@article {pmid40849301,
year = {2025},
author = {Buonaiuto, G and Desideri, F and Setti, A and Palma, A and D'Angelo, A and Storari, G and Santini, T and Laneve, P and Trisciuoglio, D and Ballarino, M},
title = {LncRNA HSCHARME is altered in human cardiomyopathies and promotes stem cell-derived cardiomyogenesis via splicing regulation.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {7880},
pmid = {40849301},
issn = {2041-1723},
support = {RM123188F6B80CE4//Sapienza Università di Roma (Sapienza University of Rome)/ ; AR223188B40CB2D0//Sapienza Università di Roma (Sapienza University of Rome)/ ; B83C22002870006//Ministero dell'Istruzione, dell'Università e della Ricerca (Ministry of Education, University and Research)/ ; B53D23016090006//Ministero dell'Istruzione, dell'Università e della Ricerca (Ministry of Education, University and Research)/ ; B53D23026140001//Ministero dell'Istruzione, dell'Università e della Ricerca (Ministry of Education, University and Research)/ ; B83C22002860006//Ministero dell'Istruzione, dell'Università e della Ricerca (Ministry of Education, University and Research)/ ; B53D23016090006//Ministero dell'Istruzione, dell'Università e della Ricerca (Ministry of Education, University and Research)/ ; DBA.AD005.225-NUTRAGE-FOE2021//Consiglio Nazionale delle Ricerche (National Research Council)/ ; DSB.AD006.371-InvAt-FOE2022//Consiglio Nazionale delle Ricerche (National Research Council)/ ; },
mesh = {Humans ; *RNA, Long Noncoding/genetics/metabolism ; *Myocytes, Cardiac/metabolism/cytology ; Polypyrimidine Tract-Binding Protein/metabolism/genetics ; Animals ; Cell Differentiation/genetics ; Induced Pluripotent Stem Cells/metabolism/cytology ; Mice ; *Cardiomyopathies/genetics/metabolism/pathology ; *RNA Splicing/genetics ; Heterogeneous-Nuclear Ribonucleoproteins/metabolism/genetics ; CRISPR-Cas Systems ; },
abstract = {A growing body of evidence suggests that tissue-specific lncRNAs play pivotal roles in the heart. Here, we exploit the synteny between the mouse and human genomes to identify the human lncRNA HSCHARME and combine single-cell transcriptomics, CAGE-seq data, RNA-FISH imaging and CRISPR/Cas9 genome editing to document its role in cardiomyogenesis. By investigating the mechanism of action of HSCHARME in hiPSC-derived cardiomyocytes, we report that the locus produces the major pCHARME isoform that associates with SC35-containing speckles and interacts with the splicing regulator PTBP1. Consistently, the functional inactivation of pCHARME influences the splicing of cardiac-specific pre-mRNAs and impacts their expression, which reflects a decline in cardiomyocyte differentiation and physiology. In line with a possible association with disease, large-scale analysis of the lncRNA expression across cardiomyopathy patients reveals increased levels of pCHARME in hypertrophic and dilated hearts. We also find that HSCHARME dosage can modulate the expression of a subset of disease-associated targets. Our findings provide mechanistic insights into the role of pCHARME in cardiac cells with potential implications for disease.},
}

Humans
*RNA, Long Noncoding/genetics/metabolism
*Myocytes, Cardiac/metabolism/cytology
Polypyrimidine Tract-Binding Protein/metabolism/genetics
Animals
Cell Differentiation/genetics
Induced Pluripotent Stem Cells/metabolism/cytology
Mice
*Cardiomyopathies/genetics/metabolism/pathology
*RNA Splicing/genetics
Heterogeneous-Nuclear Ribonucleoproteins/metabolism/genetics
CRISPR-Cas Systems

@article {pmid40848256,
year = {2025},
author = {Guo, JA and Gong, D and Evans, K and Takahashi, K and Jambhale, AD and Shiau, C and Yu, P and Wang, S and Wu, WW and Chugh, S and Kapner, KS and Dilly, J and Zhao, D and Chen, P and Smith, EL and Mancias, JD and Vazquez, F and Singh, H and Hwang, WL and Aguirre, AJ},
title = {Integrative genomic identification of therapeutic targets for pancreatic cancer.},
journal = {Cell reports},
volume = {44},
number = {9},
pages = {116191},
doi = {10.1016/j.celrep.2025.116191},
pmid = {40848256},
issn = {2211-1247},
abstract = {Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease, and new therapeutic strategies are urgently needed. Here, we conduct an integrative, genome-scale examination of genetic dependencies and cell surface targets using CRISPR-Cas screening and multi-omic data, including single-nucleus and spatial transcriptomic data from patient tumors. We systematically identify clinically tractable and biomarker-linked PDAC dependencies, including CDS2 as a synthetic lethal target in cancer cells expressing signatures of epithelial-to-mesenchymal transition. We examine biomarkers and co-dependencies of the KRAS oncogene, defining gene expression signatures of sensitivity and resistance associated with response to pharmacological inhibition of KRAS. mRNA and protein profiling reveal cell surface protein-encoding genes with robust expression in patient tumors and minimal expression in non-malignant tissues. Furthermore, we define intratumoral and interpatient heterogeneity of target gene expression and identify orthogonal targets that suggest combinatorial strategies. Collectively, this work identifies multiple targets that may inform therapeutic strategies for patients with PDAC.},
}

@article {pmid40845808,
year = {2025},
author = {Yang, KK and Amini, AP},
title = {Simplifying protein engineering with deep learning.},
journal = {Cell},
volume = {188},
number = {17},
pages = {4477-4479},
doi = {10.1016/j.cell.2025.07.037},
pmid = {40845808},
issn = {1097-4172},
mesh = {*Deep Learning ; *Protein Engineering/methods ; Gene Editing/methods ; Humans ; CRISPR-Cas Systems ; Proteins/genetics ; },
abstract = {When it comes to deep learning for protein engineering, there is strength in simplicity. In this issue of Cell, through thoughtful deployment of existing fixed-backbone sequence design models, Caixia Gao and colleagues engineer diverse genome editing systems with improved functionality, enabling powerful capabilities in fine-grained and large-scale genome editing as demonstrated through strong experimental validation.},
}

*Deep Learning
*Protein Engineering/methods
Gene Editing/methods
Humans
CRISPR-Cas Systems
Proteins/genetics

@article {pmid40842235,
year = {2025},
author = {Moon, SY and Kim, MR and An, NY and Noh, MH and Lee, JY},
title = {Dual-mode CRISPRa/i for genome-scale metabolic rewiring in Escherichia coli.},
journal = {Nucleic acids research},
volume = {53},
number = {15},
pages = {},
doi = {10.1093/nar/gkaf818},
pmid = {40842235},
issn = {1362-4962},
support = {//Ministry of Science and ICT/ ; //National Research Foundation/ ; 2022M3A9B6082671//Republic of Korea: Bio & Medical Technology Development Program/ ; 2024M3J5A1045980//Artificial Cellular Organelles Project/ ; //Korea-US Collaborative Research Fund (KUCRF)/ ; RS-2024-00468410//MSIT and the Ministry of Health & Welfare, Republic of Korea/ ; },
mesh = {*Escherichia coli/genetics/metabolism ; Cyclic AMP Receptor Protein/genetics/metabolism ; *CRISPR-Cas Systems ; Gene Expression Regulation, Bacterial ; *Escherichia coli Proteins/genetics/metabolism ; *Metabolic Engineering/methods ; Genome, Bacterial ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {CRISPR (clustered regularly interspaced palindromic repeats)-mediated transcriptional regulation is a powerful and programmable approach for controlling gene expression. While CRISPR-based gene repression is well established in bacteria, simultaneous activation and repression remain challenging due to the limited availability of effective bacterial activation domains. Here, we provide an efficient dual-mode CRISPR activation and interference (CRISPRa/i) system that integrates an evolved protospacer adjacent motif (PAM)-flexible dxCas9 with an engineered Escherichia coli cAMP receptor protein (CRP). Through systematic optimization of the CRP domains and linkers, we developed a versatile effector capable of precise gene expression control when combined with dxCas9. Our dxCas9-CRP system demonstrated robust activation of upstream regulatory regions and effective repression of coding sequences, enabling targeted and programmable gene regulation. Using dual-fluorescent reporters, we validated the ability of this system to concurrently regulate multiple genes. Furthermore, with pooled guide RNA libraries, we applied the dxCas9-CRP system to increase violacein production in E. coli via genome-scale activation and repression in a coordinated manner, successfully identifying key regulatory targets that significantly increase production. Overall, this dual-mode CRISPRa/i system advances the potential for bacterial metabolic pathway rewiring, providing precise and flexible control for a wide range of biotechnological applications.},
}

*Escherichia coli/genetics/metabolism
Cyclic AMP Receptor Protein/genetics/metabolism
*CRISPR-Cas Systems
Gene Expression Regulation, Bacterial
*Escherichia coli Proteins/genetics/metabolism
*Metabolic Engineering/methods
Genome, Bacterial
RNA, Guide, CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40841511,
year = {2025},
author = {Potts, MA and Mizutani, S and Deng, Y and Vaidyanathan, S and Ting, KE and Giner, G and Sridhar, S and Shenoy, G and Liao, Y and Diepstraten, ST and Kueh, AJ and Pal, M and Healey, G and Tai, L and Wang, Z and König, C and Kaloni, D and Whelan, L and Milevskiy, MJG and Coughlan, HD and Pomilio, G and Wei, AH and Visvader, JE and Papenfuss, AT and Wilcox, S and Jeyasekharan, AD and Shi, W and Lelliott, EJ and Kelly, GL and Brown, KK and Strasser, A and Herold, MJ},
title = {Genome-wide in vivo CRISPR screens identify GATOR1 complex as a tumor suppressor in Myc-driven lymphoma.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {7582},
pmid = {40841511},
issn = {2041-1723},
mesh = {Animals ; *Lymphoma/genetics/metabolism/pathology ; Mice ; Mechanistic Target of Rapamycin Complex 1/metabolism/genetics ; *Proto-Oncogene Proteins c-myc/genetics/metabolism ; Humans ; CRISPR-Cas Systems ; TOR Serine-Threonine Kinases/metabolism ; *Genes, Tumor Suppressor ; Mice, Knockout ; Clustered Regularly Interspaced Short Palindromic Repeats ; Cell Line, Tumor ; Signal Transduction ; Tumor Suppressor Proteins/genetics/metabolism ; GTPase-Activating Proteins/genetics/metabolism ; Mice, Transgenic ; },
abstract = {Identifying tumor suppressor genes is predicted to inform on the development of novel strategies for cancer therapy. To identify new lymphoma driving processes that cooperate with oncogenic MYC, which is abnormally highly expressed in ~70% of human cancers, we use a genome-wide CRISPR gene knockout screen in Eµ-Myc;Cas9 transgenic hematopoietic stem and progenitor cells in vivo. We discover that loss of any of the GATOR1 complex components - NPRL3, DEPDC5, NPRL2 - significantly accelerates c-MYC-driven lymphoma development in mice. MYC-driven lymphomas lacking GATOR1 display constitutive mTOR pathway activation and are highly sensitive to mTOR inhibitors, both in vitro and in vivo. These findings identify GATOR1 suppression of mTORC1 as a tumor suppressive mechanism in MYC-driven lymphomagenesis and suggest an avenue for therapeutic intervention in GATOR1-deficient lymphomas through mTOR inhibition.},
}

Animals
*Lymphoma/genetics/metabolism/pathology
Mice
Mechanistic Target of Rapamycin Complex 1/metabolism/genetics
*Proto-Oncogene Proteins c-myc/genetics/metabolism
Humans
CRISPR-Cas Systems
TOR Serine-Threonine Kinases/metabolism
*Genes, Tumor Suppressor
Mice, Knockout
Clustered Regularly Interspaced Short Palindromic Repeats
Cell Line, Tumor
Signal Transduction
Tumor Suppressor Proteins/genetics/metabolism
GTPase-Activating Proteins/genetics/metabolism
Mice, Transgenic

@article {pmid40838896,
year = {2025},
author = {Losier, TT and King, KE and Rousseaux, MWC and Russell, RC},
title = {Identification of organelle-specific autophagy regulators from tandem CRISPR screens.},
journal = {The Journal of cell biology},
volume = {224},
number = {10},
pages = {},
doi = {10.1083/jcb.202405138},
pmid = {40838896},
issn = {1540-8140},
support = {#153034/CAPMC/CIHR/Canada ; PJT-169097/CAPMC/CIHR/Canada ; #2023-05587//Natural Sciences and Engineering Research Council of Canada/ ; RGPIN-2019-04133//Natural Sciences and Engineering Research Council of Canada/ ; DGECR-2019-00369//Natural Sciences and Engineering Research Council of Canada/ ; 201911CGV-434032-74238//Natural Sciences and Engineering Research Council of Canada/ ; //Natural Sciences and Engineering Research Council Postgraduate Scholarship/ ; },
mesh = {*Autophagy/genetics ; Humans ; Signal Transduction ; Endoplasmic Reticulum/metabolism/genetics ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Organelles/metabolism ; *Autophagy-Related Proteins/metabolism/genetics ; HeLa Cells ; },
abstract = {Autophagy is a conserved degradative process that promotes cellular homeostasis under stress conditions. Under nutrient starvation, autophagy is nonselective, promoting indiscriminate breakdown of cytosolic components. Conversely, selective autophagy is responsible for the specific turnover of damaged organelles. We hypothesized that selective autophagy may be regulated by signaling pathways distinct from those controlling starvation-induced autophagy, thereby promoting organelle turnover. To address this question, we conducted kinome-wide CRISPR screens to identify distinct signaling pathways responsible for the regulation of basal autophagy, starvation-induced autophagy, and two types of selective autophagy, ER-phagy and pexophagy. These parallel screens identified both known and novel autophagy regulators, some common to all conditions and others specific to selective autophagy. More specifically, CDK11A and NME3 were further characterized to be selective ER-phagy regulators. Meanwhile, PAN3 and CDC42BPG were identified as an activator and inhibitor of pexophagy, respectively. Collectively, these datasets provide the first comparative description of the kinase signaling that defines the regulation of selective autophagy and bulk autophagy.},
}

*Autophagy/genetics
Humans
Signal Transduction
Endoplasmic Reticulum/metabolism/genetics
*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*Organelles/metabolism
*Autophagy-Related Proteins/metabolism/genetics
HeLa Cells

@article {pmid40828859,
year = {2025},
author = {Saunier, M and Humbert, A and Kreis, V and Peltier, J and Tisba, A and Auxilien, S and Blum, M and Caldelari, I and Lucier, JF and Ueda, J and Gautheret, D and Toffano-Nioche, C and Andreani, J and Fortier, LC and Soutourina, O},
title = {Deciphering the RNA-based regulation mechanism of the phage-encoded AbiF system in Clostridioides difficile.},
journal = {PLoS genetics},
volume = {21},
number = {8},
pages = {e1011831},
doi = {10.1371/journal.pgen.1011831},
pmid = {40828859},
issn = {1553-7404},
mesh = {*Clostridioides difficile/genetics/virology/pathogenicity ; Prophages/genetics ; CRISPR-Cas Systems/genetics ; *Bacteriophages/genetics ; Toxin-Antitoxin Systems/genetics ; Clostridium Infections/microbiology/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Bacterial Proteins/genetics ; Humans ; RNA, Bacterial/genetics ; Gene Expression Regulation, Bacterial ; },
abstract = {Clostridioides difficile is the major cause of nosocomial infections associated with antibiotic therapy. The severity of C. difficile infections increased worldwide with the emergence of hypervirulent strains, including 027 ribotype epidemic strains. Many aspects of C. difficile adaptation strategies during pathogenesis remain poorly understood. This pathogen thrives in gut communities that are rich in microbes and phages. To regulate horizontal transfer of genetic material during its infection cycle, C. difficile relies on diverse mechanisms. More specifically, CRISPR (clustered regularly interspaced short palindromic repeats)-Cas and Toxin-Antitoxin (TA) systems contribute to prophage maintenance, prevention of phage infection, and stress response. Abortive infection (Abi) systems can provide additional lines of anti-phage defense. RNAs have emerged as key components of these systems including CRISPR RNAs and antitoxin RNAs within type I and type III TA. We report here the identification of a new AbiF-like system within a prophage of the hypervirulent C. difficile strain R20291. It is associated with an Abi_2/AbiD/F protein family largely distributed in Bacillota and Pseudomonadota with structural links to ancestral Cas13 proteins at the origin of the RNA-targeting CRISPR-Cas13 systems. We demonstrated toxic activity of the AbiFCd protein in C. difficile and in Escherichia coli and negative regulation of the abiFCd expression by an associated non-coding RNA RCd22. RCd22 contains two conserved abiF motifs and is active both in cis and in trans to neutralize the toxin by direct RNA-protein interaction, similar to RNA antitoxin in type III TA. A mass spectrometry interactomics analysis of protein fractions from MS2-Affinity Purification coupled with RNA sequencing (MAPS) revealed the AbiFCd protein among the most enriched RCd22 partners in C. difficile. Structural modeling of the RNA-protein complex and mutagenesis analysis revealed key positions on both protein and RNA partners for this interaction and toxic activity. In summary, these findings provide valuable insights into the mechanisms of interaction between bacteria and phages, which are pertinent to the advancement of phage therapy, genome editing, epidemiological surveillance, and the formulation of novel therapeutic approaches.},
}

*Clostridioides difficile/genetics/virology/pathogenicity
Prophages/genetics
CRISPR-Cas Systems/genetics
*Bacteriophages/genetics
Toxin-Antitoxin Systems/genetics
Clostridium Infections/microbiology/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Bacterial Proteins/genetics
Humans
RNA, Bacterial/genetics
Gene Expression Regulation, Bacterial

@article {pmid40728278,
year = {2025},
author = {Joung, Y and Han, DK and Jang, H and Kang, T and Chen, L and Choo, J},
title = {Dual-Pathway Lateral Flow Assay for Rapid and Sensitive SARS-CoV-2 RNA Detection via CRISPR/Cas13a-Mediated SERS.},
journal = {ACS sensors},
volume = {10},
number = {8},
pages = {6253-6262},
doi = {10.1021/acssensors.5c02084},
pmid = {40728278},
issn = {2379-3694},
mesh = {*Spectrum Analysis, Raman/methods ; *SARS-CoV-2/genetics/isolation & purification ; *RNA, Viral/analysis/genetics ; *CRISPR-Cas Systems/genetics ; Humans ; *COVID-19/diagnosis/virology ; *COVID-19 Nucleic Acid Testing/methods ; },
abstract = {Reverse transcription-polymerase chain reaction (RT-PCR) has been the gold standard for SARS-CoV-2 detection during the COVID-19 pandemic. However, its requirement for RNA-to-DNA conversion, reliance on centralized laboratory infrastructure, and lengthy turnaround times have limited its application in point-of-care (POC) settings. CRISPR/Cas13a-mediated lateral flow assays (LFAs) have emerged as promising alternatives for direct RNA analysis, yet their two-step workflows introduce procedural complexity and reduce sensitivity. To overcome these limitations, we developed a dual-pathway LFA strip based on surface-enhanced Raman scattering (SERS), which integrates CRISPR/Cas13a-mediated RNA cleavage and SERS detection into a single, portable platform. The device utilizes five vertically stacked paper layers with distinct geometries, enabling sequential CRISPR reaction and SERS quantification through two independent pathways. When tested with SARS-CoV-2 ORF1ab RNA targets, the system exhibited an 80-fold increase in sensitivity and a 10 min reduction in assay time compared to conventional fluorescence assays. Clinical validation using 18 samples (13 positives and 5 negatives) demonstrated high diagnostic accuracy, fully consistent with RT-PCR results. By unifying CRISPR-based RNA recognition and SERS signal amplification in a user-friendly format, this dual-pathway LFA strip offers a rapid, ultrasensitive, and practical diagnostic tool for infectious diseases in POC settings.},
}

*Spectrum Analysis, Raman/methods
*SARS-CoV-2/genetics/isolation & purification
*RNA, Viral/analysis/genetics
*CRISPR-Cas Systems/genetics
Humans
*COVID-19/diagnosis/virology
*COVID-19 Nucleic Acid Testing/methods

