/CAPMC/CIHR/Canada ; //Research Manitoba/ ; /AS/Autism Speaks/United States ; //Natural Sciences and Engineering Council of Canada/ ; },
mesh = {Animals ; *Disease Models, Animal ; Mice ; *Alleles ; Mice, Knockout ; *Gangliosidoses, GM2/genetics/metabolism/pathology ; Phenotype ; CRISPR-Cas Systems ; *beta-Hexosaminidase alpha Chain/genetics ; Gene Knock-In Techniques ; Mutation ; Humans ; N-Acetylgalactosaminyltransferases ; },
abstract = {The GM2 gangliosidoses are lysosomal storage disorders exhibiting a spectrum of neurological phenotypes ranging from childhood death to debilitating adult-onset neurological impairment. To date, no mouse model harbouring a specific human mutation causing GM2 gangliosidosis has been created. We used CRISPR/Cas9 to generate knockin (KI) mice with the common adult-onset Hexa Gly269Ser variant as well as knockout (KO) mice with Hexa mutations expected to cause complete HexA deficiency. We also created Neu3 KO alleles that combined with Hexa KO or KI alleles were expected to create acute and chronic models of GM2 gangliosidosis, respectively. However, both models accumulated GM2 ganglioside throughout the brain when compared to controls (CON), and exhibited progressive loss of reflexes, gait abnormalities, and premature death by 24 weeks of age. Although survival and behavioural phenotypes did not differ between KO and KI models, the KI model had substantial Hexa mRNA and evidence of GM2 turnover. This KI model will be useful for developing gene editing to correct the variant causing the Gly269Ser substitution and its novel biochemical phenotype suggests it may be suitable for testing therapies that treat partial β-hexosaminidase A deficiency.},
}

Animals
*Disease Models, Animal
Mice
*Alleles
Mice, Knockout
*Gangliosidoses, GM2/genetics/metabolism/pathology
Phenotype
CRISPR-Cas Systems
*beta-Hexosaminidase alpha Chain/genetics
Gene Knock-In Techniques
Mutation
Humans
N-Acetylgalactosaminyltransferases

@article {pmid40692527,
year = {2025},
author = {Kohso, A and Inaba, H and Kanemaki, MT and Gabazza, EC and Toyoda, H and Hirayama, M and Goto, H},
title = {Aurora-A Promotes Cell-Cycle Progression From Quiescence Through Primary Cilia Disassembly.},
journal = {Cancer science},
volume = {116},
number = {10},
pages = {2763-2773},
doi = {10.1111/cas.70153},
pmid = {40692527},
issn = {1349-7006},
support = {JP22K06219//Japan Society for the Promotion of Science/ ; JP23H04925//Japan Society for the Promotion of Science/ ; JP23K06674//Japan Society for the Promotion of Science/ ; JP23K07651//Japan Society for the Promotion of Science/ ; //Takeda Science Foundation/ ; },
mesh = {Humans ; *Cilia/metabolism ; HCT116 Cells ; *Aurora Kinase A/metabolism/genetics ; *Cell Cycle ; CRISPR-Cas Systems ; Apoptosis/genetics ; Cell Proliferation ; },
abstract = {Aurora-A (AurA) is a member of the mitotic kinase family and is highly expressed in various tumors. Inhibition of AurA generally leads to fetal mitotic errors. We previously reported that AurA inhibition induces G0/G1 cell cycle arrest in noncancerous cells by promoting the reassembly of primary cilia. However, the mechanisms by which AurA regulates cell cycle progression beyond mitosis remain largely unknown. In this study, we generated noncancerous RPE1 and cancerous HCT116 cell lines expressing endogenous AurA tagged with a minimal auxin-inducible degron (mAID) using CRISPR/Cas9-based gene editing. This system enabled specific and rapid depletion of endogenous AurA protein. By combining this approach with cell synchronization in RPE1 cells, we investigated AurA function specifically in the transition from quiescence to the proliferative cell cycle. Targeted degradation of AurA not only delayed cell cycle progression but also impaired the disassembly of primary cilia at the G0/G1 transition in RPE1 cells. Since this delay in cell cycle progression was rescued by forced deciliation via the knockout of IFT20, AurA facilitates deciliation, which in turn accelerates the transition from quiescence to the proliferative phase of the cell cycle in RPE1 cells. AurA depletion for 4 days increased apoptotic markers in HCT116 cells but not in RPE1 cells. Notably, forced deciliation in RPE1 cells partially enhanced apoptosis induced by AurA depletion. These results suggest that the ability to assemble primary cilia may serve as a protective mechanism against cell death following AurA inhibition.},
}

Humans
*Cilia/metabolism
HCT116 Cells
*Aurora Kinase A/metabolism/genetics
*Cell Cycle
CRISPR-Cas Systems
Apoptosis/genetics
Cell Proliferation

@article {pmid40675820,
year = {2025},
author = {Turn, RE and Hilgendorf, KI and Johnson, CT and Han, K and Aziz-Zanjani, MO and Swails Bollinger, S and Domizi, P and Cheng, R and Rabiee, A and Zhu, Y and Jiang, Z and Asthana, A and Demeter, J and Svensson, KJ and Bassik, MC and Jackson, PK},
title = {A genome-wide, CRISPR-based screen reveals new requirements for translation initiation and ubiquitination in driving adipogenic fate change.},
journal = {Genes & development},
volume = {39},
number = {19-20},
pages = {1241-1264},
doi = {10.1101/gad.352779.125},
pmid = {40675820},
issn = {1549-5477},
support = {P30 DK116074/DK/NIDDK NIH HHS/United States ; R01 DK125260/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; Mice ; *Ubiquitination/genetics ; *Adipogenesis/genetics ; 3T3-L1 Cells ; Lipogenesis/genetics ; Adipocytes/cytology/metabolism ; *Peptide Chain Initiation, Translational/genetics ; CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Mitosis/genetics ; },
abstract = {In response to excess nutrients, white adipose tissue expands by both generating new adipocytes and upregulating lipogenesis in existing adipocytes. Here, we performed a genome-wide functional CRISPR screen to identify regulators of adipogenesis in the mouse 3T3-L1 preadipocyte model. In this pooled screening strategy, we used FACS to isolate populations based on lipid content, gating for fluorescence intensity of lipophilic fluorescent BODIPY dye. Additionally, we categorized whether the gene functions primarily during mitotic clonal expansion, lipogenesis, or both. We found that translation initiation and ubiquitin-dependent protein stability regulators drive both adipogenic fate change and lipogenesis. We further supported these findings with proteomics, demonstrating that essential changes in protein reprogramming can drive or inhibit 3T3-L1 adipogenesis independent of transcription. Furthermore, we demonstrated that specific branches of the hypusination pathway, a conserved regulator of translation initiation, are critical for translating adipogenic inducers of mitotic clonal expansion and that the neddylation/ubiquitin pathway modulates insulin sensitivity during lipogenesis.},
}

Animals
Mice
*Ubiquitination/genetics
*Adipogenesis/genetics
3T3-L1 Cells
Lipogenesis/genetics
Adipocytes/cytology/metabolism
*Peptide Chain Initiation, Translational/genetics
CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Mitosis/genetics

@article {pmid40671314,
year = {2025},
author = {Kumar, P and Bisht, NC},
title = {High-level production of health-beneficial glucoraphanin by multiplex editing of AOP2 gene family in mustard.},
journal = {Plant biotechnology journal},
volume = {23},
number = {10},
pages = {4668-4680},
doi = {10.1111/pbi.70171},
pmid = {40671314},
issn = {1467-7652},
support = {BT/PR23893/GET/119/81/2017//Department of Biotechnology, Ministry of Science and Technology, India/ ; BT/PR53959/PBN/18/17/2024//Department of Biotechnology, Ministry of Science and Technology, India/ ; },
mesh = {Sulfoxides ; *Glucosinolates/metabolism/biosynthesis ; Oximes ; *Imidoesters/metabolism ; *Mustard Plant/genetics/metabolism ; *Gene Editing/methods ; CRISPR-Cas Systems ; Plants, Genetically Modified/metabolism ; *Plant Proteins/genetics/metabolism ; *Isothiocyanates/metabolism ; },
abstract = {Intake of glucosinolates through the consumption of cruciferous vegetables has been associated with numerous health benefits. In recent decades, glucosinolate glucoraphanin has gained a lot of attention, as its hydrolysis product (sulforaphane) is known to possess numerous health-promoting benefits, including anti-cancer and chemopreventive activities. However, due to the low availability of glucoraphanin in most of the cultivated Brassica crops (except broccoli), there is an increasing interest in many laboratories around the world to manipulate the glucosinolate profile for human benefit. Here, we report the high-level production of health-beneficial glucoraphanin by CRISPR/Cas9 editing of the ALKENYL HYDROXALKYL PRODUCING 2 (BjuAOP2) gene family, displaying distinct expression profiles in the allotetraploid mustard, Brassica juncea. Multiplex editing of five BjuAOP2 homologues, using four gRNAs, provided glucoraphanin accumulation up to 41.60, 75.10, 59.21 and 27.64 μmoles/g dry weight in sprouts, microgreens, seeds and leaves, respectively, of the transgene-free BjuAOP2-edited lines, while providing a significant reduction of the anti-nutritional and goitrogenic alkenyl glucosinolates including progoitrin. The glucoraphanin enhancement in BjuAOP2-edited lines was found to be dose-dependent, wherein loss-of-function mutations in BjuAOP2.A09 and BjuAOP2.B01 homologues had a more prominent effect. The transgene-free BjuAOP2-edited lines were stable for high glucoraphanin and performed at par with the wild-type plants for various seed quality and yield parameters when tested under containment conditions in the field. The development of high-glucoraphanin mustard will help its adoption as a global superfood with health-promoting benefits and as a bioactive source of high-value sulforaphane for industrial production.},
}

Sulfoxides
*Glucosinolates/metabolism/biosynthesis
Oximes
*Imidoesters/metabolism
*Mustard Plant/genetics/metabolism
*Gene Editing/methods
CRISPR-Cas Systems
Plants, Genetically Modified/metabolism
*Plant Proteins/genetics/metabolism
*Isothiocyanates/metabolism

@article {pmid40652545,
year = {2025},
author = {Zhu, M and Yan, L and Zhan, Z and Chen, H and Wang, D and Xu, M and Zheng, Z and Zhang, Y and Yang, N and Wu, J and Zhan, H and Tian, Y and Xiong, L and He, Y},
title = {Transgene-Killer-CRISPR version 2 (TKC2) eliminates occasional transgene escape by coupling with a RUBY reporter.},
journal = {Plant biotechnology journal},
volume = {23},
number = {10},
pages = {4621-4632},
doi = {10.1111/pbi.70257},
pmid = {40652545},
issn = {1467-7652},
support = {32200335//National Natural Science Foundation of China/ ; U21A20207//National Natural Science Foundation of China/ ; Y2023QC39//Youth innovation Program of Chinese Academy of Agricultural Sciences/ ; CARS-01//Earmarked Fund for China Agriculture Research System/ ; YBXM2405//Nanfan special project of CAAS/ ; YBXM2446//Nanfan special project of CAAS/ ; B23CJ0208//Hainan Seed Industry Laboratory/ ; },
mesh = {*Transgenes/genetics ; Plants, Genetically Modified/genetics ; *Gene Editing/methods ; Genes, Reporter/genetics ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Oryza/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {A critical step in generating gene-edited plants is the removal of CRISPR-related transgenes from T0 plants and their progenies, a process that is generally time-consuming and labour-intensive. We previously reported a Transgene Killer CRISPR (TKC) technology that enables self-elimination of transgenes after the targeted gene has been edited. However, we observed that a small number of T1 plants generated by TKC still retained the transgenes. Herein, we first integrated Cas9 and guide RNA (gRNA) with the RUBY reporter gene (RUBY-CRISPR) to monitor the Cas9/sgRNA expression and track the presence or absence of transgenes in the T0 generation and its progenies. We then combined the RUBY-CRISPR unit with several TKC variants to develop four RUBY-TKC (TKC2) systems including TKC2.1, TKC2.2, TKC2.3 and TKC2.4, to facilitate the elimination of escaped transgene plants. Compared to non-TKC, TKC alone and RUBY-CRISPR, our TKC2s were much more efficient in the generation of transgene-free edited progenies by up to 100% in the T0 generation. TKC2s not only omit the need for screening of the plants with transgenes in the T0 generation, but also enable visualisation of the escape of plants with transgenes in the following progenies. The TKC2 systems developed here provide straightforward yet highly effective approaches for the generation of transgene-free edited plants for either rice functional genomics or genetic improvement, with potential applications in gene editing of other crops.},
}

*Transgenes/genetics
Plants, Genetically Modified/genetics
*Gene Editing/methods
Genes, Reporter/genetics
*CRISPR-Cas Systems/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Oryza/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid39970341,
year = {2025},
author = {Fan, F and Wu, MY and Zhang, HQ and Li, G and Luo, C},
title = {Rapid and Simple Detection of Anilinopyrimidine Resistance in Botrytis cinerea by Combining Recombinase Polymerase Amplification with the CRISPR/Cas12a Assay.},
journal = {Plant disease},
volume = {109},
number = {9},
pages = {1831-1838},
doi = {10.1094/PDIS-11-24-2346-SR},
pmid = {39970341},
issn = {0191-2917},
mesh = {*Botrytis/drug effects/genetics ; *Drug Resistance, Fungal/genetics ; *CRISPR-Cas Systems/genetics ; *Fungicides, Industrial/pharmacology ; *Nucleic Acid Amplification Techniques/methods ; Plant Diseases/microbiology ; *Pyrimidines/pharmacology ; Mutation ; Recombinases/genetics/metabolism ; },
abstract = {Anilinopyrimidine (AP) fungicides have been widely adopted to control Botrytis cinerea since the 1990s. As a high-risk pathogen for the development of fungicide resistance, B. cinerea developed resistance to AP fungicides soon after their application. To ensure the proper use of these fungicides, it is necessary to establish a rapid and simple method for resistance detection. Our previous study demonstrated that the E407K mutation in Bcmdl1 was the major mutation conferring AP resistance in China. Based on the combination of recombinase polymerase amplification (RPA) and CRISPR/Cas12a nucleic acid detection assay (RPA/Cas12a detection assay), a simple method for the rapid detection of AP resistance was established by specifically identifying this resistance-related mutation. The new detection assay could precisely identify the E407K mutants from other mutants and wild-type isolates within 50 min, relying solely on a water/metal bath and a UV flashlight. Moreover, this assay could detect genomic DNA at a concentration as low as 1.8 × 10[6] fg/μl, which is comparable with conventional PCR, indicating its high sensitivity. High specificity among different species was also observed in this assay. Above all, this assay was compatible with a 2-min DNA extraction method, implying its feasibility for field application. In conclusion, the RPA/Cas12a detection assay developed in this study is rapid and simple, making it an ideal method for AP resistance detection in local agencies and other points of care. Instant information on resistance monitoring can provide important guidance on resistance management.},
}

*Botrytis/drug effects/genetics
*Drug Resistance, Fungal/genetics
*CRISPR-Cas Systems/genetics
*Fungicides, Industrial/pharmacology
*Nucleic Acid Amplification Techniques/methods
Plant Diseases/microbiology
*Pyrimidines/pharmacology
Mutation
Recombinases/genetics/metabolism

@article {pmid39433694,
year = {2025},
author = {Farrokhi, S and Eslahi, A and Alizadeh, F and Kerachian, MA and Mojarrad, M},
title = {Assessment the Efficacy of the CRISPR System for Inducing Mutations in the AIMP2 Gene to Create a Cell Line Model of HLD17 Disease.},
journal = {Molecular biotechnology},
volume = {67},
number = {10},
pages = {3922-3929},
pmid = {39433694},
issn = {1559-0305},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; HEK293 Cells ; *Gene Editing/methods ; *Mutation ; Plasmids/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Hypomyelinating leukodystrophy-17 is a neurodevelopmental disorder caused by autosomal recessive mutations in the AIMP2 gene, resulting in a lack of myelin deposition during brain development, leading to variable neurological symptoms. Research on brain function in these disorders is challenging due to the lack of access to brain tissue. To overcome this problem, researchers have utilized different cell and animal models. The CRISPR-Cas9 system is considered the most optimal and effective method for genetic modification and developing cell models. We studied the efficacy of the CRISPR-Cas9 technology in inducing mutations in the AIMP2 gene in HEK293 cell lines. The study involved transfecting HEK293 cells with recombinant PX458 plasmids targeting spCas-9 and AIMP2 sgRNA. The cells were evaluated using fluorescent microscopy and enriched using serial dilution. The CRISPR/Cas9 plasmids were validated through PCR and Sanger sequencing. After serial dilution, AS-PCR, Sanger sequencing, and TIDE program analysis showed the construct successfully induces an indel mutation in HEK cells. Our findings demonstrated the great efficacy of the CRISPR system and produced a construct for inducing mutations in the AIMP2 gene, which can be utilized to edit the AIMP2 gene in nerve cells and create a cellular model of the HLD17 disease.},
}

Humans
*CRISPR-Cas Systems/genetics
HEK293 Cells
*Gene Editing/methods
*Mutation
Plasmids/genetics
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid39377911,
year = {2025},
author = {Bhoomika, S and Salunkhe, SR and Sakthi, AR and Saraswathi, T and Manonmani, S and Raveendran, M and Sudha, M},
title = {CRISPR-Cas9: Unraveling Genetic Secrets to Enhance Floral and Fruit Traits in Tomato.},
journal = {Molecular biotechnology},
volume = {67},
number = {10},
pages = {3786-3799},
pmid = {39377911},
issn = {1559-0305},
mesh = {*Solanum lycopersicum/genetics/growth & development ; *Fruit/genetics/growth & development ; *CRISPR-Cas Systems ; *Flowers/genetics/growth & development ; Gene Editing/methods ; Plants, Genetically Modified/genetics ; Gene Expression Regulation, Plant ; Plant Proteins/genetics/metabolism ; Plant Breeding/methods ; },
abstract = {Tomato, a globally consumed vegetable, possesses vast genetic diversity, making it suitable for genetic manipulation using various genetic improvement techniques. Tomatoes are grown extensively for their market value and health benefits, primarily contributed by enhanced yield and nutritional value respectively, influenced by floral and fruit traits. Floral morphology is maintained by genes involved in meristem size control, regulation of inflorescence transition, and pollen development. SP (SELF-PRUNING) and SP5G (SELF-PRUNING 5G) determine growth habit and flowering time. RIN (RIPENING INHIBITOR) and PG (POLYGALACTURONASE) are responsible for the shelf life of fruits. In addition to this, nutrition-enriched tomatoes have been developed in recent times. In this review, we comprehensively discuss the major genes influencing floral morphology, flowering time, fruit size, fruit shape, shelf life, and nutritional value, ultimately resulting in enhanced yield. Additionally, we address the advances in CRISPR/Cas9 applied for the genetic improvement of tomatoes along with prospects of areas in which research development in terms of tomato genetic improvement has to be advanced.},
}

*Solanum lycopersicum/genetics/growth & development
*Fruit/genetics/growth & development
*CRISPR-Cas Systems
*Flowers/genetics/growth & development
Gene Editing/methods
Plants, Genetically Modified/genetics
Gene Expression Regulation, Plant
Plant Proteins/genetics/metabolism
Plant Breeding/methods

@article {pmid41020188,
year = {2025},
author = {Shalaby, KE and Hmila, I and Uddin, SMN and Zawia, NH and El-Agnaf, OMA and Aouida, M},
title = {Enhanced Cellular Uptake of Compact Cas Proteins: A Comparative Study of Cas12f and Cas9 in Human Cells.},
journal = {Engineering in life sciences},
volume = {25},
number = {9},
pages = {e70042},
pmid = {41020188},
issn = {1618-0240},
abstract = {The clinical translation of CRISPR genome-editing therapies is often hindered by inefficient delivery of the CRISPR-Cas RNA-protein complex into target cells. The most widely used CRISPR-Cas9 system poses a significant challenge for efficient delivery into cells due to its large size (∼1.4 kDa). Recently reported compact Cas proteins, such as Cas12f (552 Da), Cas12k (639 Da), and Cas12m (596 Da) represent attractive alternatives as cargoes for delivery. In this brief research report, we employ efficient delivery vectors to evaluate the efficiency of cellular uptake of a compact Cas protein (Cas12f) compared to the widely used larger Cas9 in human cells. Our findings demonstrate that compact Cas proteins may facilitate more efficient cellular penetration and delivery, making them a promising alternative for the development of CRISPR-based therapies. Practical Application: Our study demonstrates that compact Cas proteins significantly enhance cellular uptake compared to larger Cas proteins. This improved uptake efficiency suggests that compact Cas proteins could be more effective for clinical application, where size constraints and delivery efficiency are critical challenges. Combined with the optimization and refinement of the editing efficiencies of compact Cas systems, our study provokes further exploration of compact Cas proteins in various therapeutic contexts to advance the development of more efficient CRISPR-based therapies.},
}

@article {pmid41019300,
year = {2025},
author = {Bilger, R and Drepper, F and Knapp, B and Berndt, T and Landerer, H and Putzer, H and Huesgen, PF and Hess, WR},
title = {Involvement of RNase J in CRISPR RNA maturation in the cyanobacterium Synechocystis sp. PCC 6803.},
journal = {microLife},
volume = {6},
number = {},
pages = {uqaf022},
pmid = {41019300},
issn = {2633-6693},
abstract = {Many bacteria and archaea use CRISPR-Cas systems, which provide RNA-based, adaptive, and inheritable immune defenses against invading viruses and other foreign genetic elements. The proper processing of CRISPR guide RNAs (crRNAs) is a crucial step in the maturation of the defense complexes and is frequently performed by specialized ribonucleases encoded by cas genes. However, some systems employ enzymes associated with degradosome or housekeeping functions, such as RNase III or the endoribonuclease RNase E. Here, the endo- and 5´-exoribonuclease RNase J was identified as an additional enzyme involved in crRNA maturation, acting jointly with RNase E in the crRNA maturation of a type III-Bv CRISPR-Cas system, and possibly together with a further RNase in the cyanobacterium Synechocystis sp. PCC 6803. Co-IP experiments revealed a small set of proteins that were co-enriched with RNase J, among them the exoribonuclease polyribonucleotide nucleotidyltransferase (PNPase). Despite a measured, strong 3' exonucleolytic activity of the recombinant enzyme, PNPase was not confirmed to contribute to crRNA maturation. However, the co-IP results indicate that PNPase in Synechocystis is an enzyme that can recruit either RNase E or RNase J, together with additional proteins.},
}