@article {pmid40675426,
year = {2025},
author = {Li, W and Tang, Z and Zhu, X and Wang, B and Zhang, X and Ji, Z and Cheng, S},
title = {Ultrasensitive detection of MMP-2 via T7 RNA polymerase and CRISPR/Cas13a-Enhanced electrochemiluminescence biosensor for COPD diagnosis.},
journal = {Methods (San Diego, Calif.)},
volume = {242},
number = {},
pages = {80-88},
doi = {10.1016/j.ymeth.2025.07.005},
pmid = {40675426},
issn = {1095-9130},
mesh = {*Matrix Metalloproteinase 2/analysis/blood/genetics ; *Biosensing Techniques/methods ; Humans ; CRISPR-Cas Systems/genetics ; Limit of Detection ; Luminescent Measurements/methods ; *Electrochemical Techniques/methods ; *Pulmonary Disease, Chronic Obstructive/diagnosis/blood ; *DNA-Directed RNA Polymerases/genetics/metabolism ; *Viral Proteins/genetics/metabolism ; Metal Nanoparticles/chemistry ; Gold/chemistry ; Biomarkers ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {In this work, an electrochemiluminescence (ECL) biosensor integrating T7 RNA polymerase amplification and CRISPR/Cas13a-mediated signal enhancement was developed for the ultrasensitive detection of matrix metalloproteinase-2 (MMP-2), a key biomarker associated with chronic inflammatory diseases such as COPD. A peptide nucleic acid (PNA) probe was designed to respond specifically to MMP-2 cleavage, enabling the release of DNA templates for subsequent T7 RNA polymerase-driven transcription amplification. The generated RNA triggers the collateral cleavage activity of CRISPR/Cas13a, resulting in a significant amplification of the ECL signal. The biosensor's surface was constructed using a AuNPs/Ti3C2Tx/Ru(II)-PEI nanocomposite, which enhanced signal transduction and stability. Under optimized conditions, the proposed biosensor achieved a detection limit as low as 62.05 fM, demonstrating superior sensitivity compared to conventional methods, as summarized in Table 1. The platform also exhibited excellent specificity and anti-interference capability, ensuring reliable detection of MMP-2 in complex biological samples. This study provides a simple yet highly efficient strategy for enzymatic biomarker detection, offering great potential for clinical applications in early disease diagnosis and monitoring.},
}

*Matrix Metalloproteinase 2/analysis/blood/genetics
*Biosensing Techniques/methods
Humans
CRISPR-Cas Systems/genetics
Limit of Detection
Luminescent Measurements/methods
*Electrochemical Techniques/methods
*Pulmonary Disease, Chronic Obstructive/diagnosis/blood
*DNA-Directed RNA Polymerases/genetics/metabolism
*Viral Proteins/genetics/metabolism
Metal Nanoparticles/chemistry
Gold/chemistry
Biomarkers
Nucleic Acid Amplification Techniques/methods

@article {pmid40626419,
year = {2025},
author = {Jacobebbinghaus, N and Bigge, F and Saudhof, M and Hübner, W and Kruse, O and Baier, T},
title = {Transcriptional gene fusions via targeted integration at safe harbors for high transgene expression in Chlamydomonas reinhardtii.},
journal = {The New phytologist},
volume = {247},
number = {6},
pages = {2665-2677},
doi = {10.1111/nph.70368},
pmid = {40626419},
issn = {1469-8137},
support = {2221NR063X//Federal Ministry of Agriculture, Food and Regional Identity/ ; },
mesh = {*Chlamydomonas reinhardtii/genetics/metabolism ; *Transgenes/genetics ; *Gene Fusion ; *Transcription, Genetic ; CRISPR-Cas Systems/genetics ; Gene Editing ; Terpenes/metabolism ; },
abstract = {Conventional genetic engineering in green microalgae employs error-prone nonhomologous end joining to integrate recombinant DNA at double-strand breaks generated at random positions across the nuclear genome. This typically results in variable transcription strength and requires a labor-intensive screening procedure to identify transformants with sufficient expression. Current advances in genome editing enable scar-less integration of DNA at any desired locus for engineered bioproduction. We optimized construct design for predictable transgene expression at a high level, significantly improved scar-less integration rates into the nuclear genome via homology arm length optimization and quantified endogenous gene expression in vivo. Subsequently, endogenous genes were successfully targeted via Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein 9 (CRISPR/Cas9) to evaluate their capacity for high transgene expression and terpenoid production. Highest scar-less homology-directed repair efficiency was achieved with 50 bp homology arms. The Light harvesting Chl a/b binding protein of LHCII (LHCBM1) locus was found to be differentially expressed under several light intensities and allows an 8.6-fold increase in transgenic protein accumulation compared with random insertion approaches. Co-expression of a functional sesquiterpene synthase achieved a 60-fold increase in valencene production compared with previous attempts. We showed LHCBM1 locus constitutes a genetic safe harbor for transgene expression and demonstrates the potential of C. reinhardtii as a green cell factory.},
}

*Chlamydomonas reinhardtii/genetics/metabolism
*Transgenes/genetics
*Gene Fusion
*Transcription, Genetic
CRISPR-Cas Systems/genetics
Gene Editing
Terpenes/metabolism

@article {pmid40338215,
year = {2025},
author = {Wu, S and Yuan, J and Xi, X and Wang, L and Li, Y and Wang, Y and Lin, J},
title = {A Colorimetric Biosensor Integrating Rotifer-Mimicking Magnetic Separation with RAA/CRISPR-Cas12a for Rapid and Sensitive Detection of Salmonella.},
journal = {ACS sensors},
volume = {10},
number = {8},
pages = {5473-5483},
doi = {10.1021/acssensors.4c03356},
pmid = {40338215},
issn = {2379-3694},
mesh = {*Salmonella/isolation & purification/genetics ; *Biosensing Techniques/methods ; *Colorimetry/methods ; *CRISPR-Cas Systems ; Limit of Detection ; Milk/microbiology ; *Rotifera/chemistry ; Gold/chemistry ; Animals ; Food Microbiology ; Nucleic Acid Amplification Techniques ; Metal Nanoparticles/chemistry ; Food Contamination/analysis ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Efficient detection of foodborne bacteria is crucial for ensuring food safety, yet current methods often fall short in balancing speed, accuracy, sensitivity, and cost. This study presents an integrated biosensing platform for the rapid and sensitive detection of Salmonella in large-volume food samples. The platform incorporates a Rotifer-Mimicking Magnetic Separator (RMMS) that enhances the sample pretreatment by effectively mixing and isolating the bacteria from the sample. Coupled with this, the colorimetric biosensor utilizes a streamlined one-pot system that combines Recombinase Aided Amplification (RAA), betaine, and CRISPR-Cas12a to enable efficient pathogen detection. Initially, phenylboronic acid-modified magnetic beads (PBA-MBs) capture Salmonella, forming bacteria-PBA-MB complexes, which are then isolated using the RMMS. Target DNA amplicons activate ribonucleoprotein complexes, and Au@PtNPs-MBs with linker single DNAs are cleaved to release Au@PtNPs. The Au@PtNPs catalyze the H2O2-3,3',5,5'-tetramethylbenzidine, producing a visible blue color that indicates Salmonella concentration. This biosensor successfully detects Salmonella in 40 mL spiked milk samples within 75 min, achieving a detection limit of 89 CFU/mL. This work offers a simple, sensitive, low-cost detection method with potential applications in on-site testing, significantly enhancing food safety monitoring.},
}

*Salmonella/isolation & purification/genetics
*Biosensing Techniques/methods
*Colorimetry/methods
*CRISPR-Cas Systems
Limit of Detection
Milk/microbiology
*Rotifera/chemistry
Gold/chemistry
Animals
Food Microbiology
Nucleic Acid Amplification Techniques
Metal Nanoparticles/chemistry
Food Contamination/analysis
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40838720,
year = {2025},
author = {Ferronato, GA and Silveira, JC and Ferraz, MAMM},
title = {Potential of small extracellular vesicles as Cas9 delivery tool: A promising approach for gene editing livestock gametes and embryos.},
journal = {Biology of reproduction},
volume = {},
number = {},
pages = {},
doi = {10.1093/biolre/ioaf195},
pmid = {40838720},
issn = {1529-7268},
abstract = {Genome editing is a rapidly advancing technology with transformative potential in livestock, offering opportunities that range from enhanced production traits to the generation of biomedical models for human disease and xenotransplantation. The CRISPR/Cas9 system, originally identified as a bacterial defense mechanism, has become the most widely used tool for precise genome editing. In this review, we first summarize the potential applications of CRISPR/Cas9 in livestock and highlight notable successes to date. We then address the ongoing challenges associated with delivering CRISPR/Cas9 into gametes and embryos, as current methods such as microinjection and electroporation often result in high mosaicism and cellular damage. We subsequently introduce extracellular vesicles (EVs) as a promising alternative delivery system. Secreted by virtually all cell types, EVs can efficiently transport bioactive molecules and are readily internalized by gametes and embryos. Although EV-mediated delivery of CRISPR/Cas9 has shown success in somatic cells, its use in reproductive cells remains largely unexplored. We review emerging strategies for loading EVs with CRISPR/Cas components and discuss the potential advantages of combining this approach with recently developed smaller Cas variants to overcome delivery barriers. Collectively, these innovations support the promise of EVs as a biologically compatible, efficient, and minimally invasive system for targeted genome editing in livestock reproduction.},
}

@article {pmid40838475,
year = {2025},
author = {},
title = {RETRACTION: Spider Eye Development Editing and Silk Fiber Engineering Using CRISPR-Cas.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {},
number = {},
pages = {e202517416},
doi = {10.1002/anie.202517416},
pmid = {40838475},
issn = {1521-3773},
}

@article {pmid40835841,
year = {2025},
author = {Watson, LC and Sala, KA and Bernitz, N and Baumgärtel, L and Pallett, MA and Marzook, NB and Straker, LC and Peng, D and Collinson, L and Sateriale, A},
title = {Targeted CRISPR screens reveal genes essential for Cryptosporidium survival in the host intestine.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {7749},
pmid = {40835841},
issn = {2041-1723},
support = {CC2063/WT_/Wellcome Trust/United Kingdom ; CC2063/WT_/Wellcome Trust/United Kingdom ; CC2063/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Animals ; *Cryptosporidiosis/parasitology ; *Cryptosporidium/genetics/pathogenicity ; *CRISPR-Cas Systems ; *Intestines/parasitology ; Mice ; *Protozoan Proteins/genetics/metabolism ; *Genes, Essential ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Humans ; Gene Knockout Techniques ; Cryptosporidium parvum/genetics ; Protozoan Vaccines/immunology ; },
abstract = {The Cryptosporidium parasite is one of the leading causes of diarrheal morbidity and mortality in children, and adolescent infections are associated with chronic malnutrition. There are no vaccines available for protection and only one drug approved for treatment that has limited efficacy. A major barrier to developing new therapeutics is a lack of foundational knowledge of Cryptosporidium biology, including which parasite genes are essential for survival and virulence. Here, we iteratively improve the tools for genetically manipulating Cryptosporidium and develop a targeted CRISPR-based screening method to rapidly assess how the loss of individual parasite genes influence survival in vivo. Using this method, we examine the parasite's pyrimidine salvage pathway and a set of leading Cryptosporidium vaccine candidates. From this latter group, using inducible knockout, we determined the parasite gene known as Cp23 to be essential for survival in vivo. Parasites deficient in Cp23 were able to replicate within and emerge from infected epithelial cells, yet unable to initiate gliding motility which is required for the reinfection of neighbouring cells. The targeted screening method presented here is highly versatile and will enable researchers to more rapidly expand the knowledge base for Cryptosporidium infection biology, paving the way for new therapeutics.},
}

Animals
*Cryptosporidiosis/parasitology
*Cryptosporidium/genetics/pathogenicity
*CRISPR-Cas Systems
*Intestines/parasitology
Mice
*Protozoan Proteins/genetics/metabolism
*Genes, Essential
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Humans
Gene Knockout Techniques
Cryptosporidium parvum/genetics
Protozoan Vaccines/immunology

@article {pmid40835811,
year = {2025},
author = {Luzics, S and Baka, E and Otto, M and Kosztik, J and Szalontai, H and Bata-Vidács, I and Nagy, I and Tóth, Á and Táncsics, A and Pápai, M and Nagy, I and Orsini, M and Kukolya, J},
title = {High-quality de novo genome assembly and functional genomic insights into Thermobifida alba DSM43795[T], a mesophilic actinobacterium isolated from garden soil.},
journal = {Biologia futura},
volume = {},
number = {},
pages = {},
pmid = {40835811},
issn = {2676-8607},
support = {K142686//National Research, Development and Innovation Office/ ; EKÖP- 24- VI/MATE-3//Ministry for Culture and Innovation from the source of the National Research, Development and Innovation Fund/ ; EKÖP-MATE/2024/25/D//Ministry for Culture and Innovation from the source of the National Research, Development and Innovation Fund/ ; },
abstract = {Thermobifida alba DSM43795[T], a mesophilic actinobacterium isolated from garden soil, plays a vital role in lignocellulose degradation and holds biotechnological and pharmaceutical potential. We present a high-quality, complete de novo genome assembly of T. alba DSM43795[T] using combined PacBio long-read and Illumina short-read sequencing, resulting in a single circular chromosome of 4.9 Mbp with 72.1% GC content. Comparative genomics with the thermophilic relative T. fusca YX revealed 83.39% average nucleotide identity and extensive genome synteny alongside niche-specific differences. Functional annotation identified 4345 genes, including a rich complement of carbohydrate-active enzymes (CAZymes) such as glycoside hydrolases (GHs), esterases, and polysaccharide lyases, supporting versatile plant biomass degradation. GH gene sets were largely conserved between the species in both gene number and distribution, but T. alba uniquely encodes a novel GH10 endo-xylanase near a characterised palindrome regulatory sequence, indicating species-specific regulation. We hypothesise that thermophilic adaptation in T. fusca requires more proteins for ribosome integrity and amino acid metabolism, with reduced emphasis on carbohydrate metabolism and defence compared to T. alba. Moreover, T. alba harbours a broader array of defence-related genes and mobile genetic elements, including integrases and transposases. Although lacking a complete CRISPR-Cas system, two CRISPR arrays were detected, suggesting alternative immune strategies. Virulence factor homologs shared by both species likely reflect environmental survival rather than pathogenicity. This genomic characterisation elucidates T. alba's metabolic versatility and ecological adaptations, laying the groundwork for its potential applications in biomass conversion, environmental biotechnology, and drug discovery.},
}

@article {pmid40835333,
year = {2025},
author = {Sanders, J and Lin, S},
title = {Molecular techniques for understanding harmful algal blooms: A review.},
journal = {Harmful algae},
volume = {148},
number = {},
pages = {102909},
doi = {10.1016/j.hal.2025.102909},
pmid = {40835333},
issn = {1878-1470},
mesh = {*Harmful Algal Bloom ; Dinoflagellida/classification/genetics ; Cyanobacteria/classification/genetics ; Seawater ; Fresh Water ; *Water Pollution/analysis/prevention & control ; Molecular Probe Techniques ; Enzyme-Linked Immunosorbent Assay ; Molecular Diagnostic Techniques ; CRISPR-Cas Systems ; In Situ Hybridization, Fluorescence ; DNA Barcoding, Taxonomic ; *Molecular Typing/methods ; },
abstract = {Harmful algal blooms (HABs) are intricate ecological events caused by diverse algal species and are influenced by a myriad of biotic and abiotic factors. The urgently needed development of effective prevention and control techniques face two primary challenges. The first challenge is the technical shortfalls in rapidly identifying and monitoring the causative species. The second challenge is the absence of research frameworks and technologies for accurately diagnosing the primary drivers of these blooms. Molecular techniques offer promising solutions to these issues, and research in this field has seen significant growth over the past two decades. Previous reviews have predominantly focused on species identification and monitoring, leaving the status of bloom driver studies less clear. This review provides a comprehensive overview of molecular techniques for HAB identification and driver analysis. HAB-specific use cases of techniques and comparison between them based on technical specifications are provided. Nucleic acid-based techniques presently dominate over antibody-based techniques due to their tunable taxon-specificity and ease to prepare probes. In situ applications and monitoring platforms still have a large room for improvement. The omics approach is the most promising choice for unraveling HAB drivers but requires a framework and a quantitative model for estimating the contribution of potential responsible factors. Future prospects relating to particular needs in HAB research and emerging technologies are also discussed.},
}

*Harmful Algal Bloom
Dinoflagellida/classification/genetics
Cyanobacteria/classification/genetics
Seawater
Fresh Water
*Water Pollution/analysis/prevention & control
Molecular Probe Techniques
Enzyme-Linked Immunosorbent Assay
Molecular Diagnostic Techniques
CRISPR-Cas Systems
In Situ Hybridization, Fluorescence
DNA Barcoding, Taxonomic
*Molecular Typing/methods

@article {pmid40782800,
year = {2025},
author = {Traxler, P and Reichl, S and Folkman, L and Shaw, L and Fife, V and Nemc, A and Pasajlic, D and Kusienicka, A and Barreca, D and Fortelny, N and Rendeiro, AF and Halbritter, F and Weninger, W and Decker, T and Farlik, M and Bock, C},
title = {Integrated time-series analysis and high-content CRISPR screening delineate the dynamics of macrophage immune regulation.},
journal = {Cell systems},
volume = {16},
number = {8},
pages = {101346},
doi = {10.1016/j.cels.2025.101346},
pmid = {40782800},
issn = {2405-4720},
mesh = {*Macrophages/immunology/metabolism ; Animals ; Mice ; Signal Transduction ; CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Expression Regulation ; },
abstract = {Macrophages are innate immune cells involved in host defense. Dissecting the regulatory landscape that enables their swift and specific response to pathogens, we performed time-series analysis of gene expression and chromatin accessibility in murine macrophages exposed to various immune stimuli, and we functionally evaluated gene knockouts at scale using a combined CROP-seq and CITE-seq assay. We identified new roles of transcription regulators such as Spi1/PU.1 and JAK-STAT pathway members in immune cell homeostasis and response to pathogens. Macrophage activity was modulated by splicing proteins SFPQ and SF3B1, histone acetyltransferase EP300, cohesin subunit SMC1A, and mediator complex proteins MED8 and MED14. We further observed crosstalk among immune signaling pathways and identified molecular drivers of pathogen-induced dynamics. In summary, this study establishes a time-resolved regulatory map of pathogen response in macrophages, and it describes a broadly applicable method for dissecting immune-regulatory programs through integrative time-series analysis and high-content CRISPR screening. A record of this paper's transparent peer review process is included in the supplemental information.},
}

*Macrophages/immunology/metabolism
Animals
Mice
Signal Transduction
CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Gene Expression Regulation

@article {pmid40781772,
year = {2025},
author = {Kweon, J and Park, S and Jeon, MY and Lim, K and Jang, G and Jang, AH and Lee, M and Seok, C and Lee, C and Park, S and Ahn, J and Jang, J and Kim, N and Sung, YH and Kim, D and Kim, Y},
title = {High-efficiency base editing for nuclear and mitochondrial DNA with an optimized DYW-like deaminase.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ymthe.2025.08.007},
pmid = {40781772},
issn = {1525-0024},
abstract = {CRISPR-based cytosine base editors enable precise genome editing without inducing double-stranded DNA breaks yet traditionally depend on a limited selection of deaminases from the APOBEC/AID or TadA families. Here, we present SsCBE, a CRISPR-based cytosine base editor utilizing SsdAtox, a DYW-like deaminase derived from the toxin of Pseudomonas syringae. Strategic engineering of SsdAtox has led to remarkable improvements in the base editing efficiency (by up to 8.4-fold) and specificity for SsCBE, while concurrently reducing cytotoxicity. Exhibiting exceptional versatility, SsCBE was delivered and efficiently applied using diverse delivery methods, including engineered virus-like particles. Its application has enabled targeted cytosine base editing in mouse zygotes and pioneering edits in mitochondrial DNA. SsCBE expands the genome editing toolbox by introducing a distinct deaminase scaffold with broad utility for both basic research and potential therapeutic applications.},
}