@article {pmid41018953,
year = {2025},
author = {Ajmal, H and Nandi, S and Kebabci, N and Ryan, CJ},
title = {Benchmarking genetic interaction scoring methods for identifying synthetic lethality from combinatorial CRISPR screens.},
journal = {NAR genomics and bioinformatics},
volume = {7},
number = {3},
pages = {lqaf129},
pmid = {41018953},
issn = {2631-9268},
mesh = {*Synthetic Lethal Mutations ; Benchmarking ; Humans ; Algorithms ; *CRISPR-Cas Systems ; *Epistasis, Genetic ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Synthetic lethality (SL) is an extreme form of negative genetic interaction, where simultaneous disruption of two non-essential genes causes cell death. SL can be exploited to develop cancer therapies that target tumour cells with specific mutations, potentially limiting toxicity. Pooled combinatorial CRISPR screens, where two genes are simultaneously perturbed and the resulting impacts on fitness estimated, are now widely used for the identification of SL targets in cancer. Various scoring methods have been developed to infer SL genetic interactions from these screens, but there has been no systematic comparison of these approaches. Here, we performed a comprehensive analysis of five scoring methods for SL detection using five combinatorial CRISPR datasets. We assessed the performance of each algorithm on each screen dataset using two different benchmarks of paralog SL. We find that no single method performs best across all screens but identify two methods that perform well across most datasets. Of these two scores, Gemini-Sensitive has an available R package that can be applied to most screen designs, making it a reasonable first choice.},
}

*Synthetic Lethal Mutations
Benchmarking
Humans
Algorithms
*CRISPR-Cas Systems
*Epistasis, Genetic
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid41017815,
year = {2025},
author = {Alipanahi, R and Safari, L and Khanteymoori, A},
title = {Advancing CRISPR with deep learning: A comprehensive review of models and databases.},
journal = {Molecular therapy. Nucleic acids},
volume = {36},
number = {4},
pages = {102691},
pmid = {41017815},
issn = {2162-2531},
abstract = {CRISPR is considered a powerful tool for targeted genome editing. However, off-target effects remain a significant challenge in the CRISPR field, hindering its broader clinical application. To enhance the development of gene-editing therapies, it is essential to predict the efficiency of CRISPR-based genome editing experiments, before trying them on clinical cases. Machine learning (ML) and deep learning (DL) tools are projected to become the leading methods for predicting CRISPR on-target and off-target activity. Current prediction accuracy is limited by the amount of available training data. As more sequence features are identified and incorporated in DL tools, predictions of them are expected to better align with experimental results. Hence, the increasing focus on ML/DL approaches to predict off-target sites necessitates large and easily searchable databases. In this review, we will take a closer look at available CRISPR databases.},
}

@article {pmid41017813,
year = {2025},
author = {Sharma, S and Pokharel, YR},
title = {Measles and rubella: From global health challenges to advancements in molecular diagnostics in the elimination era.},
journal = {Molecular therapy. Nucleic acids},
volume = {36},
number = {4},
pages = {102698},
pmid = {41017813},
issn = {2162-2531},
abstract = {Measles and rubella are highly contagious viral infections with significant public health implications, particularly in low- and middle-income countries. Despite the availability of effective vaccines, these diseases continue to cause periodic outbreaks, contributing to substantial global morbidity, mortality, and economic burden. Immunization programs have drastically abridged disease incidence; however, gaps in vaccination coverage and surveillance systems deter complete elimination. The economic impact of outbreaks includes direct healthcare costs and indirect societal losses, emphasizing the need for robust disease control strategies. Accurate and timely diagnosis is pivotal to measles and rubella elimination efforts. Current diagnostic approaches range from conventional RT-PCR (including multiplex and real-time formats), ELISA, and plaque reduction neutralization test (PRNT), to emerging methods such as isothermal amplification loop-mediated isothermal amplification, recombinase polymerase amplification), CRISPR-Cas systems, next-generation sequencing (NGS), microfluidics, and lateral flow assays. Despite their sensitivity, many of these methods require complex infrastructure and skilled personnel, limiting their utility in field settings. To bridge diagnostic gaps, there is an urgent need for rapid, affordable, and field-deployable nucleic acid-based diagnostics that are simple to use with minimal training. Innovations like CRISPR-Cas and microfluidic platforms hold promise for decentralized testing and real-time surveillance, potentially transforming global measles and rubella elimination programs for the future.},
}

@article {pmid41017535,
year = {2025},
author = {Han, B and Xie, X and Zhao, Y and Zhang, J and Yang, X and Jiang, Y and Zhang, W and Zhang, X},
title = {Recent development and applications of emerging biosensing technologies and on-site analytical devices for food adulteration detection: a critical review.},
journal = {Critical reviews in food science and nutrition},
volume = {},
number = {},
pages = {1-20},
doi = {10.1080/10408398.2025.2564216},
pmid = {41017535},
issn = {1549-7852},
abstract = {The increasing incidence of food adulteration poses a significant challenge to global health and food safety. Although current detection methods can effectively complete food adulteration detection, they usually require complex pre-preparation processes and professional technicians to some extent. Therefore, the development of rapid and on-site detection technologies for food adulteration is imperative. Recently, biosensing technologies and portable devices have been developed for efficient and precise food adulteration detection. In this review, the strengths and weaknesses of conventional food adulteration detection methods were compared. The recent development of emerging biosensing technologies (i.e., antibody-based biosensors, aptamer-based biosensors, molecular imprinted polymers (MIPs)-based biosensors, and clustered regularly interspaced short palindromic repeats-associated proteins (CRISPR/Cas) systems-based biosensors) and portable analytical devices (e.g., lateral flow assays (LFAs), microfluidic devices, handheld Raman, and nanopore-based devices) for food adulteration detection has been comprehensively summarized and discussed. Remarkably, the challenges and opportunities in this field have been proposed.},
}

@article {pmid41016988,
year = {2025},
author = {Zeng, J and Luo, J and Zeng, Y},
title = {Cancer gene therapy: historical perspectives, current applications, and future directions.},
journal = {Functional & integrative genomics},
volume = {25},
number = {1},
pages = {200},
pmid = {41016988},
issn = {1438-7948},
support = {S202413705064//Innovation and Entrepreneurship Training Program for College Students/ ; 82104084//National Natural Science Foundation of China/ ; 2024qnGzn12 and 2024kjTzn08//CMC Excellent-talent Program of Chengdu Medical College/ ; },
mesh = {Humans ; *Genetic Therapy/methods/trends/history ; *Neoplasms/therapy/genetics ; Gene Editing ; CRISPR-Cas Systems ; Genetic Vectors/genetics ; Animals ; Oncolytic Virotherapy ; },
abstract = {Gene therapy has emerged as a transformative approach in cancer treatment, leveraging genetic modifications to target malignancies with enhanced precision. Early efforts faced challenges such as inefficient vector delivery (< 5% tumor transduction rates with first-generation adenoviruses), immune responses (neutralizing antibodies in ~ 30% of patients), and limited clinical efficacy (< 10% objective response rates in 1990s trials). However, advancements in viral and non-viral vectors (e.g., AAVs achieving > 50% transduction efficiency in solid tumors), alongside CRISPR-Cas9 (90% target gene knockout rates in preclinical models) and RNA interference technologies, have revolutionized the field. Presently, gene therapy strategies, including tumor suppressor gene restoration, oncogene silencing, and immune modulation, demonstrate promising clinical outcomes. Despite persistent hurdles like off-target effects and high costs, emerging innovations in personalized gene editing, oncolytic viruses, and combination therapies signal a paradigm shift in oncology. This review explores the evolution of gene therapy for cancer, highlighting key milestones, current applications, and future directions that could unlock its full therapeutic potential.},
}

Humans
*Genetic Therapy/methods/trends/history
*Neoplasms/therapy/genetics
Gene Editing
CRISPR-Cas Systems
Genetic Vectors/genetics
Animals
Oncolytic Virotherapy

@article {pmid41016806,
year = {2025},
author = {Watanabe, T},
title = {[Exploration and Functional Analysis of Epstein-Barr Virus Pathogenic Factors Using a Multidimensional Approach].},
journal = {Uirusu},
volume = {75},
number = {1},
pages = {73-86},
doi = {10.2222/jsv.75.73},
pmid = {41016806},
issn = {0042-6857},
mesh = {*Herpesvirus 4, Human/genetics/pathogenicity/physiology ; Humans ; Animals ; Mice ; *Epstein-Barr Virus Infections/virology ; Genome, Viral/genetics ; CRISPR-Cas Systems ; Disease Models, Animal ; Chromosomes, Artificial, Bacterial ; Gene Editing ; Virus Latency/genetics ; },
abstract = {Epstein-Barr virus (EBV), a member of the herpesvirus family, infects more than 90% of adults and establishes a lifelong latent infection. In addition to its involvement in a wide range of malignancies such as lymphomas, nasopharyngeal carcinoma, and gastric cancer, recent evidence has shown its potential association with autoimmune diseases, positioning EBV as an interdisciplinary research model linking virology, oncology, and immunology. Historically, EBV research has been hindered by technical limitations in viral culture systems and animal models. However, recent advances-including whole-genome cloning using bacterial artificial chromosomes (BACs), gene editing via CRISPR/Cas9, and the development of in vivo models such as humanized mice-have accelerated the elucidation of EBV' s unique life cycle and tumorigenic mechanisms. In this review, we discuss the evolution of techniques for generating recombinant EBVs and in vivo modeling, both essential for functional genetic analysis, and highlight our contributions to the advancement of these tools and their application in researching EBV-associated tumorigenesis.},
}

*Herpesvirus 4, Human/genetics/pathogenicity/physiology
Humans
Animals
Mice
*Epstein-Barr Virus Infections/virology
Genome, Viral/genetics
CRISPR-Cas Systems
Disease Models, Animal
Chromosomes, Artificial, Bacterial
Gene Editing
Virus Latency/genetics

@article {pmid41016566,
year = {2025},
author = {Hussen, BM and Abdullah, SR and Hidayat, HJ and Glassy, MC and Safarzadeh, A and Komaki, A and Samsami, M and Taheri, M},
title = {CRISPR/Cas as a Tool to Overcome Drug Resistance in Cancer: From Challenge to Opportunity.},
journal = {Molecular and cellular probes},
volume = {},
number = {},
pages = {102052},
doi = {10.1016/j.mcp.2025.102052},
pmid = {41016566},
issn = {1096-1194},
abstract = {Drug resistance remains a significant challenge in cancer therapy, often resulting in treatment failure, tumor progression, and metastasis. The underlying resistance mechanisms-including genetic mutations, epigenetic alterations, and modifications in drug efflux pathways-are complex and not yet fully understood. This review explores the application of CRISPR-Cas gene editing technology in understanding and overcoming drug resistance in cancer. It focuses on how CRISPR can identify and target resistance-associated genes to restore drug sensitivity. CRISPR-based approaches enable precise genetic modifications that offer new insights into the molecular basis of drug resistance. The technology has shown promise in dissecting resistance mechanisms and developing targeted therapeutic strategies. Nevertheless, key limitations such as inefficient delivery systems, off-target effects, and limited specificity hinder clinical translation. Current efforts focus on improving guide RNA design, creating more effective delivery vectors, and integrating CRISPR with existing treatments. CRISPR-Cas technology holds significant potential to address drug resistance in cancer by enabling targeted genetic interventions. Continued advancements are required to enhance its safety, specificity, and delivery, paving the way for its integration into future clinical applications.},
}

@article {pmid41015553,
year = {2025},
author = {Zhang, Y and Shi, Q and Xie, H and Xie, B and Li, L and Wu, W and Xie, H and Xiao, Z and Xie, D and Lai, R},
title = {Construction and phenotypic analysis of p2rx2 knockout zebrafish lines.},
journal = {Zhong nan da xue xue bao. Yi xue ban = Journal of Central South University. Medical sciences},
volume = {50},
number = {6},
pages = {919-930},
pmid = {41015553},
issn = {1672-7347},
support = {2023JJ30753//the Natural Science Foundation of Hunan Province/ ; 2023SK4030//the Innovative Construction Foundation of Hunan Province/ ; kq2208326//the Natural Science Foundation of Changsha/ ; },
mesh = {Animals ; *Zebrafish/genetics ; *Receptors, Purinergic P2X2/genetics/deficiency ; CRISPR-Cas Systems/genetics ; *Gene Knockout Techniques ; Phenotype ; *Zebrafish Proteins/genetics ; Disease Models, Animal ; },
abstract = {OBJECTIVES: The purinergic receptor P2X2 (P2RX2) encodes an ATP-gated ion channel permeable to Na[+], K[+], and especially Ca[2+]. Loss-of-function mutations in P2RX2 are known to cause autosomal dominant nonsyndromic deafness 41 (DFNA41), which manifests as high-frequency hearing loss, accelerated presbycusis, and increased susceptibility to noise-induced damage. Zebrafish, owing to their small size, rapid development, high fecundity, transparent embryos, and high gene conservation with humans, provide an ideal model for studying human diseases and developmental mechanisms. This study aims to generate a p2rx2 knockout zebrafish model using CRISPR/Cas9 gene editing system to investigate the effect of p2rx2 deficiency on the auditory system, providing a basis for understanding P2RX2-related hearing loss and developing gene therapy strategies.

METHODS: Two CRISPR targets (sgRNA1 and sgRNA2) spaced 47 bp apart were designed within the zebrafish p2rx2 gene. Synthesized sgRNAs and Cas9 protein were microinjected into single-cell stage Tübingen (TU)-strain zebrafish embryos. PCR and gel electrophoresis verified editing efficiency at 36 hours post-fertilization (hpf). Surviving embryos were raised to adulthood (F0), tail-clipped, genotyped, and screened for positive mosaics. F1 heterozygotes were generated by outcrossing, and F2 homozygous mutants were obtained by intercrossing. Polymerase chain reaction (PCR) combined with sequencing verified mutation type and heritability. At 5 days post-fertilization (dpf), YO-PRO-1 staining was used to examine hair cell morphology and count in lateral line neuromasts and the otolith region. Auditory evoked potential (AEP) thresholds at 600, 800, 1 000, and 2 000 Hz were measured in nine 4-month-old wild type and mutant zebrafish per group.

RESULTS: A stable p2rx2 knockout zebrafish line was successfully established. Sequencing revealed a 66 bp insertion at the first target site introducing a premature stop codon (TAA), leading to early termination of protein translation and loss of function. Embryos developed normally with no gross malformations. At 5 dpf, mutants exhibited significantly reduced hair cell density in the otolith region compared with wild type, although lateral line neuromasts were unaffected. AEP testing showed significantly elevated auditory thresholds at all 4 frequencies in homozygous mutants compared with wild type (all P<0.001), indicating reduced hearing sensitivity.

CONCLUSIONS: We successfully generated a p2rx2 loss-of-function zebrafish model using CRISPR/Cas9 technology. p2rx2 deficiency caused hair cell defects in the otolith region and increased auditory thresholds across frequencies, indicating its key role in maintaining zebrafish auditory hair cell function and hearing perception. The phenotype's restriction to the otolith region suggests tissue-specific roles of p2rx2 in sensory organs. This model provides a valuable tool for elucidating the molecular mechanisms of P2RX2-related hearing loss and for screening otoprotective drugs and developing gene therapies.},
}

Animals
*Zebrafish/genetics
*Receptors, Purinergic P2X2/genetics/deficiency
CRISPR-Cas Systems/genetics
*Gene Knockout Techniques
Phenotype
*Zebrafish Proteins/genetics
Disease Models, Animal

@article {pmid41013258,
year = {2025},
author = {Shelenkov, A and Slavokhotova, A and Yunusova, M and Kulikov, V and Mikhaylova, Y and Akimkin, V},
title = {Genomic typing, antimicrobial resistance gene, virulence factor and plasmid replicon database for the important pathogenic bacteria Staphylococcus aureus.},
journal = {BMC genomic data},
volume = {26},
number = {1},
pages = {65},
pmid = {41013258},
issn = {2730-6844},
mesh = {*Virulence Factors/genetics ; *Plasmids/genetics ; *Staphylococcus aureus/genetics/classification/pathogenicity/drug effects ; *Replicon ; *Drug Resistance, Bacterial/genetics ; Genome, Bacterial ; *Databases, Genetic ; Humans ; Genomics ; Staphylococcal Infections/microbiology ; Anti-Bacterial Agents/pharmacology ; },
abstract = {BACKGROUND: Bacterial infections pose a global health threat across clinical and community settings. Over the past decade, the alarming expansion of antimicrobial resistance (AMR) has progressively narrowed therapeutic options, particularly for healthcare-associated infections. This critical situation has been formally recognized by the World Health Organization as a major public health concern. Epidemiological studies have demonstrated that the dissemination of AMR is frequently mediated by specific high-risk bacterial lineages, often designated as "global clones" or "clonal complexes." Consequently, surveillance of these epidemic clones and elucidation of their pathogenic mechanisms and AMR acquisition pathways have become essential research priorities. The advent of whole genome sequencing has revolutionized these investigations, enabling comprehensive epidemiological tracking and detailed analysis of mobile genetic elements responsible for resistance gene transfer. However, despite the exponential increase in available bacterial genome sequences, significant challenges persist. Current genomic datasets often suffer from uneven representation of clinically relevant strains and inconsistent availability of accompanying metadata. These limitations create substantial obstacles for large-scale comparative studies and hinder effective surveillance efforts.

DESCRIPTION: This database represents a comprehensive genomic analysis of 98,950 Staphylococcus aureus isolates, a high-priority bacterial pathogen of global clinical significance. We provide detailed isolate characterization through several established typing schemes including multilocus sequence typing (MLST), clonal complex (CC) assignments, spa typing results, and core genome MLST (cgMLST) profiles. The dataset also documents the presence of CRISPR-Cas systems in these isolates. Beyond fundamental typing data, our resource incorporates the distribution of antimicrobial resistance determinants, virulence factors, and plasmid replicons. These systematically curated genomic features offer researchers valuable insights into isolate epidemiology, resistance mechanisms, and horizontal gene transfer patterns in this highly concerning pathogen.

CONCLUSION: This database is freely available under CC BY-NC-SA at https://doi.org/10.5281/zenodo.14833440 . The data provided enables researchers to identify optimal reference isolates for various genomic studies, supporting critical investigations into S. aureus epidemiology and antimicrobial resistance evolution. This resource will ultimately inform the development of more effective prevention and control measures against this high-priority pathogen.},
}

*Virulence Factors/genetics
*Plasmids/genetics
*Staphylococcus aureus/genetics/classification/pathogenicity/drug effects
*Replicon
*Drug Resistance, Bacterial/genetics
Genome, Bacterial
*Databases, Genetic
Humans
Genomics
Staphylococcal Infections/microbiology
Anti-Bacterial Agents/pharmacology

@article {pmid41013219,
year = {2025},
author = {Zhao, C and Xia, J and Liang, B and Lin, S and Song, Y and Hong, D and Gu, J},
title = {Large-scale screening of genes responsible for silique length and seed size in Brassica Napus via pooled CRISPR library.},
journal = {BMC genomics},
volume = {26},
number = {1},
pages = {829},
pmid = {41013219},
issn = {1471-2164},
support = {2022ZD04008//Biological Breeding-National Science and Technology Major Project/ ; 2022ZD04008//Biological Breeding-National Science and Technology Major Project/ ; XGKJ2024020003//Natural Science Foundation of Xiaogan Municipality/ ; },
mesh = {*Brassica napus/genetics/growth & development ; *Seeds/genetics/growth & development/anatomy & histology ; *CRISPR-Cas Systems ; Gene Editing ; Gene Library ; *Genes, Plant ; Phenotype ; Mutation ; Gene Expression Regulation, Plant ; },
abstract = {BACKGROUND: Enhancing rapeseed (Brassica napus, B. napus) yield is critical for ensuring global vegetable oil security. However, yield is heavily influenced by silique development and seed size, the enhancement of which is limited by scarce genetic resources. The CRISPR/Cas9 system has emerged as a powerful tool for constructing genome-wide mutant libraries, even in polyploid crops with complex genomes.

RESULTS: The transcriptome-wide association study (TWAS) data, tissue-specific expression profiles data and reported genes were integrated to identify candidate genes regulating silique development and seed size. We constructed a sgRNA library targeting these genes and generated a CRISPR/Cas9 editing mutant library through genetic transformation. Specifically, 6124 sgRNAs were designed for 1739 candidate genes with ≦ 4 orthologues. 681 T0 plants were obtained through genetic transformation, which harbor 453 sgRNAs. Of 408 T0 plants analyzed, 151 (37.00%) exhibited successful gene editing events, targeting 84 candidate genes. Ten homozygous mutant plants were isolated and preliminary phenotypic analysis was performed in mutants targeting the BnaHRDs. The results suggest that mutations in BnaHRD.A03 and BnaHRD.C03 may modulate plant height (PH), main inflorescence length (MIL), silique length (SL), effective silique number per plant (ENS), seed number per silique (SNPS), and thousand-seed weight (TSW).

CONCLUSIONS: This study harnessed the CRISPR/Cas9 technology to establish a preliminary library of gene-edited mutants in B. napus, thereby laying a robust foundation for the future screening of candidate genes pertaining to silique development and seed size. Furthermore, this study provides a methodological framework for rapid functional gene discovery in B. napus through CRISPR-based approaches.},
}

*Brassica napus/genetics/growth & development
*Seeds/genetics/growth & development/anatomy & histology
*CRISPR-Cas Systems
Gene Editing
Gene Library
*Genes, Plant
Phenotype
Mutation
Gene Expression Regulation, Plant

@article {pmid41012695,
year = {2025},
author = {Madirov, A and Iksat, N and Masalimov, Z},
title = {Tomato Bushy Stunt Virus (TBSV): From a Plant Pathogen to a Multifunctional Biotechnology Platform.},
journal = {Viruses},
volume = {17},
number = {9},
pages = {},
pmid = {41012695},
issn = {1999-4915},
support = {No. BR21882269//Science Committee of the Ministry of Science and Higher Education of the Republic of Kazakhstan/ ; },
mesh = {*Biotechnology/methods ; *Tombusvirus/genetics/physiology ; Genetic Vectors ; Gene Editing ; *Plant Diseases/virology ; CRISPR-Cas Systems ; Gene Silencing ; Nanotechnology ; },
abstract = {Plant viruses have evolved from being viewed exclusively as pathogens into versatile and powerful tools for modern biotechnology. Among them, Tomato bushy stunt virus (TBSV) holds a special place due to its well-studied molecular biology and unique structural properties. This review systematizes the knowledge on TBSV's dual role as a multifunctional platform. On one hand, we cover its application as a viral vector for the highly efficient expression of recombinant proteins in plants, as well as a tool for functional genomics, including Virus-Induced Gene Silencing (VIGS) and the delivery of CRISPR/Cas9 gene-editing components. On the other hand, we provide a detailed analysis of the use of the stable and monodisperse TBSV virion in nanobiotechnology. Its capsid serves as an ideal scaffold for creating next-generation vaccine candidates, platforms for targeted drug delivery to tumor cells, and as a building block for the programmable self-assembly of complex nanoarchitectures. In conclusion, key challenges limiting the widespread adoption of the platform are discussed, including the genetic instability of vectors and difficulties in scalable purification, along with promising strategies to overcome them.},
}

*Biotechnology/methods
*Tombusvirus/genetics/physiology
Genetic Vectors
Gene Editing
*Plant Diseases/virology
CRISPR-Cas Systems
Gene Silencing
Nanotechnology