@article {pmid40763746,
year = {2025},
author = {Sun, Q and Ma, X and Ning, Q and Li, S and Wang, P and Tan, X and Jin, Q and Zheng, J and Li, Y and Dong, D},
title = {Systematic screening for functional exon-skipping isoforms using the CRISPR-RfxCas13d system.},
journal = {Cell systems},
volume = {16},
number = {8},
pages = {101351},
doi = {10.1016/j.cels.2025.101351},
pmid = {40763746},
issn = {2405-4720},
mesh = {Humans ; *Exons/genetics ; Protein Isoforms/genetics ; *Alternative Splicing/genetics ; Colorectal Neoplasms/genetics/pathology ; *CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Animals ; Mice ; RNA, Guide, CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cell Proliferation/genetics ; },
abstract = {Exon skipping (ES) is the most prevalent form of alternative splicing and a hallmark of tumorigenesis, yet its functional roles remain underexplored. Here, we present a CRISPR-RfxCas13d-based platform for transcript-specific silencing of ES-derived isoforms using guide RNAs (gRNAs) targeting exon-exon junctions. We designed a transcriptome-wide gRNA library against 3,744 human ES events and conducted loss-of-function screens in colorectal cancer (CRC) cells in vitro and in vivo. This screen uncovered multiple ES events essential for CRC growth, notably HMGN3 Δ6, an isoform arising from exon 6 skipping, which enhanced tumor proliferation. Functional validation confirmed the oncogenic role of HMGN3 Δ6 and its necessity for CRC progression. Our study establishes CRISPR-RfxCas13d as a powerful tool for isoform-specific functional genomics and reveals a widespread, previously uncharacterized layer of tumor biology driven by ES. These findings position ES-derived transcripts as promising targets for therapeutic intervention in cancer.},
}

Humans
*Exons/genetics
Protein Isoforms/genetics
*Alternative Splicing/genetics
Colorectal Neoplasms/genetics/pathology
*CRISPR-Cas Systems/genetics
Cell Line, Tumor
Animals
Mice
RNA, Guide, CRISPR-Cas Systems/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Cell Proliferation/genetics

@article {pmid40623538,
year = {2025},
author = {Peng, Z and Yang, T and Xu, S and Yang, B and Zhang, Z and Ding, M and Gu, W and Zheng, L},
title = {Aerobic exercise ameliorates skeletal muscle atrophy in atic knockout zebrafish through the oxidative phosphorylation pathway.},
journal = {Free radical biology & medicine},
volume = {238},
number = {},
pages = {653-668},
doi = {10.1016/j.freeradbiomed.2025.07.007},
pmid = {40623538},
issn = {1873-4596},
mesh = {Animals ; Zebrafish/genetics ; *Muscular Atrophy/genetics/metabolism/pathology/therapy ; Oxidative Phosphorylation ; *Muscle, Skeletal/metabolism/pathology ; Aminoimidazole Carboxamide/analogs & derivatives/metabolism ; Reactive Oxygen Species/metabolism ; Ribonucleotides/metabolism ; *Physical Conditioning, Animal ; *Zebrafish Proteins/genetics/metabolism ; *Hydroxymethyl and Formyl Transferases/genetics/metabolism ; Gene Knockout Techniques ; *Nucleotide Deaminases/genetics/metabolism ; Mice ; Mitochondria/metabolism ; CRISPR-Cas Systems ; Purines/metabolism ; Multienzyme Complexes ; },
abstract = {The mechanisms linking purine metabolism disorders to skeletal muscle pathology are unclear. This study constructed a CRISPR/Cas9-mediated zebrafish atic knockout model and a siRNA-interfered C2C12 myoblast cell model. We revealed a novel mechanism by which ATIC (5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase) deletion drove the atrophy of skeletal muscle through the downregulation of the oxidative phosphorylation of mitochondria (OXPHOS) pathway. It was found that atic/Atic knockout/knockdown led to the interruption of purine de novo synthesis, abnormal 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) accumulation, and blockage of inosine monophosphate (IMP) synthesis, which in turn triggered mitochondrial structural damage, dysfunction of complex I-V function, and a burst of reactive oxygen species (ROS), and ultimately triggered muscle atrophy through activation of the ubiquitin-proteasome system. The progressive aerobic intervention revealed that 8 weeks of training significantly restored skeletal muscle function in zebrafish atic[-/-] mutants, and the mechanism was related to the enhancement of mitochondrial biogenesis, up-regulation of the core complex expression of the OXPHOS pathway, and the improvement of ROS scavenging ability. These findings reveal that ATIC deficiency disrupts mitochondrial function through purine metabolism dysregulation, linking aberrant AICAR accumulation to OXPHOS impairment, which provides a theoretical basis for the early warning of muscular toxicity of targeted purine metabolizing drugs and lays a molecular foundation for the exercise rehabilitation strategy of metabolic myopathies.},
}

Animals
Zebrafish/genetics
*Muscular Atrophy/genetics/metabolism/pathology/therapy
Oxidative Phosphorylation
*Muscle, Skeletal/metabolism/pathology
Aminoimidazole Carboxamide/analogs & derivatives/metabolism
Reactive Oxygen Species/metabolism
Ribonucleotides/metabolism
*Physical Conditioning, Animal
*Zebrafish Proteins/genetics/metabolism
*Hydroxymethyl and Formyl Transferases/genetics/metabolism
Gene Knockout Techniques
*Nucleotide Deaminases/genetics/metabolism
Mice
Mitochondria/metabolism
CRISPR-Cas Systems
Purines/metabolism
Multienzyme Complexes

@article {pmid40456437,
year = {2025},
author = {Yoshida, S and Onozawa, M and Yokoyama, S and Matsukawa, T and Goto, H and Hirabayashi, S and Kondo, T and Hashimoto, D and Onodera, Y and Teshima, T},
title = {Peposertib suppresses generation of FLT3-internal tandem duplication formed by contralateral double nicks.},
journal = {Experimental hematology},
volume = {149},
number = {},
pages = {104819},
doi = {10.1016/j.exphem.2025.104819},
pmid = {40456437},
issn = {1873-2399},
mesh = {*fms-Like Tyrosine Kinase 3/genetics/antagonists & inhibitors ; Humans ; Mice ; Animals ; Phthalazines/pharmacology ; *Gene Duplication/drug effects ; *Leukemia, Myeloid, Acute/genetics/drug therapy ; *Tandem Repeat Sequences/drug effects ; Piperazines/pharmacology ; *DNA Breaks, Double-Stranded/drug effects ; CRISPR-Cas Systems ; Cell Line, Tumor ; },
abstract = {Fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) is the most frequent gene mutation in acute myeloid leukemia. The consequences of FLT3-ITD have been analyzed in detail; however, the molecular mechanisms underlying the generation of FLT3-ITD remain to be elucidated. We analyzed FLT3-ITDs in clinical samples using deep sequencing and identified not only oligoclonal ITDs but also rare deletion clones clustered at the palindrome-like sequence at FLT3 exon 14. We hypothesized that FLT3 exon 14 is genetically unstable due to the palindrome-like sequence at the region and that genomic damage at the site initiates FLT3-ITD formation. We used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 to induce DNA damage for creating artificial FLT3-ITDs in human and mouse cell lines. We found that double nicks on the adjacent contralateral strand most efficiently generate ITDs. The artificial ITDs resembled clinical ITDs in the length distribution and characteristics at the joint. We further compared the inhibitory effects of olaparib and peposertib, specific inhibitors of single-strand break (SSB) and double-strand break (DSB) repair, respectively. Peposertib remarkably reduced ITD formation, but olaparib did not affect the mutation pattern. The findings indicated that nonhomologous end joining has a crucial role in the generation of ITDs. Our data shed light to the new role of peposertib, which potentially suppresses the generation of de novo FLT3-ITDs caused by mis-repair events of the DNA damages in a clinical course.},
}

*fms-Like Tyrosine Kinase 3/genetics/antagonists & inhibitors
Humans
Mice
Animals
Phthalazines/pharmacology
*Gene Duplication/drug effects
*Leukemia, Myeloid, Acute/genetics/drug therapy
*Tandem Repeat Sequences/drug effects
Piperazines/pharmacology
*DNA Breaks, Double-Stranded/drug effects
CRISPR-Cas Systems
Cell Line, Tumor

@article {pmid40397098,
year = {2025},
author = {Williams, PJ},
title = {The New Old Colonialism.},
journal = {The CRISPR journal},
volume = {8},
number = {4},
pages = {296-299},
doi = {10.1089/crispr.2025.0059},
pmid = {40397098},
issn = {2573-1602},
mesh = {Humans ; *Colonialism ; *Gene Editing/legislation & jurisprudence/ethics ; Genome, Human ; CRISPR-Cas Systems ; },
abstract = {The question of how law should regulate the manipulation of the human genome or germline is inflected by the interconnected, intersectional parrying among different systems of moral value. Contract law and constitutional law reflect two poles of interest: the transactional aspects of market valuation and the relational aspects of the web of life that acknowledge "pricelessness." In the decades from the initial decoding of the human genome in 2000 to the emergence of CRISPR technologies, powerful companies and powerful individuals now all but own the fate of our species and the health of our planet. The destructive effects of the realignments we are undergoing are still largely invisible (if not for long) and largely unresponsive to conventional checks.},
}

Humans
*Colonialism
*Gene Editing/legislation & jurisprudence/ethics
Genome, Human
CRISPR-Cas Systems

@article {pmid40397097,
year = {2025},
author = {Musunuru, K and Urnov, F},
title = {Moving Therapeutic Genome Editing into Global Clinical Trials and Medicine.},
journal = {The CRISPR journal},
volume = {8},
number = {4},
pages = {228-231},
doi = {10.1089/crispr.2025.0049},
pmid = {40397097},
issn = {2573-1602},
mesh = {Humans ; Clinical Trials as Topic ; CRISPR-Cas Systems ; *Gene Editing/methods/trends ; *Genetic Therapy/methods/trends ; },
abstract = {Moving CRISPR-based therapies from discovery to dosing patients in clinical trials and ultimately to approval involves navigating a challenging terrain of highs and lows. In this interview, physician-scientist Kiran Musunuru and genome editor Fyodor Urnov reflect on the past 20 years of their nonclinical and clinical programs in the field, the current landscape of innovation, and what they see on the horizon.},
}

Humans
Clinical Trials as Topic
CRISPR-Cas Systems
*Gene Editing/methods/trends
*Genetic Therapy/methods/trends

@article {pmid40397096,
year = {2025},
author = {Chandru, V and Gupta, V and Hegde, V and Venkatesan, A and Arora, R},
title = {Intent to Cure: The Need for a Rare Disease Platform in India and Across the Global South.},
journal = {The CRISPR journal},
volume = {8},
number = {4},
pages = {277-281},
doi = {10.1089/crispr.2025.0046},
pmid = {40397096},
issn = {2573-1602},
mesh = {Humans ; India ; *Rare Diseases/therapy/genetics ; *Genetic Therapy/methods ; Gene Editing/methods ; Genomics/methods ; Artificial Intelligence ; CRISPR-Cas Systems ; },
abstract = {The democratization of genomic technologies presents substantial opportunities for addressing rare genetic diseases, particularly in collaborations between the Global South and North. In this Perspective, we describe the current progress in gene therapy, including CRISPR, in India and see an upward trajectory of innovation. We propose the establishment of a rare disease platform in India and across the Global South designed to bridge scientific, clinical, and economic gaps, transforming untapped genetic diversity into shared opportunities for therapeutic innovation and health care equity. This platform would encompass a comprehensive data infrastructure capturing clinical, genomic, and biosample data, complemented by an artificial intelligence-powered analytics layer to enhance patient engagement and clinical trial matching, ultimately enabling cost-effective research and development (R&D) of novel therapies.},
}

Humans
India
*Rare Diseases/therapy/genetics
*Genetic Therapy/methods
Gene Editing/methods
Genomics/methods
Artificial Intelligence
CRISPR-Cas Systems

@article {pmid40397095,
year = {2025},
author = {Qiu, J},
title = {From 'Frankenstein Science' to Cosmopolitan Ethics: Overlooked Perspectives on the 'CRISPR Babies' Scandal.},
journal = {The CRISPR journal},
volume = {8},
number = {4},
pages = {257-261},
doi = {10.1089/crispr.2025.0057},
pmid = {40397095},
issn = {2573-1602},
mesh = {Humans ; *Gene Editing/ethics ; *Bioethics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Cas Systems ; China ; },
abstract = {In November 2018, Chinese biophysicist He Jiankui stunned the world by announcing that he had created the first genetically-modified babies. Is he a rogue scientist? What are the socio-cultural contexts that motivated him to commit an act widely regarded as morally indefensible? What does it say about Chinese bioethics? How should we determine whether it can ever be justified to permanently alter the human gene pool? This article highlights the global institutional failures that enabled this unfortunate episode, including the prevailing international scientific culture and the persistent Western bias against scientific work originated in the Global South. It calls for systemic efforts-including regulatory reforms, increased transparency, public engagement, and international cooperation-to strengthen ethics governance both within nations and across borders. Finally, it advocates for decolonizing bioethics, advancing the sociology of bioethics, and fostering a cosmopolitan approach to ethics grounded in diversity, equity, inclusion, and our shared humanity.},
}

Humans
*Gene Editing/ethics
*Bioethics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*CRISPR-Cas Systems
China

@article {pmid40397093,
year = {2025},
author = {Jasanoff, S and Hurlbut, JB and Saha, K and Moses, JD and Affsprung, D and Austin, H and Baylis, F and Evans, JH and Hunt, T and Kysar, DA and Lwoff, L and Mills, P and Phalkey, J and Porteus, MH and Snead, OC and Sunder Rajan, K and Wolinetz, CD},
title = {A Reset for Bioethics: A Statement from the Global Observatory for Genome Editing.},
journal = {The CRISPR journal},
volume = {8},
number = {4},
pages = {232-238},
doi = {10.1089/crispr.2025.0058},
pmid = {40397093},
issn = {2573-1602},
mesh = {*Gene Editing/ethics ; Humans ; *Bioethics/trends ; CRISPR-Cas Systems ; },
abstract = {How should we govern our increasing power to intervene in the processes of life? Genome editing, especially of the human germline, has brought this question to the forefront of global debate. We must seek to rectify shortcomings of earlier deliberative approaches by setting aside a science-and-technology first approach; expanding the range of questions for deliberation; revisiting the distribution of innovation's benefits and risks; and reimagining the limits of research. This Perspective from the Organizing Committee of the 2025 Global Observatory for Genome Editing International Summit calls for a new social compact, recognizing and rendering accountable the constitutive role of science and technology in shaping the meaning of human life in the 21st century.},
}

*Gene Editing/ethics
Humans
*Bioethics/trends
CRISPR-Cas Systems

@article {pmid40397087,
year = {2025},
author = {Newman, SA},
title = {Opposing Human Genetic Engineering.},
journal = {The CRISPR journal},
volume = {8},
number = {4},
pages = {252-256},
doi = {10.1089/crispr.2025.0043},
pmid = {40397087},
issn = {2573-1602},
mesh = {Humans ; *Genetic Engineering/ethics/methods ; *Gene Editing/ethics ; CRISPR-Cas Systems ; Germ Cells ; },
abstract = {The past five decades have been a time of substantial change in the technological capacity to modify genetic material. During this period, I have maintained an unwavering stance against human germline modification. As a biologist who has researched the complexities of genotype-phenotype relationships, I remain convinced embryo-stage human genetic modification will always remain in the realm of uncontrolled experimentation. Based on my observations and participation in the twists and turns of genetics and society, I point to the limits of calls for "broad societal consensus."},
}

Humans
*Genetic Engineering/ethics/methods
*Gene Editing/ethics
CRISPR-Cas Systems
Germ Cells

@article {pmid40397084,
year = {2025},
author = {Baylis, F},
title = {Summitting CRISPR for Human Heritable Genome Editing.},
journal = {The CRISPR journal},
volume = {8},
number = {4},
pages = {239-244},
doi = {10.1089/crispr.2025.0051},
pmid = {40397084},
issn = {2573-1602},
mesh = {Humans ; *Gene Editing/ethics/methods ; *CRISPR-Cas Systems/genetics ; *Genome, Human ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {The ethical issues of human heritable genome editing have been discussed at international summits held since 2015. In this Perspective, I consider how the discussions evolved over three summits held in Washington, DC (2015), Hong Kong (2018), and London (2023). The significance of safety and efficacy, meanings of a moratorium, and place of broad societal consensus are traced through publications produced surrounding these summits. Looking ahead, I highlight the difference between two fundamentally distinct ethical questions: Is human heritable genome editing ethical? Can human heritable genome editing be done ethically?},
}

Humans
*Gene Editing/ethics/methods
*CRISPR-Cas Systems/genetics
*Genome, Human
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid39428567,
year = {2025},
author = {Liu, Y and Yang, X and Wu, P and Guo, X and Liu, Z and Huang, Y and Xu, X},
title = {Multi-targets cleavage of BmNPV genome through genome-wide repeat sequence using CRISPR/Cas9 antiviral system.},
journal = {Insect science},
volume = {32},
number = {4},
pages = {1174-1184},
doi = {10.1111/1744-7917.13462},
pmid = {39428567},
issn = {1744-7917},
support = {32100381//National Natural Science Foundation of China/ ; 31830093//National Natural Science Foundation of China/ ; 32021001//National Natural Science Foundation Innovation Group Project/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Bombyx/virology/genetics ; *Nucleopolyhedroviruses/genetics/physiology ; *Genome, Viral ; Gene Editing ; Animals, Genetically Modified ; },
abstract = {The escalating severity of Bombyx mori nuclear polyhedrosis virus (BmNPV) infections poses significant challenges to the silkworm industry, especially when massive production shifts occur from the eastern regions to western regions with lower labor costs. Education and experience levels are different and disease control is badly needed. To solve the problems, we have developed an innovative CRISPR/Cas9 system specifically targeting BmNPV to enhance viral resistance. For the system, we selected BmNPV genes linked to virus replication and proliferation as targets, designing 2 sites for each gene. Mutating the target sequence renders the system incapable of efficiently cleaving the virus genome, hence decreasing cleavage efficiency. We conducted a search for "NGG" or "CCN" target sequences in the BmNPV genome, excluding non-recurring and potential targets in the B. mori genome. We successfully identified 2 distinct target sequences in the BmNPV genome-one being repeated 12 times and the other three times. These sequences lead to fragmentation of virus genome into multiple large segments that are difficult to repair. Transgenic silkworms demonstrate robust resistance to viruses, significantly boosting their survival rates compared with wild-type silkworms under various virus infection concentrations. Our system efficiently targets dozens of viral genomes with just 2 sequences, minimizing transposable elements while ensuring cutting effectiveness. This marks a pioneering advancement by using repetitive elements within the virus genome for targeted CRISPR cleavage, aiming for antiviral effects through genome fragmentation rather than disrupting essential viral genes. Our research introduces innovative concepts to CRISPR antiviral investigations and shows promise for the practical application of gene editing in industrial silkworm strains.},
}

Animals
*CRISPR-Cas Systems
*Bombyx/virology/genetics
*Nucleopolyhedroviruses/genetics/physiology
*Genome, Viral
Gene Editing
Animals, Genetically Modified