@article {pmid41012691,
year = {2025},
author = {Zhao, X and Jiang, G and Ruan, Q and Qu, Y and Yang, X and Shi, Y and Wang, D and Zhou, J and Liu, J and Hou, L},
title = {Rapid Visual Detection of Senecavirus A Based on RPA-CRISPR/Cas12a System with Canonical or Suboptimal PAM.},
journal = {Viruses},
volume = {17},
number = {9},
pages = {},
pmid = {41012691},
issn = {1999-4915},
support = {National Key Research and Development Program of China (2023YFD1800501)//Lei Hou/ ; Introduction Program of High-Level Innovation and Entrepreneurship Talents in Jiangsu Province//Jue Liu/ ; the 111 Project D18007 (D18007)//Jue Liu/ ; Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD)//Jue Liu/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Swine ; *Picornaviridae/genetics/isolation & purification ; *Swine Diseases/virology/diagnosis ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; *Picornaviridae Infections/diagnosis/veterinary/virology ; Recombinases/metabolism/genetics ; },
abstract = {Senecavirus A (SVA) is an emerging pathogen responsible for vesicular lesions and neonatal mortality in swine. In the absence of effective vaccines or therapeutics, early and accurate diagnosis is essential for controlling SVA outbreaks. Although nucleic acid-based detection methods are commonly employed, there remains a pressing need for rapid, convenient, highly sensitive, and specific diagnostic tools. Here, we developed a two-pot assay combining recombinase polymerase amplification (RPA) with CRISPR/Cas12a containing crRNA targeting canonical protospacer adjacent motifs (PAMs) for simple, rapid, and visual identification of SVA in clinical samples. Subsequently, we successfully streamlined this system into a one-pot assay by selecting a specially designed crRNA targeting suboptimal PAM and integrating RPA amplification reagents and CRISPR/Cas12a detection components into a single reaction system in one tube. The developed methods exhibited diagnostic specificity, showing no cross-reactivity with four major swine viruses, while showing remarkable sensitivity with a lower detection limit of just two copies. Clinical validation in field samples using these two methods revealed perfect agreement (100% concordance) with conventional quantitative PCR (qPCR) results (sample size, n = 28), with both assays completing detection within 30 min. These results demonstrate that both the one-pot and two-pot RPA-CRISPR/Cas12a assays offer a reliable and efficient method for detecting SVA in this pilot study. Despite the limited sample size, the assays combine rapid reaction time with high sensitivity and specificity, showing great potential for future diagnostic applications.},
}

Animals
*CRISPR-Cas Systems
Swine
*Picornaviridae/genetics/isolation & purification
*Swine Diseases/virology/diagnosis
*Nucleic Acid Amplification Techniques/methods
Sensitivity and Specificity
*Picornaviridae Infections/diagnosis/veterinary/virology
Recombinases/metabolism/genetics

@article {pmid41012605,
year = {2025},
author = {Lee, MF and Tham, SK and Poh, CL},
title = {Antiviral Strategies Targeting Enteroviruses: Current Advances and Future Directions.},
journal = {Viruses},
volume = {17},
number = {9},
pages = {},
doi = {10.3390/v17091178},
pmid = {41012605},
issn = {1999-4915},
mesh = {*Antiviral Agents/pharmacology/therapeutic use ; Humans ; *Enterovirus Infections/drug therapy/virology ; *Enterovirus/drug effects/genetics ; Animals ; Virus Replication/drug effects ; },
abstract = {Enteroviruses, a diverse genus within the Picornaviridae family, are responsible for a wide range of human infections, including hand, foot, and mouth disease, respiratory disease, aseptic meningitis, encephalitis, myocarditis, and acute flaccid paralysis. Despite their substantial global health burden and the frequent emergence of outbreaks, no specific antiviral therapies are currently approved for clinical use against non-polio enteroviruses. This review provides a comprehensive overview of the current landscape of antiviral strategies targeting enteroviruses, including direct-acting antivirals such as capsid binders, protease inhibitors, and viral RNA polymerase inhibitors. We also examine the potential of host-targeting agents that interfere with virus-host interactions essential for replication. Emerging strategies such as immunotherapeutic approaches, RNA interference, CRISPR-based antivirals, and peptide-based antivirals are also explored. Furthermore, we address key challenges, including viral diversity, drug resistance, and limitations in preclinical models. By highlighting recent advances and ongoing efforts in antiviral development, this review aims to guide future research and accelerate the discovery of effective therapies against enterovirus infections.},
}

*Antiviral Agents/pharmacology/therapeutic use
Humans
*Enterovirus Infections/drug therapy/virology
*Enterovirus/drug effects/genetics
Animals
Virus Replication/drug effects

@article {pmid41011478,
year = {2025},
author = {Cortés, M and Olate, P and Rodriguez, R and Diaz, R and Martínez, A and Hernández, G and Sepulveda, N and Paz, EA and Quiñones, J},
title = {Human Microbiome as an Immunoregulatory Axis: Mechanisms, Dysbiosis, and Therapeutic Modulation.},
journal = {Microorganisms},
volume = {13},
number = {9},
pages = {},
doi = {10.3390/microorganisms13092147},
pmid = {41011478},
issn = {2076-2607},
support = {N° 21231033//Agencia Nacional de Investigación y Desarrollo/ ; },
abstract = {The human microbiome plays a central role in modulating the immune system and maintaining immunophysiological homeostasis, contributing to the prevention of immune-mediated diseases. In particular, the gut microbiota is a key ecosystem for immune system maturation, especially in early life. This review aimed to analyze the molecular and cellular mechanisms linking the microbiome to immune and neuronal functions, as well as the impact of dysbiosis and emerging therapeutic strategies targeting the microbiome. The analysis was based on scientific databases, prioritizing studies published since 2000, with special emphasis on the past decade. The microbiome influences immune signaling through microorganism-associated molecular patterns (MAMPs) and pattern recognition receptors (PRRs), including Toll-like receptors (TLRs). Additionally, microbial metabolites-such as short-chain fatty acids (SCFAs), tryptophan derivatives, and secondary bile acids-exert significant immunomodulatory effects. The intestinal epithelial barrier is also described as an active immunological interface contributing to systemic regulation. The literature highlights innovative therapies, including fecal microbiota transplantation (FMT), probiotics, and microbiome editing with CRISPR-Cas technologies. These strategies aim to restore microbial balance and improve immune outcomes. The growing body of evidence positions the microbiome as a valuable clinical and diagnostic target, with significant potential for application in personalized medicine.},
}

@article {pmid41011338,
year = {2025},
author = {Meng, S and Zhao, Z and Huang, L and Peng, X and Chen, H and Tang, X},
title = {CRISPR/Cas Technology for the Diagnosis of Animal Infectious Diseases.},
journal = {Microorganisms},
volume = {13},
number = {9},
pages = {},
doi = {10.3390/microorganisms13092006},
pmid = {41011338},
issn = {2076-2607},
support = {2023174004//Beihai Science and Technology Program/ ; AB241484035//Guangxi Key R&D Program Project/ ; YCSW2024140//Innovation Project of Guangxi Graduate Education/ ; },
abstract = {Increasingly complex epidemics of animal infectious diseases have emerged as a major risk to livestock production and human health. However, current detection methods for animal infectious diseases suffer from shortcomings such as insufficient sensitivity, complicated operation, and reliance on skilled personnel, highlighting the urgent need for novel sensing platforms. CRISPR/Cas systems are adaptive immune systems found in many prokaryotes. Owing to their ability to precisely and reliably target and cleave nucleic acids, the CRISPR/Cas-based nucleic acid detection technology is considered a promising new detection method. When leveraged with a pre-amplification step and established readout methods, CRISPR/Cas-based sensing platforms can achieve a high sensitivity of single-base resolution or attomolar levels on-site. In this review, we first outline the history, working principles, and nucleic acid detection platforms derived from various CRISPR/Cas systems. Next, we evaluate the advantages and limitations of different nucleic acid pre-amplification methods integrated with CRISPR/Cas systems, followed by a discussion of readout methods employed in CRISPR/Cas-based sensing platforms. Additionally, we highlight recent applications of CRISPR/Cas-based sensing platforms in identifying animal infectious diseases. Finally, we address the challenges and prospects of CRISPR/Cas-based sensing platforms for the early and accurate diagnosis of animal infectious diseases.},
}

@article {pmid41010035,
year = {2025},
author = {Angelis, KJ and Holá, M and Vágnerová, R and Vaculíková, J and Paleček, JJ},
title = {The Phenotype of Physcomitrium patens SMC6 Mutant with Interrupted Hinge Interactions.},
journal = {Genes},
volume = {16},
number = {9},
pages = {},
doi = {10.3390/genes16091091},
pmid = {41010035},
issn = {2073-4425},
support = {GA20-05095S//Czech Science Foundation/ ; GA23-05284S)//Czech Science Foundation/ ; },
mesh = {*Bryopsida/genetics/metabolism ; *Cell Cycle Proteins/genetics/metabolism ; *Plant Proteins/genetics/metabolism/chemistry ; Phenotype ; DNA Breaks, Double-Stranded ; CRISPR-Cas Systems ; Mutation ; *Chromosomal Proteins, Non-Histone/genetics ; },
abstract = {Background/Objectives: The Structural Maintenance of Chromosomes (SMC) proteins form essential heterocomplexes for the preservation of DNA structure and its functions, and hence cell viability. The SMC5/6 dimer is assembled by direct interactions of ATP heads via the kleisin NSE4 bridge and by SMC hinges. The structure might be interrupted by a single point mutation within a conserved motif of the SMC6-hinge. We describe the phenomena associated with the impairment of the SMC5/6 complex with morphology, repair of DNA double strand breaks (DSB), mutagenesis, recombination and gene targeting (GT) in the moss Physcomitrium patens (P. patens). Methods: Using CRISPR/Cas9-directed oligonucleotide replacement, we have introduced two close G to R point mutations in the hinge domain of SMC6 of P. patens and show that both mutations are not toxic and allow viability of mutant lines. Results: The G514R mutation fully prevents the interaction of SMC6 not only with SMC5, but also with NSE5 and NSE6, while the mutation at G517R has no effect. The Ppsmc6_G514R line has aberrant morphology, spontaneous and bleomycin-induced mutagenesis, and maintenance of the number of rDNA copies. The most unique feature is the interference with gene targeting (GT), which is completely abolished. In contrast, the Ppsmc6_G517R line is close to WT in many aspects. Surprisingly, both mutations have no direct effect on the rate of DSB repair in dividing and differentiated cells. Conclusions: Abolished interactions of SMC6 with SMC5 and NSE5,6 partners, which allow DSB repair, but impair other repair and recombination functions, suggests also regulatory role for SMC6.},
}

*Bryopsida/genetics/metabolism
*Cell Cycle Proteins/genetics/metabolism
*Plant Proteins/genetics/metabolism/chemistry
Phenotype
DNA Breaks, Double-Stranded
CRISPR-Cas Systems
Mutation
*Chromosomal Proteins, Non-Histone/genetics

@article {pmid41009844,
year = {2025},
author = {Haval, M and Unakal, C and Ghagane, SC and Pandit, BR and Daniel, E and Siewdass, P and Ekimeri, K and Rajamanickam, V and Justiz-Vaillant, A and Lootawan, KA and Oliveira, FM and Bashetti, N and Naqvi, TA and Shettar, A and Bhasme, P},
title = {Biofilms Exposed: Innovative Imaging and Therapeutic Platforms for Persistent Infections.},
journal = {Antibiotics (Basel, Switzerland)},
volume = {14},
number = {9},
pages = {},
doi = {10.3390/antibiotics14090865},
pmid = {41009844},
issn = {2079-6382},
abstract = {Biofilms constitute a significant challenge in the therapy of infectious diseases, offering remarkable resistance to both pharmacological treatments and immunological elimination. This resilience is orchestrated through the regulation of extracellular polymeric molecules, metabolic dormancy, and quorum sensing, enabling biofilms to persist in both clinical and industrial environments. The resulting resistance exacerbates chronic infections and contributes to mounting economic burdens. This review examines the molecular and structural complexities that drive biofilm persistence and critically outlines the limitations of conventional diagnostic and therapeutic approaches. We emphasize advanced technologies such as super-resolution microscopy, microfluidics, and AI-driven modeling that are reshaping our understanding of biofilm dynamics and heterogeneity. Further, we highlight recent progress in biofilm-targeted therapies, including CRISPR-Cas-modified bacteriophages, quorum-sensing antagonists, enzyme-functionalized nanocarriers, and intelligent drug-delivery systems responsive to biofilm-specific cues. We also explore the utility of in vivo and ex vivo models that replicate clinical biofilm complexity and promote translational applicability. Finally, we discuss emerging interventions grounded in synthetic biology, such as engineered probiotic gene circuits and self-regulating microbial consortia, which offer innovative alternatives to conventional antimicrobials. Collectively, these interdisciplinary strategies mark a paradigm shift from reactive antibiotic therapy to precision-guided biofilm management. By integrating cutting-edge technologies with systems biology principles, this review proposes a comprehensive framework for disrupting biofilm architecture and redefining infection treatment in the post-antibiotic era.},
}

@article {pmid41009658,
year = {2025},
author = {Zhang, H and Teng, C and Lyu, S and Fan, Y},
title = {High-Frequency Generation of Homozygous/Biallelic Mutants via CRISPR/Cas9 Driven by AtKu70/80 Promoters.},
journal = {International journal of molecular sciences},
volume = {26},
number = {18},
pages = {},
doi = {10.3390/ijms26189094},
pmid = {41009658},
issn = {1422-0067},
support = {ZR2023MC070//Natural Science Foundation of Shandong province/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Promoter Regions, Genetic ; *Arabidopsis/genetics ; *Gene Editing/methods ; *Ku Autoantigen/genetics ; Homozygote ; *Arabidopsis Proteins/genetics ; Plants, Genetically Modified/genetics ; *Mutation ; Glycine max/genetics ; Alleles ; DNA End-Joining Repair ; },
abstract = {CRISPR/Cas9 gene editing technology is widely used in plant gene editing to verify gene function or improve agronomic traits. In the CRISPR/Cas9 system, Cas9 expression hinges on promoter choice, and CRISPR/Cas9 driven by a strong promoter or cell division-specific promoter has a higher editing efficiency. The CRISPR/Cas9 mechanism involves the CAS9 enzyme, which, directed by guide RNA, cleaves target double-stranded DNA and subsequently induces insertions or deletions (InDels) through the non-homologous end joining (NHEJ) repair pathway. The Ku protein plays a central role in the NHEJ repair process. It remains unclear whether driving Cas9 with promoters of AtKu70 and AtKu80, which are subunits of the Ku protein, will enhance gene editing efficiency. In this study, the promoters of AtKu70 and AtKu80 were cloned and used to drive Cas9 in the CRISPR/Cas9 system. Four different genes, GmRj7, GmNNL1, AtPDS3, and AtBRI1, were designed for soybean hairy root transformation and Arabidopsis transformation. The results showed that the CRISPR/Cas9 systems driven by the promoters of AtKu70 and AtKu80 achieved higher homozygous/biallelic mutation efficiencies than the CRISPR/Cas9 system driven by the 35S promoter in hairy root transformation by Rhizobium rhizogenes and stable genetic transformation with Rhizobium tumefaciens.},
}

*CRISPR-Cas Systems/genetics
*Promoter Regions, Genetic
*Arabidopsis/genetics
*Gene Editing/methods
*Ku Autoantigen/genetics
Homozygote
*Arabidopsis Proteins/genetics
Plants, Genetically Modified/genetics
*Mutation
Glycine max/genetics
Alleles
DNA End-Joining Repair

@article {pmid41006228,
year = {2025},
author = {Maric, M and Segura-Bayona, S and Kuthethur, R and Takaki, T and Borel, V and Stanage, TH and Ivanov, MP and Parnandi, N and Hewitt, G and Millar, R and Fonseca, CS and Patel, H and Llorian, M and Warchal, S and Howell, M and Chaudhuri, AR and Kotsantis, P and Boulton, SJ},
title = {EXO1 as a therapeutic target for Fanconi Anaemia, ZRSR2 and BRCA1-A complex deficient cancers.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {8476},
pmid = {41006228},
issn = {2041-1723},
mesh = {*Exodeoxyribonucleases/genetics/metabolism ; Humans ; *BRCA1 Protein/genetics/metabolism/deficiency ; *Fanconi Anemia/genetics/metabolism ; DNA Repair/genetics ; *Neoplasms/genetics/metabolism ; DNA Replication/genetics ; *DNA Repair Enzymes/genetics/metabolism ; DNA Damage ; Cell Line, Tumor ; Cisplatin/pharmacology ; Synthetic Lethal Mutations ; Poly(ADP-ribose) Polymerase Inhibitors/pharmacology ; CRISPR-Cas Systems ; },
abstract = {Exonuclease EXO1 performs multiple roles in DNA replication and DNA damage repair (DDR). However, EXO1 loss is well-tolerated, suggesting the existence of compensatory mechanisms that could be exploited in DDR-deficient cancers. Using CRISPR screening, we find EXO1 loss as synthetic lethal with many DDR genes somatically inactivated in cancers, including Fanconi Anaemia (FA) pathway and BRCA1-A complex genes. We also identify the spliceosome factor and tumour suppressor ZRSR2 as synthetic lethal with loss of EXO1 and show that ZRSR2-deficient cells are attenuated for FA pathway activation, exhibiting cisplatin sensitivity and radial chromosome formation. Furthermore, FA or ZRSR2 deficiencies depend on EXO1 nuclease activity and can be potentiated in combination with PARP inhibitors or ionizing radiation. Finally, we uncover dysregulated replication-coupled repair as the driver of synthetic lethality between EXO1 and FA pathway attributable to defective fork reversal, elevated replication fork speeds, post-replicative single stranded DNA exposure and DNA damage. These findings implicate EXO1 as a synthetic lethal vulnerability and promising drug target in a broad spectrum of DDR-deficient cancers unaddressed by current therapies.},
}

*Exodeoxyribonucleases/genetics/metabolism
Humans
*BRCA1 Protein/genetics/metabolism/deficiency
*Fanconi Anemia/genetics/metabolism
DNA Repair/genetics
*Neoplasms/genetics/metabolism
DNA Replication/genetics
*DNA Repair Enzymes/genetics/metabolism
DNA Damage
Cell Line, Tumor
Cisplatin/pharmacology
Synthetic Lethal Mutations
Poly(ADP-ribose) Polymerase Inhibitors/pharmacology
CRISPR-Cas Systems

@article {pmid41006213,
year = {2025},
author = {Chen, PR and Qin, PP and Wang, YN and Liu, PF and Zhang, XY and Qian, T and Ye, BC and Yin, BC},
title = {De novo design of hypercompact transcript degraders by engineering substrate-specific toxins and Cas6-CBS system.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {8446},
pmid = {41006213},
issn = {2041-1723},
mesh = {Escherichia coli/genetics/metabolism ; Humans ; *Escherichia coli Proteins/genetics/metabolism ; *Endoribonucleases/metabolism/genetics ; CRISPR-Cas Systems ; RNA Stability ; Protein Engineering/methods ; Binding Sites ; HEK293 Cells ; Bacterial Toxins/genetics/metabolism ; *CRISPR-Associated Proteins/metabolism/genetics ; DNA-Binding Proteins ; },
abstract = {Artificial assembly of small functional proteins provides effective strategies for development of compact RNA degradation systems, which overcome the challenges associated with delivery. Here, we excavate and evolve three small toxin endoribonucleases with simple RNA cleavage motifs (barnase, MqsR, and MaZF), and integrate catalytically dead Cas6 (dCas6) along with its cognate stem-loop RNA (Cas6 binding site, termed CBS) from Escherichia coli (E. coli) to create hypercompact transcript degraders (317 ~ 430 amino acids), named STAR (small toxin- and dEcCas6-CBS-based RNA degraders). We experimentally find that CBS can be fine-tuned for EcCas6 processing but exhibits high conservatism in EcCas6 and dEcCas6 binding, laying a foundation for the design of CBS guides to effectively recruit dEcCas6-toxins. STAR exhibits high-efficiency knockdown of both cytoplasmic and nuclear transcripts in the tested mammalian cells, with significantly reduced off-target activities compared to established CRISPR and RNA interference (RNAi) technologies. Moreover, the small size of STAR enables delivery via a single adeno-associated virus (AAV) for ease of multiplex RNA knockdown, including effective silencing of the oncogenic RNA MYC in human cancer cells. Together, STAR unlocks new territory for employing toxin to design miniature, efficacious and safer RNA degraders.},
}

Escherichia coli/genetics/metabolism
Humans
*Escherichia coli Proteins/genetics/metabolism
*Endoribonucleases/metabolism/genetics
CRISPR-Cas Systems
RNA Stability
Protein Engineering/methods
Binding Sites
HEK293 Cells
Bacterial Toxins/genetics/metabolism
*CRISPR-Associated Proteins/metabolism/genetics
DNA-Binding Proteins

@article {pmid41005481,
year = {2025},
author = {Gong, Z and Lu, T and Ruan, Z and Zhang, R and Zhu, S and Xia, Z and Zhong, J and Wang, G and Li, Y and He, Q and Liu, R and Che, J},
title = {A multiplexed TSA/CRISPR-mediated one-pot system for rapid detection of high-risk animal-derived infectious diseases.},
journal = {Journal of microbiological methods},
volume = {238},
number = {},
pages = {107277},
doi = {10.1016/j.mimet.2025.107277},
pmid = {41005481},
issn = {1872-8359},
abstract = {The importance of rapid and convenient pathogen detection has been emphasized by the alarming threat of the Coronavirus Disease 2019 (COVID-19) pandemic since 2019. Point-of-care testing (POCT) provides rapid diagnostic results directly at the sampling site. However, isothermal amplification-based POCT faces technical challenges including primer design complexity and false-positive rates. To address these limitations, we developed the Thermostatic Step Amplification (TSA)/Clustered regularly interspaced short palindromic repeats (CRISPR) One-Pot System (TCOPS). This sensitive, rapid, and efficient platform specifically detects Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Mpox virus (MPXV) and Rabíes virus (RV) through integrated amplification and CRISPR-based detection. Our integrated TCOPS overcomes the technical challenges through single-tube reactions combining thermostatic amplification and CRISPR detection, reducing contamination while maintaining high accuracy for field applications. TCOPS enables single-tube CRISPR detection of high-risk viruses, with 10 copies/μL sensitivity shown using cloned DNA template for RV. In evaluations against Quantitative Polymerase Chain Reaction (qPCR) using 50 clinical samples, TCOPS incorporating freeze-dried reagents and a newly developed miniature fluorescence system (Q max) demonstrated >90 % sensitivity and 100 % specificity. Combined with the portable Q max device and its lyophilized reagent kit, TCOPS enables simple, rapid detection of multiple zoonotic viruses (SARS-CoV-2, MPXV, and RV) at the point of care. This integrated system achieves high sensitivity and specificity while establishing a practical, field-deployable prototype for next-generation POCT applications in resource-limited settings.},
}