@article {pmid39300921,
year = {2025},
author = {Liu, ZL and Zhou, YY and Xu, QX and Wang, XC and Liu, TX and Tian, HG},
title = {Efficient CRISPR-mediated genome editing can be initiated by embryonic injection but not by ovarian delivery in the beetle Tribolium castaneum.},
journal = {Insect science},
volume = {32},
number = {4},
pages = {1185-1200},
doi = {10.1111/1744-7917.13447},
pmid = {39300921},
issn = {1744-7917},
support = {2017YFD0200900//National Key Research and Development Program of China/ ; 31101432//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Tribolium/genetics/embryology ; *Gene Editing/methods ; Female ; *CRISPR-Cas Systems ; Ovary ; Embryo, Nonmammalian ; Microinjections ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {The clustered regularly interspaced small palindromic repeats (CRISPR) / CRISPR-associated nuclease 9 (Cas9)-mediated gene editing technology has revolutionized the study of fundamental biological questions in various insects. Diverse approaches have been developed to deliver the single-guide RNA (sgRNA) and Cas9 to the nucleus of insect embryos or oocytes to achieve gene editing, including the predominant embryonic injection methods and alternative protocols through parental ovary delivery. However, a systematic comparative study of these approaches is limited, especially within a given insect. Here, we focused on revealing the detailed differences in CRISPR/Cas9-mediated gene editing between the embryo and ovary delivery methods in the beetle Tribolium castaneum, using the cardinal and tyrosine hydroxylase (TH) as reporter genes. We demonstrated that both genes could be efficiently edited by delivering Cas9/sgRNA ribonucleoproteins to the embryos by microinjection, leading to the mutant phenotypes and indels in the target gene sites. Next, the Cas9/sgRNA complex, coupled with a nanocarrier called Branched Amphiphilic Peptide Capsules (BAPC), were delivered to the ovaries of parental females to examine the efficacy of BAPC-mediated gene editing. Although we observed that a small number of beetles' progeny targeting the cardinal exhibited the expected white-eye phenotype, unexpectedly, no target DNA indels were found following subsequent sequencing analysis. In addition, we adopted a novel approach termed "direct parental" CRISPR (DIPA-CRISPR). However, we still failed to find gene-editing events in the cardinal or TH gene-targeted insects. Our results indicate that the conventional embryonic injection of CRISPR is an effective method to initiate genome editing in T. castaneum. However, it is inefficient by the parental ovary delivery approach.},
}

Animals
*Tribolium/genetics/embryology
*Gene Editing/methods
Female
*CRISPR-Cas Systems
Ovary
Embryo, Nonmammalian
Microinjections
RNA, Guide, CRISPR-Cas Systems

@article {pmid40838063,
year = {2023},
author = {Maddalena, A and Kleinlogel, S},
title = {CRISPR-mediated optogene expression from a cell-specific endogenous promoter in retinal ON-bipolar cells to restore vision.},
journal = {Frontiers in drug delivery},
volume = {3},
number = {},
pages = {934394},
pmid = {40838063},
issn = {2674-0850},
abstract = {Retinitis pigmentosa, an inherited form of retinal degeneration, is characterized by a progressive loss of rods and subsequent degeneration of cones, leading to blindness. However, the remaining neural portion of the retina (bipolar and ganglion cells) remains anatomically and functionally intact for an extended time. A possible treatment to restore the light sensitivity of the retina consists of rendering the remaining retinal cells photosensitive using optogenetic tools like, for example, Opto-mGluR6, a light-sensitive mGluR6 receptor. We have previously demonstrated that AAV vector-mediated expression of Opto-mGluR6 in ON-bipolar cells restores visual function in otherwise blind mice. However, classical gene supplementation therapy still suffers from high off-target expression rates and uncontrollable target gene expression levels that may lead to either cytotoxicity or lack of functional restoration. To address these issues and achieve cell-specific and endogenously controlled Opto-mGluR6 expression, we employed the CRISPR/Cas technology-in particular, homology-independent targeted integration (HITI) and microhomology-dependent targeted integration (MITI)-to knock-in the Opto-mGluR6 gene behind the ON-bipolar cell-specific GRM6 promoter. We compared four Cas systems in vitro and show that SpCas9 for HITI and LbCpf1 for MITI are well suited to promoting knock-in. As AAV2-mediated ON-bipolar cell transduction resulted in inefficiency, we evaluated Exo-AAVs as delivery vehicles and found Exo-AAV1 efficient for targeting ON-bipolar cells. We demonstrate that intravitreal injection of Exo-AAV1 carrying vectors that promote MITI significantly improved visual acuity in otherwise blind rd1 mice. We conclude by confirming and providing a qualitative evaluation of the MITI-mediated knock-in in the correct genomic locus.},
}

@article {pmid40835744,
year = {2025},
author = {Sinaga, DS and Huang, PY and Huang, CK and Lu, CA and Huang, LF},
title = {A single donor cassette enables site-specific knock-in at either the αAmy3 or αAmy8 locus in rice cells via CRISPR/Cas9.},
journal = {Applied microbiology and biotechnology},
volume = {109},
number = {1},
pages = {190},
pmid = {40835744},
issn = {1432-0614},
support = {112-2313-B-008-002-MY3//the National Science and Technology Council of the Republic of China/ ; 112-2313-B-008-002-MY3//the National Science and Technology Council of the Republic of China/ ; 111-2313-B-155-001-MY3//the National Science and Technology Council of the Republic of China/ ; },
mesh = {*Oryza/genetics ; *CRISPR-Cas Systems ; Gene Editing/methods ; *Gene Knock-In Techniques/methods ; Promoter Regions, Genetic ; Introns ; },
abstract = {CRISPR/Cas9 gene editing is widely used to manipulate gene expression and integrate transgenes into specific target sites, making it a powerful tool for recombinant protein expression. In this study, we generated a single donor cassette for CRISPR/Cas9-mediated knock-in at either the αAmy3 or αAmy8 locus in rice cells. The transgene was inserted downstream of the promoter and first exon of the rice αAmy3 or αAmy8 genes, which are highly expressed under sugar-starved conditions in rice suspension cultures. We constructed a simple vector with the homologous intron sequences of the αAmy3 and αAmy8, along with rice codon-optimized recombinant receptor binding domain (rcRBD) of the SARS-CoV-2 spike protein, a functional domain responsible for binding to the angiotensin-converting enzyme 2 (ACE2) receptor on host cells. Using this construct, rcRBD was successfully integrated into the intron 1 of either the αAmy3 or αAmy8 genes. As a result, rcRBD expression was driven by endogenous αAmy3 or αAmy8 promoter-signal peptide. Under the control of αAmy3-signal peptide, rcRBD proteins was detected in both the soluble cellular protein fraction and culture medium, whereas expression driven by the αAmy8 promoter-signal peptide was exclusively detected in the culture medium of rice suspension cells. The highest secreted protein yield of rcRBD in the rice culture medium under the control of αAmy8 endogenous promoter reached 20.7 mg/L, demonstrating a production efficiency comparable to that driven by the endogenous αAmy3 promoter.},
}

*Oryza/genetics
*CRISPR-Cas Systems
Gene Editing/methods
*Gene Knock-In Techniques/methods
Promoter Regions, Genetic
Introns

@article {pmid40835660,
year = {2025},
author = {Dong, T and Zhao, Y and Zhang, M and Lang, WY and Liu, DY and Zhang, KS and Wang, YJ and Li, L and Lian, J and Yao, HB and Zhang, HY and Jin, HF and Lu, T and Shen, L and Yue, LL and Lin, Y},
title = {SNTA1-deficient human cardiomyocytes show shorter field potential duration and slower conduction velocity.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {30600},
pmid = {40835660},
issn = {2045-2322},
support = {2022-KYYWF-0808//Basic scientific research expenses of colleges and universities in Heilongjiang Province/ ; },
mesh = {Humans ; *Myocytes, Cardiac/metabolism/physiology ; NAV1.5 Voltage-Gated Sodium Channel/metabolism/genetics ; *Calcium-Binding Proteins/genetics/deficiency ; Arrhythmias, Cardiac/genetics ; CRISPR-Cas Systems ; Action Potentials ; Gene Knockout Techniques ; Cell Differentiation ; Cell Line ; *Membrane Proteins/genetics/deficiency ; *Muscle Proteins/genetics/deficiency ; },
abstract = {In clinical settings, patients with α-1-syntrophin point mutations are often associated with rare arrhythmias, including Long QT syndrome, Brugada syndrome, and sudden infant death syndrome. Previous studies on α-1-syntrophin have predominantly utilized nonhuman cardiomyocyte models. This study aims to elucidate the phenotype of α-1-syntrophin deficiency using human cardiomyocytes. Using CRISPR/Cas9 technology, we generated SNTA1 knockout (KO) embryonic stem cell line, which were subsequently differentiated into cardiomyocytes using 2D differentiation method. Genotype analysis identified an adenine (A) insertion in the second exon of SNTA1, resulting in a premature stop codon at the 149th amino acid position and truncation within the PDZ domain. SNTA1-deficient cardiomyocytes exhibited a shortened field potential duration (FPD) and slower conduction velocity, as detected by micro electrode array analysis. Immunofluorescence analysis further revealed disorganized distribution of Nav1.5 in SNTA1-deficient cardiomyocytes. SNTA1 is a susceptibility locus for arrhythmias and plays a critical role as an essential auxiliary protein in the proper localization of Nav1.5 in human cardiomyocytes.},
}

Humans
*Myocytes, Cardiac/metabolism/physiology
NAV1.5 Voltage-Gated Sodium Channel/metabolism/genetics
*Calcium-Binding Proteins/genetics/deficiency
Arrhythmias, Cardiac/genetics
CRISPR-Cas Systems
Action Potentials
Gene Knockout Techniques
Cell Differentiation
Cell Line
*Membrane Proteins/genetics/deficiency
*Muscle Proteins/genetics/deficiency

@article {pmid40833724,
year = {2025},
author = {Bhattacharjee, G and Gohil, N and Khambhati, K and Murjani, K and Chu, DT and Le Bui, N and Thi, HV and Mani, I and Bansal, A and Shamili, S and Satish, L and Ramakrishna, S and Alzahrani, KJ and Singh, V},
title = {Advancements in CRISPR-Cas Systems for Genome Editing towards Eradication of Human Microbial Pathogens.},
journal = {Molecular biotechnology},
volume = {},
number = {},
pages = {},
pmid = {40833724},
issn = {1559-0305},
support = {TU-DSPP-2024-05//Taif University/ ; 2021M3A9H3015390//National Science foundation of Korea/ ; BT/PR38411/GET/119/311/2020//Department of Biotechnology/ ; },
abstract = {CRISPR-Cas systems have been explored for targeted genome editing of several organisms. It is rapid, cost-effective, specific, and versatile technology. It requires expression of multidomain single Cas9 protein and single guide RNA (sgRNA) that targets desired nucleic acids in the presence of a protospacer adjacent motif (PAM). This generates a double-stranded break that is repaired by either non-homologous end joining or a homology-directed repair pathway. Currently, several Cas protein variants have been discovered and being used for several biotechnological applications. This review highlights the recent progress of CRISPR-Cas systems for genome editing of mainly human pathogenic microorganisms for their controlling infections.},
}

@article {pmid40833532,
year = {2025},
author = {Tang, K and Wu, L and Hu, Y and Xue, T and Jin, Y and Zhou, X and Luo, C and Zhao, Y and Tong, L and Dai, J and Feng, D and Zeng, Z and Pan, D},
title = {Perturbation of calreticulin potentiates CD8+ T cell-mediated antitumor immunity.},
journal = {The Journal of experimental medicine},
volume = {222},
number = {10},
pages = {},
doi = {10.1084/jem.20242360},
pmid = {40833532},
issn = {1540-9538},
support = {82341026//National Natural Science Foundation of China/ ; 82073163//National Natural Science Foundation of China/ ; 2022YFC2505400//National Key Research and Development Program of China/ ; //Tsinghua University Initiative Scientific Research Program/ ; //Tsinghua-Peking University Center of Life Science/ ; //Tsinghua-Peking Joint Centre for Life Sciences/ ; },
mesh = {*Calreticulin/metabolism/immunology/genetics ; Animals ; *CD8-Positive T-Lymphocytes/immunology ; Humans ; Mice ; Histocompatibility Antigens Class I/immunology/metabolism ; Cell Line, Tumor ; Mice, Inbred C57BL ; *Neoplasms/immunology ; CRISPR-Cas Systems ; Immunotherapy ; *Immunity, Cellular ; },
abstract = {Effective immunotherapy relies on the presentation of tumor-derived neoantigens on the major histocompatibility complex class I (MHC-I) to activate CD8+ T cells. Deficiencies in this process are a key mechanism of immune evasion and resistance to checkpoint blockade. In this study, using an in vivo CRISPR-Cas9 screen, we unexpectedly found that inactivation of calreticulin (CALR), and other selected components of the peptide-loading complex (PLC), induced robust CD8+ T cell-mediated immune responses. We show that this effect is dependent on the expression of classical MHC-I on tumor cells. Mechanistically, loss of CALR reshaped the MHC-I peptide repertoire, favoring the presentation of low-affinity peptides in murine and human cell lines. Genetic or pharmacological inhibition of PDIA3, another PLC component, similarly induced antitumor effects. These findings reveal a previously unrecognized role of CALR and the PLC in regulating antitumor immunity and suggest that targeting this pathway could be a promising strategy to overcome immune resistance and improve the efficacy of cancer immunotherapies.},
}

*Calreticulin/metabolism/immunology/genetics
Animals
*CD8-Positive T-Lymphocytes/immunology
Humans
Mice
Histocompatibility Antigens Class I/immunology/metabolism
Cell Line, Tumor
Mice, Inbred C57BL
*Neoplasms/immunology
CRISPR-Cas Systems
Immunotherapy
*Immunity, Cellular

@article {pmid40831701,
year = {2025},
author = {Liu, W and Jiang, Y and Wang, C and Wang, M and Zhang, W and Ren, H and Xu, S and Qin, J and Liu, P and Jin, L and Zhao, D},
title = {Towards the elimination of infectious HPV: exploiting CRISPR/Cas innovations.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1627668},
pmid = {40831701},
issn = {2235-2988},
mesh = {Humans ; *CRISPR-Cas Systems ; *Papillomavirus Infections/therapy/prevention & control/virology ; *Papillomaviridae/genetics ; *Gene Editing/methods ; *Genetic Therapy/methods ; Genome, Viral ; },
abstract = {HPV has been conclusively associated with various human malignancies, making the development of prevention and treatment strategies for HPV-induced diseases a high priority. Currently, primary prevention methods include HPV immunization and routine screening, which significantly reduce the risk of HPV transmission. However, for patients diagnosed with invasive, advanced, or recurrent malignancies, non-virus-specific therapies frequently lead to drug resistance and adverse effects, resulting in minimal improvement in treatment efficacy for numerous patients. Viral genome-targeting therapy is emerging as a promising avenue for the future management of HPV infections. With the rapid advancement of genetic modification technologies, the CRISPR/Cas system has demonstrated significant potential in treating viral infections. Its ability to selectively target and edit viral genomes for elimination positions it as a highly effective approach for combating HPV. This review will explore the functions and applications of the CRISPR/Cas system as an innovative therapy for HPV. We will illustrate the prospective efficacy of CRISPR/Cas as a groundbreaking and promising cure for HPV infections, while also addressing the opportunities and challenges associated with this novel approach.},
}

Humans
*CRISPR-Cas Systems
*Papillomavirus Infections/therapy/prevention & control/virology
*Papillomaviridae/genetics
*Gene Editing/methods
*Genetic Therapy/methods
Genome, Viral

@article {pmid40831643,
year = {2025},
author = {Meza, C and Sepulveda, B and Flores-Castañón, N and Valenzuela, F and Ormeño, C and Castillo, A and Echeverría-Vega, A and Jasem Mohammed Breig, S and Alardhi, SM and Gonzalez, A and Mora-Lagos, B and Banerjee, A},
title = {Genomic basis and functional characterization of the exopolysaccharide production by a thermotolerant Bacillus isolated from Tolhuaca hot spring.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1622325},
pmid = {40831643},
issn = {1664-302X},
abstract = {Bacillus licheniformis Tol1, a thermotolerant bacterial strain isolated from the Tolhuaca hot spring in Chile, was investigated for its genomic features and the functional properties of its exopolysaccharide (EPS). The whole-genome sequencing revealed ∼4.25 Mbp genome with a GC content of 45.9% and a rich repertoire of genes associated with environmental stress adaptation, antibiotic resistance, sporulation, biofilm formation, and EPS biosynthesis, including the presence of epsD and epsC. The strain also harbored intact prophage elements and a Type I-A CRISPR-Cas system, indicating potential horizontal gene transfer and genome plasticity. Confocal microscopy revealed robust biofilm formation at 45-55°C under neutral to slightly alkaline pH, with strong EPS matrix development. EPS production was optimized using OFAT and Response Surface Methodology (RSM), achieving a yield of 2.11 g L[-1] under optimized conditions, which was further validated using an Artificial Neural Network (ANN) model (R [2] = 0.9909). The EPS exhibited promising antioxidant activity and significant emulsification potential across various vegetable oils, which were comparable or superior to commercial bacterial EPS xanthan gum. Notably, the EPS also showed cytotoxic effects against AGS gastric adenocarcinoma cells, reducing viability by 38.38 and 37% at 50-100 μg μL[-1] concentrations, respectively, suggesting potential anticancer activity. Altogether, the study highlights B. licheniformis Tol1 as a multifunctional thermophile with valuable biotechnological potential, particularly for applications in food, pharmaceutical, and biomedical industries.},
}

@article {pmid40830872,
year = {2025},
author = {Saffari Natanzi, A and Poudineh, M and Karimi, E and Khaledi, A and Haddad Kashani, H},
title = {Innovative approaches to combat antibiotic resistance: integrating CRISPR/Cas9 and nanoparticles against biofilm-driven infections.},
journal = {BMC medicine},
volume = {23},
number = {1},
pages = {486},
pmid = {40830872},
issn = {1741-7015},
mesh = {*Biofilms/drug effects ; *CRISPR-Cas Systems ; Humans ; *Nanoparticles ; *Anti-Bacterial Agents/administration & dosage/pharmacology ; Gene Editing/methods ; *Drug Resistance, Bacterial/genetics ; *Bacterial Infections/drug therapy ; Pseudomonas aeruginosa/drug effects ; },
abstract = {The increasing prevalence of antibiotic-resistant bacterial infections is a major global health concern, with biofilms playing a key role in bacterial persistence and resistance. Biofilms provide a protective matrix that limits antibiotic penetration, enhances horizontal gene transfer, and enables bacterial survival in hostile environments. Conventional antimicrobial therapies are often ineffective against biofilm-associated infections, necessitating the development of novel therapeutic strategies. The CRISPR/Cas9 gene-editing system has emerged as a revolutionary tool for precision genome modification, offering targeted disruption of antibiotic resistance genes, quorum sensing pathways, and biofilm-regulating factors. However, the clinical application of CRISPR-based antibacterials faces significant challenges, particularly in efficient delivery and stability within bacterial populations. Nanoparticles (NPs) present an innovative solution, serving as effective carriers for CRISPR/Cas9 components while exhibiting intrinsic antibacterial properties. Nanoparticles can enhance CRISPR delivery by improving cellular uptake, increasing target specificity, and ensuring controlled release within biofilm environments. Recent advances have demonstrated that liposomal CRISPR-Cas9 formulations can reduce Pseudomonas aeruginosa biofilm biomass by over 90% in vitro, while gold nanoparticle carriers enhance editing efficiency up to 3.5-fold compared to non-carrier systems. These hybrid platforms also enable co-delivery with antibiotics, producing synergistic antibacterial effects and superior biofilm disruption. Additionally, they can facilitate co-delivery of antibiotics or antimicrobial peptides, further enhancing therapeutic efficacy. This review explores the synergistic integration of CRISPR/Cas9 and nanoparticles in combating biofilm-associated antibiotic resistance. We discuss the mechanisms of action, recent advancements, and current challenges in translating this approach into clinical practice. While CRISPR-nanoparticle hybrid systems hold immense potential for next-generation precision antimicrobial therapies, further research is required to optimize delivery platforms, minimize off-target effects, and assess long-term safety. Understanding and overcoming these challenges will be critical for developing effective biofilm-targeted antibacterial strategies.},
}

*Biofilms/drug effects
*CRISPR-Cas Systems
Humans
*Nanoparticles
*Anti-Bacterial Agents/administration & dosage/pharmacology
Gene Editing/methods
*Drug Resistance, Bacterial/genetics
*Bacterial Infections/drug therapy
Pseudomonas aeruginosa/drug effects

@article {pmid40829801,
year = {2025},
author = {Mohseni, A and Nia, RG and Tafrishi, A and López, ML and Liu, XZ and Stajich, JE and Lonardi, S and Wheeldon, I},
title = {Kingdom-wide CRISPR guide design with ALLEGRO.},
journal = {Nucleic acids research},
volume = {53},
number = {15},
pages = {},
doi = {10.1093/nar/gkaf783},
pmid = {40829801},
issn = {1362-4962},
support = {NSF-2225878//Chemical, Bioengineering, Environmental and Transport Systems/ ; DBI-2400327//National Science Foundation/ ; EF-2125066//National Science Foundation/ ; CA-R-PPA-211-5062-H//U.S. Department of Agriculture, National Institute of Food and Agriculture/ ; },
mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems ; Genome, Fungal ; *Gene Editing/methods ; *Algorithms ; Kluyveromyces/genetics ; Saccharomyces cerevisiae/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Yarrowia/genetics ; Rhodotorula/genetics ; Gene Library ; Saccharomycetales ; },
abstract = {Designing CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) single guide RNA (sgRNA) libraries targeting entire kingdoms of life will significantly advance genetic research in diverse and underexplored taxa. Current sgRNA design tools are often species-specific and fail to scale to large, phylogenetically diverse datasets, limiting their applicability to comparative genomics, evolutionary studies, and biotechnology. Here, we introduce ALLEGRO, a combinatorial optimization algorithm designed to compose minimal, yet highly effective sgRNA libraries targeting thousands of species at the same time. Leveraging integer linear programming, ALLEGRO identified compact sgRNA sets simultaneously targeting multiple genes of interest for over 2000 species across the fungal kingdom. We experimentally validated sgRNAs designed by ALLEGRO in Kluyveromyces marxianus, Komagataella phaffii, Yarrowia lipolytica, and Saccharomyces cerevisiae, confirming successful genome edits. Additionally, we employed a generalized Cas9-ribonucleoprotein delivery system to apply ALLEGRO's sgRNA libraries to untested fungal genomes, such as Rhodotorula araucariae. Our experimental findings, together with cross-validation, demonstrate that ALLEGRO facilitates efficient CRISPR genome editing, enabling the development of universal sgRNA libraries applicable to entire taxonomic groups.},
}

*RNA, Guide, CRISPR-Cas Systems/genetics
*CRISPR-Cas Systems
Genome, Fungal
*Gene Editing/methods
*Algorithms
Kluyveromyces/genetics
Saccharomyces cerevisiae/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats
Yarrowia/genetics
Rhodotorula/genetics
Gene Library
Saccharomycetales

@article {pmid40792428,
year = {2025},
author = {Zhou, T and Yu, D and Wu, L and Tan, Y and Li, X and Sun, Z and Sun, X and Chen, J and Duan, M and Yuan, D},
title = {Improvement of Rice Seed Storability by Regulating Lipid Metabolism Using a CRISPR/Cas9 System.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {33},
pages = {20972-20983},
doi = {10.1021/acs.jafc.5c06276},
pmid = {40792428},
issn = {1520-5118},
mesh = {*Oryza/genetics/metabolism/chemistry ; *Seeds/metabolism/genetics/chemistry ; *Lipid Metabolism ; CRISPR-Cas Systems ; *Plant Proteins/genetics/metabolism ; Reactive Oxygen Species/metabolism ; Food Storage ; Plants, Genetically Modified/metabolism/genetics ; Fatty Acids/metabolism ; Gene Expression Regulation, Plant ; },
abstract = {Long-term storage of rice grain is critical for global food security, yet rice is inherently susceptible to deterioration during storage. Herein, rice seed storability was improved by targeting three key enzyme genes in the lipid metabolism pathway via CRISPR/Cas9 technology, and the mechanism underlying this was analyzed by an untargeted lipidomic approach. Our findings demonstrate that the significantly inferior seed storability in the Yu-Zhen-Xiang (YZX) cultivar compared with the Xi-Li-Gong-Mi (XLGM) cultivar arises from accelerated lipid catabolism and reactive oxygen species (ROS) overproduction. Moreover, a fad2-1/lox3/pldα1 triple mutant in the YZX background was rapidly generated by FMPKC systems, and the mutant exhibited lower fatty acid accumulation and reduced ROS content, along with improved grain quality and nutritional value after accelerated aging. Lipidomic analysis indicated that diminished lipid hydrolysis and peroxidation collectively accounted for enhanced storability of the flp mutant. Collectively, this study establishes a robust strategy for rapidly and significantly improving rice aging tolerance, with potential applicability to other cereal crops for addressing critical challenges of grain storage.},
}

*Oryza/genetics/metabolism/chemistry
*Seeds/metabolism/genetics/chemistry
*Lipid Metabolism
CRISPR-Cas Systems
*Plant Proteins/genetics/metabolism
Reactive Oxygen Species/metabolism
Food Storage
Plants, Genetically Modified/metabolism/genetics
Fatty Acids/metabolism
Gene Expression Regulation, Plant

@article {pmid40780629,
year = {2025},
author = {Du, X and Quinn, A and Menzies, M and Porto-Neto, LR},
title = {Optimizing the delivery of CRISPR/Cas9 ribonucleoproteins for efficient genome editing in bovine embryos.},
journal = {Gene},
volume = {966},
number = {},
pages = {149715},
doi = {10.1016/j.gene.2025.149715},
pmid = {40780629},
issn = {1879-0038},
mesh = {Animals ; *Gene Editing/methods ; Cattle ; *CRISPR-Cas Systems/genetics ; *Ribonucleoproteins/genetics ; Electroporation/methods ; Blastocyst/metabolism ; Receptors, Prolactin/genetics ; Female ; Transfection/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {To assist in establishing a streamlined and efficient workflow for generating gene-edited bovine embryos, we evaluated three transfection approaches for the delivery of CRISPR Cas9-sgRNA ribonucleoproteins into bovine zygotes: lipofection with Lipofectamine CRISPRMAX, and electroporation using either Neon or NEPA21 electroporation systems. Bos taurus prolactin receptor (PRLR) was used as the target gene. The PRLR editing outcomes were analysed by PCR genotyping and Sanger sequencing of individual embryos at day 8 post-fertilization. CRISPRMAX transfection generated up to 30 % PRLR-edited blastocysts (8 % homozygous deletion), without affecting the embryo cleavage (93 %) and blastocyst rate (39 %) relative to non-transfected controls. For both NEPA21 and Neon electroporation, we found that increasing the voltage, length and number of pulses resulted in enhanced gene editing efficiency but compromised embryo cleavage and blastocyst rates. NEPA21 electroporation with a commercial electroporation enhancer reagent produced up to 47.6 % transfected embryos with the PRLR deletion, but with decreased embryo cleavage (62 %) and blastocyst (18 %) rates. Combining NEPA21 electroporation with CRISPRMAX lipofection enhanced the gene editing efficiency to 50 % (23 % homozygous editing), with 64 % embryo cleavage rate and 18 % blastocyst rate. Notably, Neon electroporation resulted in 65.2 % PRLR-edited blastocysts with 21 % homozygous editing (50% cleavage rate and 10 % blastocyst rate). Additional studies may be necessary to further optimize electroporation parameters to achieve an optimal balance between embryo viability and gene editing efficiency. These outcomes will provide valuable insights for improving gene editing workflows for bovines and may help to promote and accelerate the widespread implementation of genome editing technology in livestock.},
}

Animals
*Gene Editing/methods
Cattle
*CRISPR-Cas Systems/genetics
*Ribonucleoproteins/genetics
Electroporation/methods
Blastocyst/metabolism
Receptors, Prolactin/genetics
Female
Transfection/methods
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid40538131,
year = {2025},
author = {Li, Z and Khan, WU and Bai, G and Dong, C and Wang, J and Zhang, Y and Wang, C and Zhang, H and Wang, W and Luo, M and Chen, F},
title = {From Code to Life: The AI-Driven Revolution in Genome Editing.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {30},
pages = {e17029},
doi = {10.1002/advs.202417029},
pmid = {40538131},
issn = {2198-3844},
support = {32172614//National Natural Science Foundation of China/ ; ZDYF2023XDNY050//Hainan Province Science and Technology Special Fund/ ; 324RC452//Hainan Provincial Natural Science Foundation of China/ ; 325QN234//Hainan Provincial Natural Science Foundation of China/ ; NKLTCB202337//Project of National Key Laboratory for Tropical Crop Breeding/ ; Y2021094//South China Botanical Garden, Chinese Academy of Sciences/ ; 2022B1515020026//Guangdong Natural Science Funds for Distinguished Young Scholars/ ; Y2021094//Youth Innovation Promotion Association, Chinese Academy of Sciences/ ; },
mesh = {*Gene Editing/methods/trends ; *Artificial Intelligence/trends ; Humans ; CRISPR-Cas Systems/genetics ; },
abstract = {Genome editing has revolutionized modern biotechnology, enabling precise modifications to DNA sequences with far-reaching applications in medicine, agriculture, and synthetic biology. Recent advancements in artificial intelligence (AI) have significantly enhanced genome editing by improving target selection, minimizing off-target effects, and optimizing CRISPR-associated systems. AI-driven models, such as deep learning-based predictors and protein language models, enable more accurate sgRNA design, novel Cas protein discovery, and enhanced gene regulatory network analysis. Additionally, AI-powered tools facilitate large-scale data integration, accelerating functional genomics and therapeutic genome editing. This review explores the intersection of AI and genome editing, highlighting key innovations, challenges, and future prospects. Despite its transformative potential, AI-driven genome editing raises ethical concerns regarding data bias, algorithmic transparency, and unintended genetic modifications. Addressing these challenges requires interdisciplinary collaboration between AI researchers, molecular biologists, and policymakers. As AI continues to evolve, its integration with genome editing will pave the way for groundbreaking advancements in precision medicine, genetic disease treatment, and sustainable agriculture.},
}

*Gene Editing/methods/trends
*Artificial Intelligence/trends
Humans
CRISPR-Cas Systems/genetics

@article {pmid40369100,
year = {2025},
author = {Correia, L and Shalygin, A and Erbacher, A and Zaisserer, J and Gudermann, T and Chubanov, V},
title = {TRPM7 underlies cadmium cytotoxicity in pulmonary cells.},
journal = {Archives of toxicology},
volume = {99},
number = {8},
pages = {3269-3281},
pmid = {40369100},
issn = {1432-0738},
support = {TRR 152 (P15)//Deutsche Forschungsgemeinschaft (DFG)/ ; Research Training Group 2338 (P10)//Deutsche Forschungsgemeinschaft (DFG)/ ; },
mesh = {*TRPM Cation Channels/genetics/metabolism/antagonists & inhibitors ; Humans ; Animals ; A549 Cells ; Mice ; *Cadmium/toxicity ; Cell Survival/drug effects ; *Lung/drug effects/cytology/metabolism ; *Protein Serine-Threonine Kinases/genetics/metabolism ; Mice, Knockout ; CRISPR-Cas Systems ; Gene Knockout Techniques ; },
abstract = {TRPM7 is a kinase-coupled ion channel that exhibits high activity in the immune and epithelial cells of different organs, including the lung. Electrophysiological studies have established that the TRPM7 channel displays high permeability to Mg[2+], Zn[2+], and Ca[2+], as well as trace metal cations. While the critical role of TRPM7 in the cellular balance of Mg[2+], Zn[2+], and Ca[2+] is well-documented, its contribution to the cellular uptake of trace metal cations, frequent respiratory pollutants, remains unclear. Here, we performed an electrophysiological assessment of pulmonary A549 cells revealing endogenous TRPM7 currents, which were eliminated by knockout (KO) of the TRPM7 gene using the CRISPR/Cas9 approach or by administration of NS8593 and VER155008, two structurally unrelated inhibitors of the TRPM7 channel. Unlike prior studies with various cell lines showing that TRPM7 KO mutation induces cell growth arrest, we observed that A549 cells maintained normal viability after genetic and pharmacological inactivation of TRPM7. Consequently, we used A549 cells to examine the impact of Cd[2+] on cell viability and found that TRPM7 KO mutation and both pharmacological agents mitigated the Cd[2+] cytotoxicity. Analogous to A549 cells, electrophysiological analysis of mouse primary alveolar type 2 (ATII) cells revealed endogenous TRPM7 currents and Cd[2+] exposure reduced the cell viability of ATII cells in a TRPM7-dependent fashion. Hence, the TRPM7 channel contributes to Cd[2+] cytotoxicity in pulmonary cells and can serve as a therapeutic target to alleviate the toxic effects of trace metal exposure.},
}

*TRPM Cation Channels/genetics/metabolism/antagonists & inhibitors
Humans
Animals
A549 Cells
Mice
*Cadmium/toxicity
Cell Survival/drug effects
*Lung/drug effects/cytology/metabolism
*Protein Serine-Threonine Kinases/genetics/metabolism
Mice, Knockout
CRISPR-Cas Systems
Gene Knockout Techniques

@article {pmid37407027,
year = {2025},
author = {Cutter, AD},
title = {Guerrilla eugenics: gene drives in heritable human genome editing.},
journal = {Journal of medical ethics},
volume = {51},
number = {9},
pages = {627-635},
doi = {10.1136/jme-2023-109061},
pmid = {37407027},
issn = {1473-4257},
mesh = {Humans ; *Gene Editing/ethics ; *Eugenics ; *CRISPR-Cas Systems ; *Genome, Human ; *Gene Drive Technology/ethics ; },
abstract = {CRISPR-Cas9 genome editing can and has altered human genomes, bringing bioethical debates about this capability to the forefront of philosophical and policy considerations. Here, I consider the underexplored implications of CRISPR-Cas9 gene drives for heritable human genome editing. Modification gene drives applied to heritable human genome editing would introduce a novel form of involuntary eugenic practice that I term guerrilla eugenics. Once introduced into a genome, stealth genetic editing by a gene drive genetic element would occur each subsequent generation irrespective of whether reproductive partners consent to it and irrespective of whether the genetic change confers any benefit. By overriding the ability to 'opt in' to genome editing, gene drives compromise the autonomy of carrier individuals and their reproductive partners to choose to use or avoid genome editing and impose additional burdens on those who hope to 'opt out' of further genome editing. High incidence of an initially rare gene drive in small human communities could occur within 200 years, with evolutionary fixation globally in a timeframe that is thousands of times sooner than achievable by non-drive germline editing. Following any introduction of heritable gene drives into human genomes, practices intended for surveillance or reversal also create fundamental ethical problems. Current policy guidelines do not comment explicitly on gene drives in humans. These considerations motivate an explicit moratorium as being warranted on gene drive development in heritable human genome editing.},
}

Humans
*Gene Editing/ethics
*Eugenics
*CRISPR-Cas Systems
*Genome, Human
*Gene Drive Technology/ethics

@article {pmid40829593,
year = {2025},
author = {Margolis, SR and Meeske, AJ},
title = {Crosstalk between three CRISPR-Cas types enables primed type VI-A adaptation in Listeria seeligeri.},
journal = {Cell host & microbe},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.chom.2025.05.020},
pmid = {40829593},
issn = {1934-6069},
abstract = {CRISPR-Cas systems confer adaptive immunity to their prokaryotic hosts through the process of adaptation, where sequences are captured from foreign nucleic acids and integrated as spacers in the CRISPR array, thereby enabling crRNA-guided interference against new threats. While the Cas1-2 integrase is critical for adaptation, it is absent from many CRISPR-Cas loci, rendering the mechanism of spacer acquisition unclear for these systems. In this study, we show that the RNA-targeting type VI-A CRISPR system of Listeria seeligeri acquires spacers from DNA substrates through the action of a promiscuous Cas1-2 integrase encoded by a co-occurring type II-C system, in a transcription-independent manner. We further demonstrate that the type II-C integration complex is strongly stimulated by preexisting spacers in a third CRISPR system (type I-B), which imperfectly match phage targets and prime type VI-A adaptation. Altogether, our results reveal an unprecedented degree of communication among CRISPR-Cas loci encoded by a single organism.},
}

@article {pmid40829592,
year = {2025},
author = {Smith, LM and Fineran, PC},
title = {Type I CRISPR-Cas immunity primes type III spacer acquisition.},
journal = {Cell host & microbe},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.chom.2025.07.021},
pmid = {40829592},
issn = {1934-6069},
abstract = {CRISPR-Cas systems are diverse, with microbes harboring multiple classes and subtypes. Type I DNA-targeting and type III RNA-targeting systems often co-occur, but their interactions remain unclear. Prodigiosinella has three CRISPR-Cas systems (I-E, I-F, and III-A) with independent adaptation machinery. Type III systems can trigger cell death, yet it is unknown how functional spacers are acquired. We found that type I interference generates substrates acquired by the type III adaptation machinery. Despite reducing type I interference efficiency, type III contributed to plasmid loss and provided an advantage when DNA-targeting systems failed. Type I priming influenced type III spacer length and source, with more spacers acquired near the type I target site. Invader DNA clearance by type I interference enabled retention of cytotoxic type III spacers that would otherwise be lost. This study reveals how RNA-targeting CRISPR-Cas systems function as a backup in multi-system hosts, bolstering population-level protection.},
}

@article {pmid40823824,
year = {2025},
author = {Moreno-Rodríguez, A and Rubio, A and Garzón, A and Smani, Y and Pérez-Pulido, AJ},
title = {The evolutionary replacement of restriction-modification by Ssp antiviral systems is associated with the distribution of prophages in the major clonal group of Acinetobacter baumannii.},
journal = {mBio},
volume = {},
number = {},
pages = {e0213525},
doi = {10.1128/mbio.02135-25},
pmid = {40823824},
issn = {2150-7511},
abstract = {Bacteriophages kill almost half of the world's bacterial cells every day. In response, bacteria have developed anti-phage defense mechanisms that number in the dozens. Individual defense systems are gained and lost by genomes of the same species, depending on their fitness advantage. Thus, the combination of defense systems presented by the individual genomes may differ, and the collection of all of them is what is known as the pan-immune system of the species. Here, we have analyzed thousands of genomes of the bacterium Acinetobacter baumannii, an opportunistic pathogen of humans of great clinical concern, and we have found 81 different defense systems. By analyzing how these systems combine, we have found that more than half of the genomes lack the universal DNA-methylating restriction-modification systems and harbor an alternative innate Ssp system that performs DNA phosphorothioate modification. The presence of one or the other innate system may modulate the evolution of the genomes of this species, causing them to present a different profile of phages integrated into the bacterial genome. In fact, the most widespread strain of this bacterium worldwide, global clone 2, showed an Ssp system as its distinctive feature. We have also observed that the presence of many other defense systems is associated with the presence of a higher number of prophages, which could be because the prophage carries the system, or because the bacterium would not need these systems in environments where the phage is absent.IMPORTANCEAcinetobacter baumannii is a bacterium of great concern in clinical contexts due to the plasticity of its genome and its resistance to antibiotics. Its cells are infected by a multitude of bacteriophages, and the bacterium defends itself with dozens of different defense systems. Here, we have analyzed the complete defensome of thousands of genomes of the species and found that more than half of the genomes do not have universal restriction-modification systems, which are replaced by another innate labeling and restriction system. Furthermore, these genomes belong to the international clone of the bacterium that causes the most concern in hospitals.},
}

@article {pmid40822281,
year = {2025},
author = {Teichmann, B and Melchior, F and Beyreuther, K and Chorianopoulou, MK},
title = {Moral judgment of genetic technologies: validation of the genetic technologies questionnaire in the German-speaking population.},
journal = {Frontiers in genetics},
volume = {16},
number = {},
pages = {1620962},
pmid = {40822281},
issn = {1664-8021},
abstract = {INTRODUCTION: The development of modern life sciences has expanded our biomedical capabilities to an unprecedented degree. For example, genetic testing can be used to predict hereditary predisposition or susceptibility to certain diseases. The development of gene scissors such as CRISPR/Cas makes it possible to repair the disease gene or introduce a protective gene in somatic cells but also in germline cells, leading to permanent changes of the genome. But is everything we "can" do morally justifiable? To what extent does the moral status of the living being, autonomy, and privacy influence the decision of whether something is morally "good" or "bad"? There is a lack of valid instruments to study the moral judgment of genetic technologies. Therefore, the aim of this study is to translate and validate the "Genetic Technologies Questionnaire" (GTQ) and the short version of the "Conventional Technologies Questionnaire" (CTQ5) into German.

METHODS: Convenience sampling (N = 317) was used to conduct a cross-sectional online study. Analyses included internal consistency, structural validity, known group construct validity, tests for floor and ceiling effects, and retest reliability with a subset of n = 69. Correlational analyses were conducted with education, age, prior knowledge of genetics, religiosity, conventional technologies, and prior genetic testing. This study used the STROBE checklist for reporting.