@article {pmid41004579,
year = {2025},
author = {Gao, Q and Gao, Y and Cao, Y and Xu, H and Ma, Y and Zu, M and Yang, Q and Yang, K and Zhu, Z and Liu, C and Shi, X and Reis, RL and Kundu, SC and Ma, S and Han, H and Xiao, B},
title = {Reinforced plant-derived lipid nanoparticles for oral precise epigenome editing in colonic diseases.},
journal = {Science advances},
volume = {11},
number = {39},
pages = {eadw9275},
pmid = {41004579},
issn = {2375-2548},
mesh = {*Nanoparticles/chemistry/administration & dosage ; *Gene Editing/methods ; Animals ; Administration, Oral ; Humans ; Mice ; *Lipids/chemistry ; CRISPR-Cas Systems ; *Epigenome ; *Colonic Diseases/genetics/therapy ; Ribonucleoproteins/genetics ; Colitis/genetics ; *Epigenesis, Genetic ; Histone Demethylases/genetics ; Epigenome Editing ; Liposomes ; },
abstract = {The clinical application of CRISPR-Cas9 remains limited by delivery challenges, particularly for oral administration. Lysine-specific demethylase 1 (Lsd1) plays a key role in colonic inflammation and tumorigenesis. Here, we developed an oral genome-editing platform (TPGS-RNP@LNP), where Lsd1-targeting ribonucleoproteins (RNPs) were encapsulated in mulberry leaf lipid nanoparticles (LNPs) and formulated with d-α-tocopherol polyethylene glycol succinate (TPGS). TPGS reinforced the lipid bilayer of LNPs, enhanced gastrointestinal stability, and facilitated colonic mucus penetration. Upon the galactose receptor-mediated endocytosis of TPGS-RNP@LNPs by macrophages, their fusion with the endosomal membrane and the presence of nuclear localization signals ensured the nuclear delivery of RNPs. TPGS-RNP@LNPs achieved 59.7% Lsd1 editing efficiency in macrophages, surpassing the commercial CRISPRMAX (43.0%). Oral TPGS-RNP@LNPs promoted H3K4 methylation to modulate epigenetic states, achieving inflammation mitigation, epithelial barrier restoration, and retardation of colitis and its associated tumorigenesis. As an LNP-based oral RNP delivery system, TPGS-RNP@LNPs provide a promising platform for precise treatment of colorectal diseases.},
}

*Nanoparticles/chemistry/administration & dosage
*Gene Editing/methods
Animals
Administration, Oral
Humans
Mice
*Lipids/chemistry
CRISPR-Cas Systems
*Epigenome
*Colonic Diseases/genetics/therapy
Ribonucleoproteins/genetics
Colitis/genetics
*Epigenesis, Genetic
Histone Demethylases/genetics
Epigenome Editing
Liposomes

@article {pmid41004518,
year = {2025},
author = {Wilbie, D and Eising, S and Amo-Addae, V and Walther, J and Bosman, E and Lei, Z and de Jong, OG and Molenaar, JJ and Mastrobattista, E},
title = {Anti-cancer compound screening identifies Aurora Kinase A inhibition as a means to favor CRISPR/Cas9 gene correction over knock-out.},
journal = {PloS one},
volume = {20},
number = {9},
pages = {e0332617},
pmid = {41004518},
issn = {1932-6203},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Aurora Kinase A/antagonists & inhibitors/genetics ; HEK293 Cells ; *Antineoplastic Agents/pharmacology ; Gene Knockout Techniques ; *Protein Kinase Inhibitors/pharmacology ; Gene Editing ; Pyrimidines/pharmacology ; Drug Screening Assays, Antitumor ; Recombinational DNA Repair/drug effects ; Cell Line, Tumor ; Azepines ; },
abstract = {CRISPR gene therapy holds the potential to cure a variety of genetic diseases by causing a targeted DNA break, which is repaired by host DNA damage responses. One option to introduce precise gene corrections is via the homology-directed repair (HDR) pathway. The problem in utilizing this pathway is that CRISPR-induced double stranded DNA breaks are more likely to be erroneously repaired by the non-homologous end joining (NHEJ) pathway, which may introduce random insertions or deletions at the cut site. We screened a small library of oncological drug compounds to steer the DNA repair process towards preferential HDR activation. We included forty compounds in the screen based on their mechanism of action. After optimizing the toxicity and adding these compounds during gene editing, nine showed a potential benefit for HDR activation. Three were shown to be beneficial after validation: rucaparib, belinostat and alisertib. The Aurora Kinase A inhibitor alisertib in particular led to an over 4-fold increase in preferential gene correction over gene knock-out in two cell models (HEK293T and Hepa 1-6) at sub-micromolar dosages on the eGFP locus, prompting further validation. On the long term this pathway did show cytotoxicity especially in the HEK293T cells, indicating further mechanistic investigation is needed, but this toxicity was less pronounced in primary hepatocytes.},
}

Humans
*CRISPR-Cas Systems/genetics
*Aurora Kinase A/antagonists & inhibitors/genetics
HEK293 Cells
*Antineoplastic Agents/pharmacology
Gene Knockout Techniques
*Protein Kinase Inhibitors/pharmacology
Gene Editing
Pyrimidines/pharmacology
Drug Screening Assays, Antitumor
Recombinational DNA Repair/drug effects
Cell Line, Tumor
Azepines

@article {pmid41004222,
year = {2025},
author = {Taweechai, S and Totañes, FIG and Westhead, D and Herrera-Arozamena, C and Foster, R and McConkey, GA},
title = {Validated antimalarial drug target discovery using genome-scale metabolic modeling.},
journal = {Antimicrobial agents and chemotherapy},
volume = {},
number = {},
pages = {e0045925},
doi = {10.1128/aac.00459-25},
pmid = {41004222},
issn = {1098-6596},
abstract = {Given the rapid resistance of Plasmodium falciparum to antimalarial drugs, there is a continual need for new treatments. A genome-scale metabolic (GSM) model was developed with integrated metabolomics and constraint-based, experimental flux-balance data to predict genes essential for P. falciparum growth as drug targets. We selected the highly ranked P. falciparum UMP-CMP kinase (UCK) to test its necessity and the ability to inhibit parasite growth in the presence of inhibitors. Conditional deletion mutants using the DiCre recombinase system, generated by CRISPR-Cas genome editing, exhibited defective asexual growth and stage-specific developmental arrest. Based on in silico and in vitro screening, inhibitors were identified that are selective for P. falciparum UCK and exhibit antiparasitic activity. This study, for the first time, shows assertions from a GSM model identifying novel, validated "druggable" targets. These findings show a role for GSM models in antimalarial drug discovery and identify P. falciparum UCK as a novel, valid malaria drug target.},
}

@article {pmid41003754,
year = {2025},
author = {Zubair, A and Ali, M and Ahmad, F and Althobaiti, SA},
title = {Unlocking the role of transcription activator-like effector nuclease (TALENs) and zinc finger nuclease (ZFN) in the treatment of HIV.},
journal = {Molecular biology reports},
volume = {52},
number = {1},
pages = {948},
pmid = {41003754},
issn = {1573-4978},
mesh = {Humans ; *Transcription Activator-Like Effector Nucleases/genetics/metabolism/therapeutic use ; *Zinc Finger Nucleases/genetics/metabolism/therapeutic use ; *HIV Infections/therapy/genetics ; Gene Editing/methods ; HIV-1/genetics ; CRISPR-Cas Systems ; Receptors, CCR5/genetics ; Zinc Fingers ; },
abstract = {Some nucleases may be programmed to break just certain portions of DNA; examples of such enzymes include zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Insertions and deletions are used by cellular machinery to repair damaged DNA. By specifically targeting long terminal repeats (LTRs), zinc-finger nucleases (ZFNs) efficiently and accurately remove HIV-1 proviral DNA from inactive human T cells, offering a new and different way to eradicate HIV-1 infections. This paper examines the potential, evaluates the current situation, and draws attention to the challenges surrounding the use of TALENs and ZFNs as therapeutic tools for the treatment of HIV infection, to mitigate the adverse off-target effects that result from their extended expression. There is less off-target editing and higher success in targeting HIV escape mutations using TALENs and ZFNs than with CRISPR/Cas-9. The use of ZFNs and TALEN has resulted in changes to many host genes. These include the entrance receptors CCR5 and CXCR4, as well as the proviral integration protein LEDGF/p75. One of the viral targets is the big terminal repeats of proviral DNA. The advancement of gene therapy from the laboratory to the clinic is hindered by the need to reduce immunogenicity, cytotoxicity, and off-target editing while simultaneously enhancing cleavage efficiency and dispersion. However, TALENs technology and breakthroughs in ZFNs are making cleavage more efficient and selective. The strategy for treating HIV might be drastically changed, and maybe even eradicated, by the creation of synthetic nucleases like ZFNs and TALENs. This review explores the current developments about ZFNs and TALENs for the treatment of HIV.},
}

Humans
*Transcription Activator-Like Effector Nucleases/genetics/metabolism/therapeutic use
*Zinc Finger Nucleases/genetics/metabolism/therapeutic use
*HIV Infections/therapy/genetics
Gene Editing/methods
HIV-1/genetics
CRISPR-Cas Systems
Receptors, CCR5/genetics
Zinc Fingers

@article {pmid41003718,
year = {2025},
author = {Behera, B and Singh, R and Sharma, K and Rai, A and Singh, S and Balan, B},
title = {A comprehensive review of advanced strategies to combat antimicrobial resistance.},
journal = {Archives of microbiology},
volume = {207},
number = {11},
pages = {281},
pmid = {41003718},
issn = {1432-072X},
mesh = {Humans ; *Anti-Bacterial Agents/pharmacology/therapeutic use ; Gene Editing ; *Drug Resistance, Bacterial ; *Bacteria/drug effects/genetics ; CRISPR-Cas Systems ; Antimicrobial Peptides/pharmacology/therapeutic use ; Phage Therapy ; *Bacterial Infections/drug therapy/microbiology/therapy ; },
abstract = {Antimicrobial Resistance (AMR) is a growing global issue, as many first-line antibiotics are becoming less effective due to their overuse and misuse. Recent advances in novel antibiotic derivatives reveal mechanisms designed to counteract AMR. Even though conventional antimicrobial therapy has failed, no new antibiotic class has been developed in the past decade. Consequently, various innovative alternative tactics have been discovered to counteract drug-resistant pathogens. The article reviews novel approaches in combating AMR, which include antimicrobial peptides, phage therapy, CRISPR-Cas gene editing, nanomaterial-based antimicrobials, immunomodulatory agents, innovative physicochemical strategies, and combination therapy. Collectively, these approaches utilize cutting edge technologies that mark a shift from the traditional paradigm of antibiotics to integrated next-generation therapeutics. AMR remains a serious issue despite all of the noted advancements, and hence, a collaborative and multidisciplinary action involving researchers, healthcare professionals, policymakers, and pharmaceutical sector is urgently required. The emergence and burden of AMR can be better tackled by inventiveness, cooperation, and proactive approaches.},
}

Humans
*Anti-Bacterial Agents/pharmacology/therapeutic use
Gene Editing
*Drug Resistance, Bacterial
*Bacteria/drug effects/genetics
CRISPR-Cas Systems
Antimicrobial Peptides/pharmacology/therapeutic use
Phage Therapy
*Bacterial Infections/drug therapy/microbiology/therapy