RESULTS: The GTQ30 (Cronbach's α = 0.938) and GTQ20 (α = 0.940) are reliable and stable instruments for testing the moral judgment of lay people, while the GTQ5 (α = 0.857) and CTQ5 (α = 0.697) showed some weaknesses. Conventional technologies were judged morally better than genetic technologies, and genetic testing considered better than genome editing. Two additional versions were validated: the GTQ-Human (GTQ-H), using all items relating to humans, and the GTQ-Moral Status (GTQ-MS), including one item per different group of living beings for genetic testing and one for genome editing.

CONCLUSION: The GTQ is a valid instrument that is now available in shorter versions for different areas of research: the GTQ-MS for philosophical questions addressing moral status and the GTQ-H for biomedical and psychological questions related to research, prognosis, diagnosis, and therapy in humans.},
}

@article {pmid40820704,
year = {2025},
author = {Chen, F and Chen, D and Deng, T and Li, J and Jiang, JH},
title = {CRISPR-Cas-Mediated Ultrasensitive Detection of Viral Nucleic Acids via Singlet Oxygen-Activated Chemiluminescence.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.5c03994},
pmid = {40820704},
issn = {1520-6882},
abstract = {Herein, we report a novel clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-mediated chemiluminescence assay (CRISPR-Cas-CLA) for the ultrasensitive detection of viral nucleic acids of HPV18 and HPV16. The CRISPR-Cas-CLA comprises a CRISPR/Cas12a system that specifically recognizes the target nucleic acid, a signal-conducting nanoconjugate (MB-ssDNA-PSNP) formed by coupling singlet-oxygen ([1]O2)-generating photosensitive nanoparticles (PSNPs) to magnetic beads (MBs) via a single-stranded DNA (ssDNA) linker, and [1]O2-activated chemiluminescence nanoparticles (CLNPs). In the presence of the target nucleic acid, the ssDNA linker of the nanoconjugate is cleaved by the target-activated CRISPR/Cas12a system, and the PSNPs are dissociated from the MBs. The PSNP-containing supernatant obtained by magnetic separation is added to the CLNP-coated detection plate. Upon light irradiation of the CLNP-PSNP mixture in the well, strong chemiluminescence is generated with the subsequent addition of hydrogen peroxide, enabling the detection of the target nucleic acids. The proposed CRISPR-Cas-CLA system offers ultrahigh sensitivity (∼1.04 aM), simple operation, and low cost, providing a new direction for the development of PCR-free detection strategies for ultralow abundance nucleic acids.},
}

@article {pmid40819925,
year = {2025},
author = {Zhou, X and Zhou, SJ and Liu, J and Wang, YX},
title = {CRISPR/Cas system targeting RNA and its derivative technology.},
journal = {Yi chuan = Hereditas},
volume = {47},
number = {8},
pages = {842-860},
doi = {10.16288/j.yczz.24-311},
pmid = {40819925},
issn = {0253-9772},
abstract = {RNA editing is one of the important research directions in the field of epigenetics. With further research, scientists have discovered that the CRISPR/Cas system can target not only DNA but also RNA, thereby achieving precise gene editing at the transcriptional level. Moreover, using the CRISPR/Cas system for RNA editing can also avoid damage to genome. At present, a variety of derivative technologies based on RNA-targeting CRISPR systems have been developed, including RNA knockdown and editing, nucleic acid detection and imaging, and RNA tracking. The emergence of these derivative technologies provides powerful tools for deciphering biological genetic mechanisms and disease treatment. In this review, we summarize the structure, function, mechanisms, and derived technologies of RNA-targeting CRISPR/Cas systems, aiming to enrich people's understanding of CRISPR/Cas-mediated RNA editing.},
}

@article {pmid40818496,
year = {2025},
author = {Masurkar, P and Meher, J and Thapa, S and Singh, RK and Bag, MK and Gautam, V and Maurya, S},
title = {Exploring Ustilaginoidea virens, the Causal Agent of False Smut of Rice Disease: A Comprehensive Study of Infection Dynamics, Effectors, and Genetic Structure.},
journal = {Microbial pathogenesis},
volume = {},
number = {},
pages = {107987},
doi = {10.1016/j.micpath.2025.107987},
pmid = {40818496},
issn = {1096-1208},
abstract = {Villosiclava virens (Anamorph: Ustilaginoidea virens) is an important and enigmatic pathogen that causes rice false smut. Some similarities between Claviceps and Ustilaginoidea genera have been found, but according to recent genomic sequence comparison research, they have different sequences. U. virens secretes mycotoxins, which make the infected grains unfavorable for human consumption. The transcriptomic analysis and genome sequencing of U. virens showed 52,554,142 clean reads assembled into 36,496 transcripts, representing 18,534 unigenes. U. virens also contains the UvNLP protein as an NLP-specific NPP1 domain that belongs to a MAMPs class protein that acts as an elicitor for defence responses in resistant plants. Unique proteins, UvCGBP1, UvPRO1, and UvBI-1, are associated with the virulence, growth, and sporulation of U. virens. Several host QTLs- qFsr1, qFsr2, qFsr4, qFsr8, qFsr10, qFsr11 and qFsr12 have been validated in diverse background (IR28, Tequing NILs, MR183-2), and are crucial for resistance breeding. The genetic diversity of U. virens was measured using molecular markers, including RAPD, AFLP, SSR, ISSR, and, more recently, SNPs. The genetic diversity of U. virens isolates was higher among the isolates than in the geographical population. This review integrates advances in molecular biology, genomics, and host-pathogen interactions to inform sustainable management strategies. Further research is needed in early detection, chlamydospore germination, targeted fungicides, and resistant hybrid rice development.},
}

@article {pmid40817028,
year = {2025},
author = {Bouzetos, E and Ganar, KA and van der Oost, J and Deshpande, S},
title = {Cell-free screening of CRISPR-Cas activity by microfluidics-assisted in vitro compartmentalization.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2025.07.007},
pmid = {40817028},
issn = {1879-3096},
abstract = {CRISPR-Cas systems are responsible for antiviral immunity of prokaryotic cells and have been repurposed as powerful genome-editing tools. Cell-free gene expression has been applied for the rapid characterization of CRISPR-Cas systems in microtiter plates. In vitro compartmentalization makes use of artificial microcompartments that individually act as bioreactors. Here, we performed cell-free reactions of CRISPR-Cas activity into microtiter plates, which we proceeded to encapsulate into double emulsion (DE) droplets generated by on-chip microfluidics. Emulsion droplets were screened for CRISPR-Cas activity based on relative fluorescence levels using a common cell sorter, and enrichment for the expected guide (g)RNA genotype was observed. Encapsulation of single gene copies per droplet is an important prerequisite for applying this technique to complex gene libraries. We show a proof-of-principle assay for efficient, compartmentalized gene amplification using magnetic microbeads. In conclusion, we demonstrate the feasibility of microfluidics-based, high-throughput, cell-free screening of CRISPR-Cas activity.},
}

@article {pmid40816052,
year = {2025},
author = {Ma, W and Liu, Q and Zhou, N and Wang, LJ and Jiao, Z and Wang, T and Zhang, CY},
title = {Engineering of a multi-modular CRISPR biomachine for ultrasensitive monitoring of β-glucosyltransferase activity.},
journal = {Biosensors & bioelectronics},
volume = {289},
number = {},
pages = {117878},
doi = {10.1016/j.bios.2025.117878},
pmid = {40816052},
issn = {1873-4235},
abstract = {β: [note: ":" is changed to "-"] Glucosyltransferase (β-GT) is a pivotal enzymatic tool for 5-hydroxymethylcytosine (5-hmC) detection, and it can specifically catalyze the glycosylation of 5-hmC. This enzymatic reaction plays a crucial role in modulating bacteriophage-specific gene expression and facilitating the survival of bacteriophages and parasites within host cells. Herein, we engineer a multi-modular and structurally ordered CRISPR/Cas-based biomachine by integrating 5-hmC glycosylation-triggered palindrome-primed hyperbranched rolling circle amplification (PP-HRCA) for ultrasensitive analysis of exogenous β-GT activity. This biomachine integrates multiple functional modules including a β-GT-catalyzed protection module, a palindrome primer-initiated amplification module, and a CRISPR system-mediated cleavage module to improve the assay sensitivity and specificity toward exogenous β-GT. β-GT-catalyzed 5-hmC glycosylation effectively protects the dumbbell probe from MspI endonuclease-mediated digestion. The intact dumbbell probe subsequently serves as a template to initiate PP-HRCA, generating abundant long-tandem double-stranded DNA products. The amplification products can activate CRISPR/Cas-mediated cleavage of signal probes, releasing numerous Cy5 molecules that can be accurately quantified by both fluorescence spectroscopy and single-molecule imaging. This biomachine enables sensitive and zero-background detection of β-GT with a detection limit (LOD) of 2.75 × 10[-6] U/mL and a broad linear range spanning seven orders of magnitude. Moreover, it can analyze β-GT kinetic parameters and screen β-GT inhibitors, providing a robust platform for in-depth exploration of β-GT biological mechanisms and study of β-GT-related epigenetics.},
}

@article {pmid40814131,
year = {2025},
author = {Liu, P and Li, S and Zeng, J and Wang, M and Du, J and Wang, HH and Xiong, E and Yang, R},
title = {Creating a Modular Activatable CRISPR-Cas12a System by Engineering crRNA Scaffold with a Steric Hindrance Effector.},
journal = {Journal of the American Chemical Society},
volume = {},
number = {},
pages = {},
doi = {10.1021/jacs.5c11227},
pmid = {40814131},
issn = {1520-5126},
abstract = {CRISPR-Cas systems inherently lack spatiotemporal control over cleavage events, compromising their accuracy and efficiency in biotechnological applications. Although advances have been made in regulating these systems, developing modular activatable platforms adaptable to diverse stimuli remains challenging. To address this limitation, we developed an innovative modular activatable CRISPR-Cas12a system by engineering a crRNA scaffold with a steric hindrance effector (SHE) motif at its 3'-terminus, attached via a stimulus-cleavable linker. Through systematic evaluation of SHEs with varied sizes and structures─including linear, duplex, hairpin, and triplex─we identified their steric hindrance-dependent inhibitory effects on CRISPR-Cas12a functionality. Notably, the effective SHEs capable of completely inhibiting CRISPR-Cas12a functionality were successfully released upon ultraviolet light exposure, restoring system functionality. Furthermore, the crRNA scaffold-engineering strategy proved applicable to other type V-A Cas12a orthologs. Importantly, the system was adapted to respond to enzymatic stimuli (such as human apurinic/apyrimidinic endonuclease 1 [APE1]) and chemical stimuli (such as glutathione [GSH]). Finally, the modular activatable CRISPR-Cas12a system enabled light-activatable, one-pot nucleic acid diagnostics and APE1-activatable, tumor cell-selective microRNA imaging. Our study highlights the innovative use of steric hindrance to manipulate CRISPR-Cas12a functionality and provides valuable insights for the rational design of modular activatable CRISPR-Cas systems.},
}

@article {pmid40813265,
year = {2025},
author = {Zhuang, S and Bai, B and Liu, Y},
title = {CRISPR-based SNP detection technologies advance from classical methods to cutting-edge innovations.},
journal = {Chemical communications (Cambridge, England)},
volume = {},
number = {},
pages = {},
doi = {10.1039/d5cc03655k},
pmid = {40813265},
issn = {1364-548X},
abstract = {Single nucleotide polymorphisms (SNPs) constitute the most prevalent form of genetic variations, critically influencing human disease susceptibility, drug response, and pathogen evolution. Conventional SNP detection methods, however, face significant limitations: they often lack the necessary balance of precision, speed, and deployability required for diverse applications, ranging from point-of-care clinical diagnostics to rapid pathogen surveillance. The advent of CRISPR/Cas systems, particularly the discovery of the trans-cleavage activity of Cas nucleases, has revolutionized this field by offering unparalleled single-nucleotide specificity, isothermal operation, and signal amplification capabilities. In this review, we first systematically examine the foundational CRISPR-based SNP detection platforms, with a focused analysis of pioneering systems including SHERLOCK, HOLMES, and Cas14-DETECTR. Subsequently, we delve into the transformative technical advancements that have propelled these platforms towards cutting-edge innovations, emphasizing three critical pathways: (1) novel strategies for achieving ultra-high specificity in single-nucleotide discrimination, (2) breakthroughs in overcoming protospacer adjacent motif (PAM) sequence constraints, and (3) innovative approaches for optimizing sensitivity to meet stringent clinical detection thresholds. Finally, we critically evaluate the persistent challenges hindering the widespread adoption of current CRISPR-based SNP detection frameworks and propose actionable research trajectories aimed at advancing CRISPR technologies for high-precision SNP genotyping. This review provides a comprehensive overview of the remarkable evolution of CRISPR-based SNP detection, from its classical origins to its current status as a frontier innovation. It also sheds light on future directions essential for realizing the full potential of CRISPR as a transformative tool in precision medicine and global health surveillance.},
}

@article {pmid40812128,
year = {2025},
author = {Liu, J and Gao, Y},
title = {Hybrid strand displacement circuit-controlled CRISPR-Cas13a activation for one-pot sensing of Non-RNA analytes.},
journal = {Biosensors & bioelectronics},
volume = {289},
number = {},
pages = {117865},
doi = {10.1016/j.bios.2025.117865},
pmid = {40812128},
issn = {1873-4235},
abstract = {The exceptionally sensitive and specific CRISPR-Cas systems have revolutionized molecular diagnostics, but their application remains largely confined to nucleic acid targets. To overcome this limitation and enable one-pot, mix-and-read detection of non-nucleic acid analytes, we present a generalizable CRISPR-Cas13a sensing platform that utilizes a hybrid DNA:RNA strand displacement-based switch to conditionally activate Cas13a. The switch leverages Cas13a's strict requirement for single-stranded RNA activators to mediate non-RNA target-triggered activation while protecting functional DNA elements from collateral cleavage. By integrating the modular Cas13a switch with various functional DNAs, including DNAzyme, aptazyme, and duplexed aptamer, we constructed a suite of one-pot sensors capable of detecting non-RNA targets such as lead ion, ATP, and thrombin. These CRISPR-Cas13a sensors demonstrate enhanced sensitivity, high selectivity, and robust performance in complex biological matrices. The high modularity of the CRISPR-Cas13a sensor allows rapid reconfiguration for new targets by simply altering the output sequence of functional DNA without extensive re-engineering. This versatile and robust sensing strategy expands the application landscape of CRISPR-Cas systems and lays the foundation for flexible, sensitive, and one-pot detection of a wide array of clinically and environmentally relevant targets.},
}

@article {pmid40777486,
year = {2025},
author = {Taboada, VP and Wu, Y and Cassidy, R and Medvedev, KE and Loeff, L and Nemudraia, A and Nemudryi, A},
title = {Bacterial Schlafens mediate anti-phage defense.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {40777486},
issn = {2692-8205},
abstract = {Human Schlafen proteins restrict viral replication by cleaving tRNA, thereby suppressing protein synthesis. Although the ribonuclease domain of Schlafen proteins is conserved across all domains of life, its function in prokaryotes has remained unclear. Here, we show that prokaryotic Schlafen nucleases (pSlfns) are widespread antiviral effectors that protect bacteria from phages. These nucleases are fused to diverse protein domains that sense phage infection. We focus on a system where Schlafen nuclease is fused to a previously unknown immunoglobulin-like sensor domain and demonstrate that it recognizes T5-like phage tail assembly chaperones and cleaves both bacterial and viral tRNA, triggering abortive infection. Our findings redefine Schlafens as an ancient, mechanistically conserved family of immune effectors, revealing the deep evolutionary origin of tRNA-targeting antiviral immunity in humans.},
}

@article {pmid40811506,
year = {2025},
author = {Pernomian, L and Parente, JM and McCarthy, CG and Wenceslau, CF},
title = {Orphan Under Pressure: GPR146 as a Mechanotransduction Modulator.},
journal = {Circulation research},
volume = {137},
number = {5},
pages = {625-627},
doi = {10.1161/CIRCRESAHA.125.327056},
pmid = {40811506},
issn = {1524-4571},
}

@article {pmid40810510,
year = {2025},
author = {Chen, DF and Roe, LT and Yuping, L and Borges, AL and Zhang, JY and Babbar, P and Maji, S and Stevens, MGV and Correy, GJ and Diolaiti, ME and Smith, DH and Ashworth, A and Stroud, RM and Kelly, MJS and Bondy-Denomy, J and Fraser, JS},
title = {AcrIF11 is a potent CRISPR-specific ADP-ribosyltransferase encoded by phage and plasmid.},
journal = {mBio},
volume = {},
number = {},
pages = {e0169825},
doi = {10.1128/mbio.01698-25},
pmid = {40810510},
issn = {2150-7511},
abstract = {Phage-encoded anti-CRISPR (Acr) proteins inhibit CRISPR-Cas systems, allowing phage replication and lysogeny maintenance. Most of the Acrs characterized to date are stable stoichiometric inhibitors. While enzymatic Acrs have been characterized biochemically, little is known about their potency, specificity, and reversibility. Here, we examine AcrIF11, a widespread phage and plasmid-encoded ADP-ribosyltransferase (ART) that inhibits the Type I-F CRISPR-Cas system. We present a nuclear magnetic resonance (NMR) structure of an AcrIF11 homolog that reveals chemical shift perturbations consistent with NAD (cofactor) binding. In experiments that model both lytic phage replication and MGE/lysogen stability under high targeting pressure, AcrIF11 is a highly potent CRISPR-Cas inhibitor and more robust to Cas protein-level fluctuations than stoichiometric inhibitors. Furthermore, we demonstrate that AcrIF11 is remarkably specific, predominantly ADP-ribosylating Csy1 when expressed in P. aeruginosa. Given the reversible nature of ADP-ribosylation, we hypothesized that ADPr eraser enzymes (macrodomains) could remove ADPr from Csy1, a potential limitation of PTM-based CRISPR inhibition. We demonstrate that a human macrodomain can indeed remove the modification from Csy1 in P. aeruginosa lysate. Together, these experiments connect the in vitro observations of AcrIF11's enzymatic activity to its potent and specific effects in vivo, clarifying the advantages and drawbacks of enzymatic Acrs in the evolutionary arms race between phages and bacteria.IMPORTANCEBacteria have evolved diverse immune systems to prevent phage infection, and, consequently, phages have evolved diverse methods of evading bacterial immune systems. To evade the bacterial CRISPR-Cas immune system, phages encode anti-CRISPR proteins (Acrs). Acrs disable CRISPR-Cas by either stably binding to the CRISPR-Cas complex or by enzymatic modification. However, Acr enzymes have not been characterized in vivo during lytic infection or lysogenic maintenance. Here, we report the benefits and drawbacks of enzymatic inhibition with AcrIF11, an ADP-ribosyltransferase. Under "high pressure" scenarios such as high CRISPR targeting or CRISPR-Cas overexpression, AcrIF11 outperforms a strong, stable binding Acr by very specifically modifying the Cas8 protein, but nothing else in the cell. AcrIF11 additionally stabilizes lysogeny effectively, but the ADP-ribose modification can potentially be removed by macrodomains, which are ADP-ribose eraser enzymes. AcrIF11 is therefore a potent and widespread plasmid/phage-encoded inhibitor of Type I-F CRISPR-Cas systems with catalytic activity.},
}

@article {pmid40808298,
year = {2025},
author = {Andersen, S and Wolff, JH and Skov, TW and Janns, JH and Davis, LJ and Haldrup, JH and Haslund, D and Revenfeld, AL and Relkovic, D and Møller, BK and Lund, J and Bak, RO and Thomsen, EA and Mikkelsen, JG},
title = {Gene editing in hematopoietic stem cells by co-delivery of Cas9/sgRNA ribonucleoprotein and templates for homology-directed repair in 'all-in-one' lentivirus-derived nanoparticles.},
journal = {Nucleic acids research},
volume = {53},
number = {15},
pages = {},
doi = {10.1093/nar/gkaf767},
pmid = {40808298},
issn = {1362-4962},
support = {//Innovation Fund Denmark/ ; //Novo Nordisk A/S/ ; 8056-00010A//Innovation Fund Denmark/ ; NNF22OC0080684//Novo Nordisk Foundation/ ; 4285-00102B//Independent Research Fund Denmark/ ; CF21-0363//Carlsberg Foundation/ ; },
abstract = {Repair of double-strand DNA breaks generated by site-directed endonucleases, like Cas9, is the hallmark of gene editing based on homology-directed repair (HDR). HDR uses an exogenous DNA template to restore the cleaved DNA sequence and can facilitate specific gene corrections as well as insertion of genes or partial complementary DNA (cDNA) sequences. For CRISPR/Cas-directed gene editing, co-administration of the Cas9/single guide RNA (sgRNA) ribonucleoprotein (RNP) complex and a DNA template typically involves two different delivery strategies or different types of vehicles. This requires exquisite timing of delivery and may potentially challenge safety and therapeutic applicability. There is a need therefore for technologies that can ferry complete editing tool kits into cells. Here, we demonstrate the use of lentivirus-derived nanoparticles (LVNPs) to transport both RNP complexes and vector RNA, which upon reverse transcription serves as a repair template for HDR-directed gene editing. Such 'all-in-one' LVNPs support targeted gene insertion with reduced off-target effects relative to nucleofection procedures. We show potent editing in the HBB gene in human erythroid progenitor cells as well as HDR-directed editing in hematopoietic stem and progenitor cells. Our findings mark a first step toward using a single virus-derived vehicle for delivering a full HDR gene editing kit.},
}

@article {pmid40807307,
year = {2025},
author = {Alessa, O and Aiba, Y and Arbaah, M and Hidaka, Y and Watanabe, S and Miyanaga, K and Wannigama, DL and Cui, L},
title = {Synthetic and Functional Engineering of Bacteriophages: Approaches for Tailored Bactericidal, Diagnostic, and Delivery Platforms.},
journal = {Molecules (Basel, Switzerland)},
volume = {30},
number = {15},
pages = {},
doi = {10.3390/molecules30153132},
pmid = {40807307},
issn = {1420-3049},
support = {(grant No. JP25ae0121045, JP25gm1610002, and JP25fk0108698), (Grant No. JP24H00662, JP24K22027, JP25K13511, and JP25K19273) and (JPJ009237).//This research was funded by the Japan Agency for Medical Research and Development JSPS KAKENHI and partially by Moonshot R&D Program for Agriculture, Forestry, Fisheries/ ; },
abstract = {Bacteriophages (phages), the most abundant biological entities on Earth, have long served as both model systems and therapeutic tools. Recent advances in synthetic biology and genetic engineering have revolutionized the capacity to tailor phages with enhanced functionality beyond their natural capabilities. This review outlines the current landscape of synthetic and functional engineering of phages, encompassing both in-vivo and in-vitro strategies. We describe in-vivo approaches such as phage recombineering systems, CRISPR-Cas-assisted editing, and bacterial retron-based methods, as well as synthetic assembly platforms including yeast-based artificial chromosomes, Gibson, Golden Gate, and iPac assemblies. In addition, we explore in-vitro rebooting using TXTL (transcription-translation) systems, which offer a flexible alternative to cell-based rebooting but are less effective for large genomes or structurally complex phages. Special focus is given to the design of customized phages for targeted applications, including host range expansion via receptor-binding protein modifications, delivery of antimicrobial proteins or CRISPR payloads, and the construction of biocontained, non-replicative capsid systems for safe clinical use. Through illustrative examples, we highlight how these technologies enable the transformation of phages into programmable bactericidal agents, precision diagnostic tools, and drug delivery vehicles. Together, these advances establish a powerful foundation for next-generation antimicrobial platforms and synthetic microbiology.},
}

@article {pmid40806631,
year = {2025},
author = {Sapakhova, Z and Kanat, R and Choi, K and Daurov, D and Daurova, A and Zhambakin, K and Shamekova, M},
title = {CRISPR-Cas Gene Editing Technology in Potato.},
journal = {International journal of molecular sciences},
volume = {26},
number = {15},
pages = {},
doi = {10.3390/ijms26157496},
pmid = {40806631},
issn = {1422-0067},
support = {BR21882269//The Science Committee of the Ministry of Science and Higher Education, Republic of Kazakhstan/ ; },
abstract = {Potato (Solanum tuberosum L.) is one of the most important food crops in the world, ranking fourth after rice, maize, and wheat. Potatoes are exposed to biotic and abiotic environmental factors, which lead to economic losses and increase the possibility of food security threats in many countries. Traditional potato breeding faces several challenges, primarily due to its genetic complexity and the time-consuming nature of the process. Therefore, gene editing-CRISPR-Cas technology-allows for more precise and rapid changes to the potato genome, which can speed up the breeding process and lead to more effective varieties. In this review, we consider CRISPR-Cas technology as a potential tool for plant breeding strategies to ensure global food security. This review summarizes in detail current and potential technological breakthroughs that open new opportunities for the use of CRISPR-Cas technology for potato breeding, as well as for increasing resistance to abiotic and biotic stresses, and improving potato tuber quality. In addition, the review discusses the challenges and future perspectives of the CRISPR-Cas system in the prospects of the development of potato production and the regulation of gene-edited crops in different countries around the world.},
}

@article {pmid40806610,
year = {2025},
author = {Shaposhnikov, LA and Rozanov, AS and Sazonov, AE},
title = {Genome-Editing Tools for Lactic Acid Bacteria: Past Achievements, Current Platforms, and Future Directions.},
journal = {International journal of molecular sciences},
volume = {26},
number = {15},
pages = {},
doi = {10.3390/ijms26157483},
pmid = {40806610},
issn = {1422-0067},
support = {№18-03//Sirius University of Science and Technology/ ; },
abstract = {Lactic acid bacteria (LAB) are central to food, feed, and health biotechnology, yet their genomes have long resisted rapid, precise manipulation. This review charts the evolution of LAB genome-editing strategies from labor-intensive RecA-dependent double-crossovers to state-of-the-art CRISPR and CRISPR-associated transposase systems. Native homologous recombination, transposon mutagenesis, and phage-derived recombineering opened the door to targeted gene disruption, but low efficiencies and marker footprints limited throughput. Recent phage RecT/RecE-mediated recombineering and CRISPR/Cas counter-selection now enable scar-less point edits, seamless deletions, and multi-kilobase insertions at efficiencies approaching model organisms. Endogenous Cas9 systems, dCas-based CRISPR interference, and CRISPR-guided transposases further extend the toolbox, allowing multiplex knockouts, precise single-base mutations, conditional knockdowns, and payloads up to 10 kb. The remaining hurdles include strain-specific barriers, reliance on selection markers for large edits, and the limited host-range of recombinases. Nevertheless, convergence of phage enzymes, CRISPR counter-selection and high-throughput oligo recombineering is rapidly transforming LAB into versatile chassis for cell-factory and therapeutic applications.},
}

@article {pmid40806599,
year = {2025},
author = {Yang, L and Lu, Q},
title = {Beyond Cutting: CRISPR-Driven Synthetic Biology Toolkit for Next-Generation Microalgal Metabolic Engineering.},
journal = {International journal of molecular sciences},
volume = {26},
number = {15},
pages = {},
doi = {10.3390/ijms26157470},
pmid = {40806599},
issn = {1422-0067},
abstract = {Microalgae, with their unparalleled capabilities for sunlight-driven growth, CO2 fixation, and synthesis of diverse high-value compounds, represent sustainable cell factories for a circular bioeconomy. However, industrial deployment has been hindered by biological constraints and the inadequacy of conventional genetic tools. The advent of CRISPR-Cas systems initially provided precise gene editing via targeted DNA cleavage. This review argues that the true transformative potential lies in moving decisively beyond cutting to harness CRISPR as a versatile synthetic biology "Swiss Army Knife". We synthesize the rapid evolution of CRISPR-derived tools-including transcriptional modulators (CRISPRa/i), epigenome editors, base/prime editors, multiplexed systems, and biosensor-integrated logic gates-and their revolutionary applications in microalgal engineering. These tools enable tunable gene expression, stable epigenetic reprogramming, DSB-free nucleotide-level precision editing, coordinated rewiring of complex metabolic networks, and dynamic, autonomous control in response to environmental cues. We critically evaluate their deployment to enhance photosynthesis, boost lipid/biofuel production, engineer high-value compound pathways (carotenoids, PUFAs, proteins), improve stress resilience, and optimize carbon utilization. Persistent challenges-species-specific tool optimization, delivery efficiency, genetic stability, scalability, and biosafety-are analyzed, alongside emerging solutions and future directions integrating AI, automation, and multi-omics. The strategic integration of this CRISPR toolkit unlocks the potential to engineer robust, high-productivity microalgal cell factories, finally realizing their promise as sustainable platforms for next-generation biomanufacturing.},
}

@article {pmid40804951,
year = {2025},
author = {Li, G and Zhou, X and Zhu, G and Pan, Y and Yan, J and Meng, J and Ye, T and Cheng, Y and Liu, C and Gu, Z},
title = {Macrobrachium rosenbergii Genome Editing Breeding with CRISPR-Cas Nucleases, Base Editors, and Prime Editors.},
journal = {Animals : an open access journal from MDPI},
volume = {15},
number = {15},
pages = {},
doi = {10.3390/ani15152161},
pmid = {40804951},
issn = {2076-2615},
support = {2024SSYS0101//"Pioneer" and "Leading Goose" R&D Program of Zhejiang/ ; 32401241//National Natural Science Foundation of China/ ; },
abstract = {This review focuses on CRISPR genome editing technology, particularly its application in the study of Macrobrachium rosenbergii (M. rosenbergii). It first elaborates on the basic principles and mechanisms of CRISPR-Cas9 technology, base editors, and prime editors. Then, it explores the application of this technology in M. rosenbergii breeding, including improving growth rate, enhancing disease resistance, and sex control. Additionally, it introduces the progress of genome editing technology in M. rosenbergii, epidemiology and pathogenesis, diagnostic techniques, analyzes the opportunities and challenges it faces, reviews the historical evolution, and looks ahead to future development directions. CRISPR technology has brought new opportunities to the research and industrial development of M. rosenbergii, but it also needs to address numerous technical and safety challenges.},
}

@article {pmid40804123,
year = {2025},
author = {Yu, M and Ai, L and Wang, B and Lian, S and Ip, L and Liu, J and Li, L and Tsai, SQ and Kleinstiver, BP and Zheng, Z},
title = {GenomePAM directs PAM characterization and engineering of CRISPR-Cas nucleases using mammalian genome repeats.},
journal = {Nature biomedical engineering},
volume = {},
number = {},
pages = {},
pmid = {40804123},
issn = {2157-846X},
support = {202001418//Vetenskapsrådet (Swedish Research Council)/ ; 11103024//Research Grants Council, University Grants Committee (RGC, UGC)/ ; T12-101/23-N//Research Grants Council, University Grants Committee (RGC, UGC)/ ; DP2-CA281401//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; },
abstract = {Characterizing the protospacer adjacent motif (PAM) requirements of different Cas enzymes is a bottleneck in the discovery of Cas proteins and their engineered variants in mammalian cell contexts. Here, to overcome this challenge and to enable more scalable characterization of PAM preferences, we develop a method named GenomePAM that allows for direct PAM characterization in mammalian cells. GenomePAM leverages genomic repetitive sequences as target sites and does not require protein purification or synthetic oligos. GenomePAM uses a 20-nt protospacer that occurs ~16,942 times in every human diploid cell and is flanked by nearly random sequences. We demonstrate that GenomePAM can accurately characterize the PAM requirement of type II and type V nucleases, including the minimal PAM requirement of the near-PAMless SpRY and extended PAM for CjCas9. Beyond PAM characterization, GenomePAM allows for simultaneous comparison of activities and fidelities among different Cas nucleases on thousands of match and mismatch sites across the genome using a single gRNA and provides insight into the genome-wide chromatin accessibility profiles in different cell types.},
}

@article {pmid40809473,
year = {2023},
author = {Mecacci, S and Torregrosa-Barragán, L and Asin-Garcia, E and Smith, RW},
title = {Multilayered safety framework for living diagnostics in the colon.},
journal = {Frontiers in systems biology},
volume = {3},
number = {},
pages = {1240040},
pmid = {40809473},
issn = {2674-0702},
abstract = {Introduction: Colorectal cancer is the second most deadly cancer worldwide. Current screening methods have low detection rates and frequently provide false positive results, leading to missed diagnoses or unnecessary colonoscopies. To tackle this issue, the Wageningen UR iGEM team from 2022 developed "Colourectal", a living diagnostic tool for colorectal cancer. Following a synthetic biology approach, the project used an engineered Escherichia coli Nissle 1917 strain capable of binding to tumour cells that detects two distinct cancer biomarkers, and secretes a coloured protein observable in stool. Due to the utilization of genetically modified bacteria in vivo, precautionary biosafety measures were included within a three level safe-by-design strategy. Results: The first genetic safeguard ensured confinement of the living diagnostic to the colon environment by implementing auxotrophy to mucin that is abundant in the colon lining. For this, a synthetic chimeric receptor was generated to ensure expression of essential genes in the presence of mucin. The second strategy limited the viability of the engineered bacteria to the human body, preventing proliferation in open environments. The use of a temperature sensitive kill switch induced bacterial cell death at temperatures below 37°C. The third biocontainment strategy was installed as an emergency kill switch to stop the Colourectal test at any point. By inducing a highly genotoxic response through CRISPR-Cas-mediated DNA degradation, cell death of E. coli Nissle is triggered. Discussion: While the use of engineered microorganisms in human applications is not yet a reality, the safety considerations of our multi-layered strategy provide a framework for the development of future living diagnostic tools.},
}

@article {pmid40803977,
year = {2025},
author = {Li, L and Zhang, Z and Zhang, B},
title = {CRISPR meets AlphaFold: guiding SWEET10-enhanced oil production.},
journal = {Trends in plant science},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tplants.2025.08.001},
pmid = {40803977},
issn = {1878-4372},
abstract = {Enhancing seed oil content significantly benefits both human welfare and environmental sustainability. Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) and artificial intelligence (AI) are transformative tools for crop trait improvement. A recent study by Wang and colleagues reported that AlphaFold-guided CRISPR genome editing of SWEET10 boosts oil contents, highlighting a breakthrough in precision crop engineering.},
}

@article {pmid40803657,
year = {2025},
author = {Ceasar, SA and Ebeed, HT and Ramakrishnan, M and García-Caparrós, P and Ignacimuthu, S},
title = {Understanding low-phosphate stress responses in plants: Opportunities for genome editing to improve phosphorous use efficiency (PUE).},
journal = {Biotechnology advances},
volume = {},
number = {},
pages = {108686},
doi = {10.1016/j.biotechadv.2025.108686},
pmid = {40803657},
issn = {1873-1899},
abstract = {Phosphorus (P) is a critical macronutrient essential for plant growth, yet its availability in soil is often limited due to poor mobility and fixation with metal ions due to acidic or alkaline soils. Plants have evolved complex adaptive responses to overcome phosphate (Pi) deficiency. Recent advancements in genome editing, particularly CRISPR/Cas tools, offer opportunities to enhance these adaptive traits for sustainable agriculture. This review consolidates current understanding of low Pi stress signaling pathways, including morphological (root architecture changes), biochemical (hormone regulation, lipid modification, organic acid exudation), and molecular (transcription factors (TFs), phosphate transporters, and microRNAs), and identifies prime candidate genes for genome editing applications. Key regulators such as phosphate transporter (PHT, PHO1), TFs (PHR1, WRKYs) and microRNAs (miR399/827) manage Pi uptake, redistribution, and signaling. Genome editing strategies targeting root-specific traits, hormonal integration, lipid remodeling, and transcriptional regulation are discussed as viable ways for improving phosphorous use efficiency (PUE). Harnessing CRISPR/Cas tools can lead to the development of crops with optimized PUE, reduced dependency on synthetic fertilizers, and improved adaptability to Pi-deficient soils. The review provides a comprehensive roadmap for researchers and breeders to apply CRISPR/Cas technology toward building next-generation crops capable of thriving under low Pi conditions.},
}

@article {pmid40803478,
year = {2025},
author = {Jian, A and Zhao, G and Wang, Y and Wang, S and Li, N},
title = {Watershed Year of Cell and Gene Therapy (CGT): A Review of 2024 CGT Approvals.},
journal = {Cancer letters},
volume = {},
number = {},
pages = {217980},
doi = {10.1016/j.canlet.2025.217980},
pmid = {40803478},
issn = {1872-7980},
abstract = {The year 2024 is a pivotal year for therapeutic breakthroughs in human diseases, alongside with an uprising growth in precision medicine, especially in cell and gene therapies (CGTs), marked by an unprecedented approval number of 13 novel CGTs authorized by the U.S. FDA, China's NMPA, E.U. EMA, and Japan's PMDA. 2024 is also a year of many firsts: the first CRISPR therapy, the first MRI-guided intracranial AAV delivery gene therapy, and the first tumor infiltration lymphocyte therapy, opening a whole new chapter of clinical translation of innovations in gene-editing and cell technologies. CGTs represent an emerging translational modality in precision medicine through utilization of cellular or genetic materials to treat or prevent disease, offering curative potential for previously refractory diseases. Despite thriving in CGTs' development, comprehensive analyses of 2024 approvals remain absent. This review employs a quadruple axle comparative framework to analyze 2024-approved CGTs in China and USA, integrating mechanistic innovation with gradually improving regulatory advancements. Raw data was extracted from official agency databases, pivotal trials, and manufacturing reports. This work further delineates how mechanistic diversity converges with regulatory agility to redefine therapeutic development.},
}

@article {pmid40803134,
year = {2025},
author = {Wu, F and Campbell, BC and Greenfield, P and Hose, GC and Midgley, DJ and George, SC},
title = {There and back again: Genomic insights into microbial life in a recirculating petroleum refinery wastewater biotreatment system.},
journal = {Microbiological research},
volume = {301},
number = {},
pages = {128299},
doi = {10.1016/j.micres.2025.128299},
pmid = {40803134},
issn = {1618-0623},
abstract = {Petroleum refinery wastewater biotreatment relies on microbes to remediate carbon, nitrogen, and sulfur compounds, yet their life strategies and ecological roles remain unclear. This study characterises the ecological functions of 20 metagenome-assembled genomes (MAGs) from a full-scale petroleum refinery wastewater treatment plant in southern China. The taxonomic identity, nutrient metabolism genes (including C/N/S cycling), carbohydrate-active enzymes, and CRISPR-Cas systems of these MAGs were analysed. The recovered MAGs represented bacteria primarily from the Pseudomonadota and Bacteroidota phyla. The major carbon sources for the represented organisms are likely aromatic and aliphatic compounds, as well as carbohydrates including peptidoglycan, chitin, and starch. Almost all MAGs contained genes for nitrate or nitrite reduction, while metabolic pathways for sulfur metabolism were generally less prevalent. Meiothermus sp. bin.89 was the most metabolically versatile MAG. This organism possessed genes that allowed it to recycle biomass, break down aliphatic and monoaromatic compounds, and perform anaerobic respiration using nitrate. However, it was likely the most susceptible to viral predation, as indicated by the high abundance of CRISPR spacers. Overall, the results revealed that stress-tolerant ecological traits were common among organisms in this microbiome, showcasing the ability of the microbes to obtain carbon from aromatic and aliphatic compounds. This study provides a substantial contribution towards future efforts in optimising microbiome stability for pollutant removal in petroleum refinery wastewater biotreatment systems.},
}

@article {pmid40802883,
year = {2025},
author = {Song, N and Wang, L and Zhang, L and Tian, G and Yao, C and Yang, D},
title = {Precision Delivery of CRISPR/Cas Systems via DNA Nanostructures for Gene Therapy and Intracellular Detection.},
journal = {Chembiochem : a European journal of chemical biology},
volume = {},
number = {},
pages = {e202500357},
doi = {10.1002/cbic.202500357},
pmid = {40802883},
issn = {1439-7633},
support = {22225505//National Natural Science Foundation of China/ ; 22322407//National Natural Science Foundation of China/ ; 22174097//National Natural Science Foundation of China/ ; 224B2505//National Natural Science Foundation of China/ ; },
abstract = {The CRISPR/Cas system represents a transformative breakthrough in genome editing technology, featuring three principal effector proteins with distinct functionalities: Cas9, which induces site-specific double-strand breaks guided by a single guide RNA, enabling precise gene knockout and knock-in modifications; Cas12, which mediates targeted DNA cleavage through cis-activity while exhibiting nonspecific trans-cleavage of single-stranded DNA, a property exploited for ultrasensitive nucleic acid detection in molecular diagnostics; and Cas13, an RNA-guided RNase that specifically degrades complementary RNA transcripts, demonstrating significant potential for antiviral therapies and transcriptome regulation. Despite these advances, the clinical translation of CRISPR/Cas systems faces substantial challenges, particularly in achieving efficient and controllable delivery. This reviewsystematically examines current delivery modalities for CRISPR/Cas systems, with particular emphasis on the implementation of DNA-based functional materials as advanced delivery vehicles. The integration of multifunctional DNA nanostructures with diverse CRISPR/Cas systems may facilitate the development of integrated theranostic platforms, thereby advancing precision medicine through synergistic bioengineering approaches.},
}