@article {pmid41002431,
year = {2025},
author = {Vivarelli, R and Vantaggiato, C and Bassi, MT and Santorelli, FM and Marchese, M},
title = {Wings of Discovery: Using Drosophila to Decode Hereditary Spastic Paraplegia and Ataxias.},
journal = {Cells},
volume = {14},
number = {18},
pages = {},
doi = {10.3390/cells14181466},
pmid = {41002431},
issn = {2073-4409},
support = {GJC21131//Telethon Foundation/ ; Ricerca Corrente 2024-2025//Italian Minister of Health/ ; },
mesh = {Animals ; *Spastic Paraplegia, Hereditary/genetics/pathology ; Disease Models, Animal ; Humans ; *Drosophila melanogaster/genetics ; CRISPR-Cas Systems ; },
abstract = {Hereditary spastic paraplegia (HSP) and hereditary ataxias (HA) are clinically and genetically heterogeneous neurodegenerative disorders that primarily affect motor coordination and neural integrity. Despite distinct pathological features, such as pyramidal tract degeneration in HSP and spinocerebellar pathway involvement in HA, these conditions share overlapping genetic pathways and mechanisms. The fruit fly Drosophila melanogaster has emerged as a powerful model organism for investigating the molecular basis of rare diseases, including HSP and HA. Its genetic tractability, rapid life cycle, and high degree of gene conservation with humans make it a cost-effective and ethically viable platform for disease modelling. In this review, we provide a comprehensive overview of Drosophila-based models for HSP and HA. We highlight the use of advanced genetic tools, including RNA interference, CRISPR/Cas9, and the GAL4/UAS system, as well as behavioral and neuroanatomical assays to model disease features. Furthermore, we discuss the application of genetic "avatars" and high-throughput drug screening platforms to test therapeutic candidates. Collectively, these models have deepened our understanding of the pathophysiology of HSP and HA, offering valuable insights for the development of targeted therapies and approaches to personalized medicine.},
}

Animals
*Spastic Paraplegia, Hereditary/genetics/pathology
Disease Models, Animal
Humans
*Drosophila melanogaster/genetics
CRISPR-Cas Systems

@article {pmid41002335,
year = {2025},
author = {Jayakumar, S and Vengadassalapathy, S and Venkadassalapathy, S and Durairajan, S and Vijayaraj, R and Govindan, L},
title = {Advancements and Applications of Split Technology in CRISPR/Cas12a: Transforming Molecular Diagnostics and Biosensing.},
journal = {Biosensors},
volume = {15},
number = {9},
pages = {},
doi = {10.3390/bios15090595},
pmid = {41002335},
issn = {2079-6374},
mesh = {*Biosensing Techniques ; *CRISPR-Cas Systems ; Humans ; *Pathology, Molecular ; *Molecular Diagnostic Techniques ; },
abstract = {The rapid evolution of CRISPR technology has revolutionized molecular biology, and among the various systems, CRISPR/Cas12a stands out for its high specificity and efficient collateral cleavage activity. This review article focuses on the recent advancements and applications of split technology within the CRISPR/Cas12a framework, highlighting its transformative role in molecular diagnostics and biosensing. Split technology innovatively divides functional nucleic acid components into modular segments that are activated by specific targets, significantly enhancing the specificity and sensitivity of biosensors. This design addresses the inherent limitations of traditional sensor systems, enabling the direct detection of ultrashort nucleic acids and improved discrimination of single-nucleotide variants, thereby facilitating the simultaneous detection of multiple biomolecules. The versatility of split-enabled biosensors extends beyond genetic testing, making them valuable tools in diagnostics, therapeutics, and environmental science. Despite challenges such as crRNA degradation and reassembly kinetics, ongoing research and engineering solutions continue to enhance the stability and performance of these systems. This review synthesizes the foundational principles, recent advancements, and potential applications of split technology while also identifying challenges and opportunities for future exploration. Ultimately, our insights provide a comprehensive resource to leverage the full potential of CRISPR/Cas12a-based split technology in advancing biosensing methodologies and clinical applications.},
}

*Biosensing Techniques
*CRISPR-Cas Systems
Humans
*Pathology, Molecular
*Molecular Diagnostic Techniques

@article {pmid41002137,
year = {2025},
author = {Zhang, S and Ji, Z and Cheng, X and Ma, Y and Feng, M and Cai, D and Bai, T},
title = {TMTP1-Modified Small Extracellular Vesicles Target BRAF Mutation in Anaplastic Thyroid Cancer Reversing Vemurafenib Resistance With CRISPR/Cas9 Delivery.},
journal = {Journal of extracellular vesicles},
volume = {14},
number = {9},
pages = {e70170},
doi = {10.1002/jev2.70170},
pmid = {41002137},
issn = {2001-3078},
mesh = {Humans ; *Thyroid Carcinoma, Anaplastic/genetics/drug therapy/metabolism ; *Vemurafenib/pharmacology ; *Extracellular Vesicles/metabolism ; *CRISPR-Cas Systems ; *Drug Resistance, Neoplasm/genetics/drug effects ; *Proto-Oncogene Proteins B-raf/genetics ; Cell Line, Tumor ; Animals ; *Thyroid Neoplasms/genetics/drug therapy ; Mutation ; Mice ; Gene Editing ; Reactive Oxygen Species/metabolism ; Drug Delivery Systems ; },
abstract = {This study investigates a novel approach to overcome Vemurafenib resistance in BRAF-mutant Anaplastic thyroid carcinoma (ATC) using CRISPR/Cas9 gene editing and TMTP1-modified extracellular vesicles (TMTP1-sgBRAF-EVs). By knocking out the BRAF gene, the study elucidates Vemurafenib-induced ferroptosis mechanisms involving lipid peroxidation and reactive oxygen species (ROS) generation in ATC cells. The developed TMTP1-sgBRAF-EVs system demonstrates superior tumour-targeting and drug delivery capabilities, significantly enhancing Vemurafenib efficacy in both in vitro and in vivo models. This innovative combination of gene editing technology with a nanoparticle delivery system shows promising potential as a therapeutic strategy for treating aggressive BRAF-mutant ATC.},
}

Humans
*Thyroid Carcinoma, Anaplastic/genetics/drug therapy/metabolism
*Vemurafenib/pharmacology
*Extracellular Vesicles/metabolism
*CRISPR-Cas Systems
*Drug Resistance, Neoplasm/genetics/drug effects
*Proto-Oncogene Proteins B-raf/genetics
Cell Line, Tumor
Animals
*Thyroid Neoplasms/genetics/drug therapy
Mutation
Mice
Gene Editing
Reactive Oxygen Species/metabolism
Drug Delivery Systems

@article {pmid41000010,
year = {2025},
author = {Wang, Z and Wang, Q and Zhang, J and Li, B and Li, Y and Chen, Z and Guo, D and Feng, S},
title = {CRISPR-driven diagnostics: Molecular mechanisms, clinical efficacy and translational challenges.},
journal = {Clinical and translational medicine},
volume = {15},
number = {10},
pages = {e70482},
doi = {10.1002/ctm2.70482},
pmid = {41000010},
issn = {2001-1326},
support = {23ZX005//the Basic Research Project of the Key Research Program of Colleges and Universities in Henan Province/ ; 2023M731023//China Postdoctoral Science Foundation/ ; //Joint Funds of Science and Technology Research and Development Plan of Henan Province/ ; 232300421164//Natural Science Foundation of Henan Province/ ; //Basic Research Project of the Key Research Program of Colleges and Universities in Henan Province/ ; 82402600//National Natural Science Foundation of China/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Translational Research, Biomedical ; },
abstract = {BACKGROUND: In the realm of public health, among the primary perils menacing human well-being, the issue of pathogen infection persists as a significant concern. Precise and timely diagnosis of diseases constitutes the bedrock for effective therapeutic interventions and epidemiological monitoring. Hence, it is crucial to develop quick, sensitive, and highly effective methods for identifying pathogen and their variants.

MATERIAL AND METHODS: This article reviews the recent research progress in the CRISPR/Cas system for detecting nucleic acids, with an emphasis on CRISPR/Cas9, CRISPR/Cas12, and CRISPR/Cas13. Initially, we provided a concise overview of the nucleic acid detection mechanism utilizing the CRISPR/Cas system. Subsequently, we dissect the molecular mechanisms of CRISPR tools, compare their clinical efficacy against traditional methods, and explore frontier innovations such as amplification-free detection and AI integration.

CONCLUSION: Ultimately, we argue that CRISPR diagnostics must evolve beyond technical optimization to embrace ecological adaptability, ensuring that precision medicine serves as a bridge-rather than a barrier-to global health equity.

KEY POINTS: Core Mechanism: Explains the molecular basis of CRISPR-Cas (Cas9, Cas12, Cas13) for nucleic acid detection, leveraging crRNA-guided targeting and trans-cleavage activity for ultra-sensitive (aM level) and specific pathogen identification. Superior Performance: Outperforms traditional methods in speed, sensitivity, and cost, making it ideal for point-of-care use in resource-limited settings. Cutting-Edge Innovations: Covers key advances like amplification-free detection, portable device integration, and multiplex platforms. Translation Challenges: Discusses hurdles in clinical adoption, including inhibitor interference in complex samples, scalability limitations, the need for multi-center clinical data, and varying regional regulations. Future Outlook: Highlights emerging directions such as integrated "sample-to-result" systems and AI integration, while also addressing associated biosafety and ethical concerns, calling for robust regulatory frameworks.},
}

Humans
*CRISPR-Cas Systems/genetics
Gene Editing/methods
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Translational Research, Biomedical

@article {pmid40998781,
year = {2025},
author = {Porter, DF and Meyers, RM and Miao, W and Reynolds, DL and Hong, AW and Yang, X and Srinivasan, S and Mondal, S and Siprashvili, Z and Fabo, T and Zhou, R and Nguyen, T and Ducoli, L and Meyers, JM and Nguyen, DT and Ko, LA and Kellman, LN and Elfaki, I and Guo, M and Winge, MC and Jackrazi, LV and Lopez-Pajares, V and Liu, BB and Qu, Y and Porter, IE and Kim, SH and Kim, G and Tao, S and Engreitz, JM and Khavari, PA},
title = {Disease-linked regulatory DNA variants and homeostatic transcription factors in epidermis.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {8387},
pmid = {40998781},
issn = {2041-1723},
support = {AR076965, AR045192//U.S. Department of Health & Human Services | NIH | National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)/ ; HG010856//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; },
mesh = {Humans ; *Transcription Factors/metabolism/genetics ; *Epidermis/metabolism/pathology ; *Polymorphism, Single Nucleotide ; Homeostasis/genetics ; *Skin Diseases/genetics ; Genetic Predisposition to Disease ; *DNA/metabolism/genetics ; Multifactorial Inheritance/genetics ; Promoter Regions, Genetic ; Alleles ; CRISPR-Cas Systems ; },
abstract = {Identifying noncoding single nucleotide variants (SNVs) in regulatory DNA linked to polygenic disease risk, the transcription factors (TFs) they bind, and the genes they dysregulate is a goal in polygenic disease research. Here, we use massively parallel reporter analysis of 3451 SNVs linked to risk for polygenic skin diseases with disrupted epidermal homeostasis to identify 355 differentially active SNVs (daSNVs). daSNV target gene analysis, combined with daSNV editing, underscored dysregulated epidermal differentiation as a shared pathomechanism. CRISPR knockout screens of 1772 human TFs revealed 123 TFs essential for epidermal homeostasis, highlighting ZNF217 and CXXC1. Population sampling CUT&RUN of 27 homeostatic TFs identified allele-specific DNA binding (ASB) differences at daSNVs enriched near epidermal homeostasis and monogenic skin disease genes, with notable representation of SP/KLF and AP-1/2 TFs. High TF-occupancy promoters were "buffered" against ASB. This resource implicates dysregulated binding of specific homeostatic TF families in risk for diverse polygenic skin diseases.},
}

Humans
*Transcription Factors/metabolism/genetics
*Epidermis/metabolism/pathology
*Polymorphism, Single Nucleotide
Homeostasis/genetics
*Skin Diseases/genetics
Genetic Predisposition to Disease
*DNA/metabolism/genetics
Multifactorial Inheritance/genetics
Promoter Regions, Genetic
Alleles
CRISPR-Cas Systems