@article {pmid40801580,
year = {2025},
author = {Leal, AF and Prieto, LE and Pachajoa, H},
title = {CRISPR/Cas-Based Ex Vivo Gene Therapy and Lysosomal Storage Disorders: A Perspective Beyond Cas9.},
journal = {Cells},
volume = {14},
number = {15},
pages = {},
doi = {10.3390/cells14151147},
pmid = {40801580},
issn = {2073-4409},
abstract = {Lysosomal storage disorders (LSDs) are inherited metabolic conditions characterized by lysosomal enzyme deficiencies leading to substrate accumulation. As genetic diseases, LSDs can be treated with gene therapies (GT), including the CRISPR/Cas systems. The CRISPR/Cas systems enable precise and programmable genome editing, leading to targeted modifications at specific genomic loci. While the classical CRISPR/Cas9 system has been extensively used to generate LSD disease models and correct disease-associated genetic alterations through homologous recombination (HR), recently described Cas proteins as well as CRISPR/Cas9-derived strategies such as base editing, prime editing, and homology-independent targeted integration (HITI) offer a novel way to develop innovative treatments for LSDs. The direct administration of the CRISPR/Cas9 system remains the primary strategy evaluated in several LSDs; nevertheless, the ex vivo CRISPR/Cas9-based approach has been recently explored, primarily in central nervous system-affecting LSDs. Ex vivo approaches involve genetically modifying, in theory, any patient cells in the laboratory and reintroducing them into the patient to provide a therapeutic effect. This manuscript reviews the molecular aspects of the CRISPR/Cas technology and its implementation in ex vivo strategies for LSDs while discussing novel approaches beyond the classical CRISPR/Cas9 system.},
}

@article {pmid40801057,
year = {2025},
author = {Talibli, F and Voß, B},
title = {Metagenomic CRISPR Array Analysis Tool: a novel graph-based approach to finding CRISPR arrays in metagenomic datasets.},
journal = {microLife},
volume = {6},
number = {},
pages = {uqaf016},
pmid = {40801057},
issn = {2633-6693},
abstract = {Clustered Regularly Interspersed Short Palindromic Repeats and CRISPR-associated genes (CRISPR-Cas) is a bacterial immune system also famous for its use in genome editing. The diversity of known systems could be significantly increased by metagenomic data. Here we present the Metagenomic CRISPR Array Analysis Tool (MCAAT), a highly sensitive algorithm for finding CRISPR arrays in unassembled metagenomic data. It takes advantage of the properties of CRISPR arrays that form multicycles in de Bruijn graphs. We show that MCAAT reliably predicts CRISPR arrays in bacterial genome sequences and that its assembly-free graph-based strategy outperforms assembly-based workflows and other assembly-free methods on synthetic and real metagenomes. Our new approach will help to increase the diversity of known CRISPR-Cas systems and enable studies of spacer evolution within metagenomic data sets.},
}

@article {pmid40796771,
year = {2025},
author = {Wolff, JH and Skov, TW and Haslund, D and Dorset, SR and Revenfeld, ALS and Aussel, C and Jørgensen, SE and Holm, M and Thomsen, MK and Ammann, S and Cathomen, T and Mogensen, TH and Møller, BK and Bak, RO and Mikkelsen, JG},
title = {Targeted gene editing and near-universal cDNA insertion of CYBA and CYBB as a treatment for chronic granulomatous disease.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {7475},
pmid = {40796771},
issn = {2041-1723},
support = {8056-00010A//Innovationsfonden (Innovation Fund Denmark)/ ; NNF220C0080684//Novo Nordisk Fonden (Novo Nordisk Foundation)/ ; CF21-0363//Carlsbergfondet (Carlsberg Foundation)/ ; CA 311/4-1 & FANEDIT/EJPRD20-209//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
abstract = {Chronic granulomatous disease (CGD) is a severe inborn error of immunity caused by NADPH oxidase defects. Here, we develop CRISPR/Cas9-based gene editing strategies for correction of variants in the CYBA and CYBB genes causing CGD. For X-linked CGD, we also develop a near-universal gene editing strategy by targeted integration of a truncated CYBB cDNA in CD34[+] hematopoietic stem and progenitor cells (HSPCs). Throughout, off-target editing and chromosomal translocations are evident, which negatively impact the ability of gene-edited HSPCs to engraft in immunodeficient mice. However, by employing a high-fidelity Cas9 to minimize off-target editing, we demonstrate restoration of the multilineage engraftment potential of gene-edited HSPCs. Moreover, to further improve safety, we develop a D10A Cas9n editing approach with no detectable off-target activity or chromosomal translocations. Collectively, through risk assessments of different gene editing approaches, we present a D10A Cas9n-based strategy with improved safety, offering a potentially curative treatment for CGD patients.},
}

@article {pmid40796304,
year = {2025},
author = {Qing, Y and Liao, Z and An, D and Zeng, Y and Zhu, Q and Zhang, X},
title = {Comparative genomics reveals the genetic diversity and plasticity of Clostridium tertium.},
journal = {Journal of applied microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1093/jambio/lxaf201},
pmid = {40796304},
issn = {1365-2672},
abstract = {AIMS: Clostridium tertium, increasingly recognized as the emerging human pathogen frequently isolated from environmental and clinical specimens, remains genetically underexplored despite its clinical relevance. This study aims to explore the genetic characteristics of C. tertium by genomic analysis.

METHODS AND RESULTS: This study presented a comprehensive genomic investigation of 45 C. tertium strains from the GenBank database. Genome sizes (3.27-4.55 Mbp) and coding gene counts varied markedly across strains. Phylogenetic analyses based on 16S rRNA gene and core genome uncovered distinct intra-species lineages, including evolutionarily divergent clusters likely shaped by niche specialization. Pan-genomic analysis confirmed an open genome, with accessory and strain-specific genes enriched in functions related to environmental adaptation and regulation. Functional annotation further identified diverse virulence factor genes (e.g. clpP, nagK) and antibiotic resistance genes (e.g. vatB, tetA(P)) co-occurring with mobile genetic elements (MGEs), suggesting that horizontal gene transfer (HGT) may be a key driver of genome plasticity in C. tertium. Notably, one-third of the strains carried CRISPR-Cas systems, indicating the defense potential against exogenous genetic elements.

CONCLUSIONS: C. tertium exhibited extensive genetic diversity and genome plasticity, probably driven by MGE-mediated HGT, defense mechanisms of CRISPR-Cas systems, and functional adaptation related to virulence and resistance. These traits may underlie its ability to colonize diverse environments and acquire pathogenicity and resistance.},
}

@article {pmid40794864,
year = {2025},
author = {Wang, F and Zhao, P and Bi, X and Zheng, R and Tian, X and Xu, J and Jiang, S and Li, G and Shen, Y and She, Q},
title = {Cyclic tetraadenylate binding induces dimerization of protein dimers to activate a CRISPR-associated PIN nuclease.},
journal = {Nucleic acids research},
volume = {53},
number = {14},
pages = {},
doi = {10.1093/nar/gkaf744},
pmid = {40794864},
issn = {1362-4962},
support = {2020YFA0906800//National Key R&D Program of China/ ; 31771380//National Natural Science Foundation of China/ ; 32270040//National Natural Science Foundation of China/ ; 32393972//National Natural Science Foundation of China/ ; SKLMTFCP-2023-05//Frontiers and Challenges/ ; ZR2024QC307//State Key Laboratory of Microbial Technology/ ; //Shandong Provincial Natural Science Foundation/ ; },
mesh = {*CRISPR-Cas Systems ; Protein Multimerization ; *Archaeal Proteins/chemistry/metabolism/genetics ; *CRISPR-Associated Proteins/metabolism/chemistry/genetics ; Protein Binding ; *Adenine Nucleotides/metabolism/chemistry ; Models, Molecular ; *Endonucleases/metabolism/chemistry/genetics ; Crystallography, X-Ray ; Protein Domains ; },
abstract = {Type III CRISPR-Cas systems synthesize cyclic oligoadenylates (cOAs), the second messengers that bind to the CARF (CRISPR-associated Rossman fold) sensor domain and allosterically activate the effector domain of CRISPR ancillary effectors to mediate antiviral defense. An arsenal of such effectors has been identified, but only a minority of them have been characterized thus far. Here, CaPN (a CRISPR-associated PIN domain nuclease), a novel effector protein encoded by Saccharolobus islandicus, was characterized. Biochemical characterization of CaPN revealed that the CARF domain senses cA4 (cyclic tetraadenylate), and its binding to the CARF domain activates the PIN domain for robust RNA cleavage. Genetic assay showed that CaPN mediates growth arrest/cell death to its archaeal host upon cA4 sensing. Determination of the crystal structures of CaPN in apo and in the cA4-bound form revealed that cA4-CARF interactions trigger the conformational changes, leading to the dimerization of the CaPN dimers. These structural changes reposition D296, one of the active site residues in the catalytic pocket, to yield an active PIN domain nuclease. Together, these results unveil a novel molecular mechanism for the activation of cOA-activated Cas ancillary RNases in the CRISPR signaling pathway.},
}

*CRISPR-Cas Systems
Protein Multimerization
*Archaeal Proteins/chemistry/metabolism/genetics
*CRISPR-Associated Proteins/metabolism/chemistry/genetics
Protein Binding
*Adenine Nucleotides/metabolism/chemistry
Models, Molecular
*Endonucleases/metabolism/chemistry/genetics
Crystallography, X-Ray
Protein Domains

@article {pmid40794863,
year = {2025},
author = {Brenker, L and Aschenbrenner, S and Bubeck, F and Staykov, K and Gebhardt, C and Wolf, B and Jendrusch, M and Kröll, AS and Becker, J and Ambiel, I and Fackler, OT and Grimm, D and Mathony, J and Niopek, D},
title = {A versatile anti-CRISPR platform for opto- and chemogenetic control of CRISPR-Cas9 and Cas12 across a wide range of orthologs.},
journal = {Nucleic acids research},
volume = {53},
number = {14},
pages = {},
doi = {10.1093/nar/gkaf752},
pmid = {40794863},
issn = {1362-4962},
support = {453202693//German Research Foundation/ ; //Aventis foundation/ ; //Rolf. M. Schwiete Stiftung/ ; //Ministry of Science, Research and Culture Baden-Württemberg/ ; },
mesh = {*CRISPR-Cas Systems/genetics/drug effects ; *Gene Editing/methods ; Humans ; *Optogenetics/methods ; *CRISPR-Associated Proteins/antagonists & inhibitors/genetics/metabolism ; CRISPR-Associated Protein 9/antagonists & inhibitors/genetics ; Ligands ; Bacterial Proteins/genetics/metabolism ; HEK293 Cells ; Chemogenetics ; },
abstract = {CRISPR-Cas technologies have revolutionized life sciences by enabling programmable genome editing across diverse organisms. Achieving dynamic and precise control over CRISPR-Cas activity with exogenous triggers, such as light or chemical ligands, remains an important need. Existing tools for CRISPR-Cas control are often limited to specific Cas orthologs or selected applications, restricting their versatility. Anti-CRISPR (Acr) proteins are natural inhibitors of CRISPR-Cas systems and provide a flexible regulatory layer but are constitutively active in their native forms. In this study, we built on our previously reported concept for optogenetic CRISPR-Cas control with engineered, light-switchable anti-CRISPR proteins and expanded it from ortholog-specific Acrs towards AcrIIA5 and AcrVA1, broad-spectrum inhibitors of CRISPR-Cas9 and CRISPR-Cas12a, respectively. We then conceived and implemented a novel, chemogenetic anti-CRISPR platform based on engineered, circularly permuted ligand receptor domains, that together respond to six clinically relevant drugs. The resulting toolbox achieves both optogenetic and chemogenetic control of genome editing in human cells with a wide range of CRISPR-Cas effectors, including type II-A and II-C CRISPR-Cas9s, and CRISPR-Cas12a. In sum, this work establishes a versatile platform for the multidimensional control of CRISPR-Cas systems, with immediate applications in basic research and biotechnology, and with the potential for therapeutic use in the future.},
}

*CRISPR-Cas Systems/genetics/drug effects
*Gene Editing/methods
Humans
*Optogenetics/methods
*CRISPR-Associated Proteins/antagonists & inhibitors/genetics/metabolism
CRISPR-Associated Protein 9/antagonists & inhibitors/genetics
Ligands
Bacterial Proteins/genetics/metabolism
HEK293 Cells
Chemogenetics

@article {pmid40794700,
year = {2025},
author = {Tagliaferri, TL and Krüttgen, A and Mendes, TAO and Gonçalves Dos Santos, S and Horz, HP},
title = {CRISPR-Cas9 targeting the blaKPC gene in a clinical isolate of Klebsiella michiganensis: Reduction of imipenem resistance and changes in genomic carbapenem resistance determinants.},
journal = {PloS one},
volume = {20},
number = {8},
pages = {e0328521},
doi = {10.1371/journal.pone.0328521},
pmid = {40794700},
issn = {1932-6203},
mesh = {*CRISPR-Cas Systems/genetics ; *Klebsiella/genetics/drug effects/isolation & purification ; *Bacterial Proteins/genetics ; *Imipenem/pharmacology ; *beta-Lactamases/genetics ; Anti-Bacterial Agents/pharmacology ; Microbial Sensitivity Tests ; Humans ; Escherichia coli/genetics/drug effects ; Plasmids/genetics ; *Drug Resistance, Bacterial/genetics ; Porins/genetics ; Klebsiella Infections/microbiology/drug therapy ; Carbapenems/pharmacology ; },
abstract = {The CRISPR-Cas technology can be used to disable drug resistance genes. Since carbapenem resistance can be mediated by multiple resistance determinants, we here investigated the extent of re-sensizitation when targeting the blaKPC carbapenemase gene and assessed possible effects on porins and efflux pumps. While full re-sensitization was achieved in a laboratory strain of Escherichia coli solely equipped with blaKPC, resistance reduction in a clinical isolate of Klebsiella michiganensis was achieved in 63% of analyzed transformants, which was a consequence of plasmid copy number reduction and decreased blaKPC gene expression. Damages in the Cas9, as well as alterations in carbapenem-resistance promoting genes including ompK36 downregulation and mutations in the acrB gene were found, likely preventing more efficient re-sensitization. Hence, interference with a single resistance gene promoted the emergence of clonal variants that exhibit alterations in outer membrane proteins. Those bacterial countermeasures, however, were not sufficient to restore the original carbapenem-resistant phenotype.},
}

*CRISPR-Cas Systems/genetics
*Klebsiella/genetics/drug effects/isolation & purification
*Bacterial Proteins/genetics
*Imipenem/pharmacology
*beta-Lactamases/genetics
Anti-Bacterial Agents/pharmacology
Microbial Sensitivity Tests
Humans
Escherichia coli/genetics/drug effects
Plasmids/genetics
*Drug Resistance, Bacterial/genetics
Porins/genetics
Klebsiella Infections/microbiology/drug therapy
Carbapenems/pharmacology

@article {pmid40794116,
year = {2025},
author = {Cai, R and Chai, N and Zhang, J and Tan, J and Liu, YG and Zhu, Q and Zeng, D},
title = {CRISPR/Cas system-mediated transgene-free or DNA-free genome editing in plants.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {138},
number = {9},
pages = {210},
pmid = {40794116},
issn = {1432-2242},
support = {32272120//National Natural Science Foundation of China/ ; 2022YFF1002800//National Key Research and Development Program of China/ ; 2024GXNSFBA010386//Guangxi Natural Science Foundation Youth Science Fund Project/ ; 2024KY0071//The Project for Enhancing Young and Middle-aged Teacher's Research Basis Ability in Colleges of Guangxi/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Plants, Genetically Modified/genetics ; *Genome, Plant ; Transgenes ; *Crops, Agricultural/genetics ; Plant Breeding ; },
abstract = {CRISPR/Cas-based genome-editing technology serves as a powerful and versatile tool for genome modification. It has been broadly utilized in crop breeding to enhance traits such as yield, various quality attributes, and biotic and abiotic stress tolerance. Because of public safety concerns over genetically modified organisms (GMOs), many countries have established stringent regulatory policies for genetically modified plants, dramatically limiting the application of related products. However, genome editing in stably transformed plants can result in transgene-free progeny through self-pollination or hybridization or yield DNA-free gene-edited plants via transient transformation. These edited plants materially differ from GMOs and are referred to as genome-edited organisms (GEOs). GEOs have the potential to alleviate regulatory burdens and aid in commercialization. Various methods have been developed to expedite the creation of transgene-free or DNA-free GEOs. This review summarizes the various strategies for creating these types of GEOs based on the CRISPR/Cas systems. It also covers the advantages and drawbacks of these strategies. Additionally, we examine off-target effects and mitigation strategies for plant genome editing and outline regulatory policies for gene-edited crops in selected countries and regions. We hope this review offers valuable references for the advancement of transgene-free and DNA-free GEOs.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
*Plants, Genetically Modified/genetics
*Genome, Plant
Transgenes
*Crops, Agricultural/genetics
Plant Breeding

@article {pmid40793766,
year = {2025},
author = {Xu, H-W and Wang, X-Y and Wei, Y and Cao, Y and Wang, S-G and Xia, P-F},
title = {Pathway crosstalk enables degradation of aromatic compounds in marine Roseobacter clade bacteria.},
journal = {Applied and environmental microbiology},
volume = {},
number = {},
pages = {e0097825},
doi = {10.1128/aem.00978-25},
pmid = {40793766},
issn = {1098-5336},
abstract = {Aromatic compounds are essential raw materials for almost all sectors of human societies but also persistent environmental pollutants recalcitrant to biodegradation. The ocean serves as a significant sink for these compounds, while their biological conversion routes remain poorly understood, hindering a comprehensive understanding of the marine carbon cycle and advancements in bioremediation and biological carbon upcycling. Here, we report the degradation pathway of aromatic molecules in the marine Roseobacter clade bacteria through multi-omics analysis and CRISPR-Cas-based genome editing. Using Roseovarius nubinhibens and 4-hydroxybenzoate (4HB) as representatives, we identified the transport of 4HB via TRAP, ABC, and MFS transporters. Then, we deciphered the integral β-ketoadipate pathway responsible for aromatic degradation. Next, we discovered a distinct pathway crosstalk at the final thiolation step, which serves as an intersection node of different pathways catalyzed by the 3-oxoadipyl-CoA thiolase from the β-ketoadipate pathway and the acetyl-CoA C-acetyltransferase and acetyl-CoA C-acyltransferase from the β-oxidation pathway. Finally, we proposed R. nubinhibens as a novel marine platform for systems-level interrogation and bioprospecting. Our study provides a foundation for leveraging Roseobacter clade bacteria as novel chassis for environmental and industrial innovations.IMPORTANCEAromatic compounds lie in an essential node of carbon cycling in both natural and engineered systems. Marine bacteria orchestrate the cycling of aromatic compounds in the ocean and, as emerging chassis, have shown unusual potentials in the degradation and valorization of aromatics. However, the corresponding metabolic pathway in marine bacteria remains poorly interpreted over decades, hindering further scientific interrogation and engineering practices. Here, we deciphered the complete degradation pathway of aromatic compounds in the marine Roseobacter clade bacteria and established a marine platform for systems and synthetic biology. Our study provides a paradigm for biological interrogation with combined multi-omics and the cutting-edge CRISPR-Cas approaches, laying a foundation for biological innovations with marine bacteria.},
}

@article {pmid40792102,
year = {2025},
author = {Chen, X and Xu, L and Luo, Z and Wang, L and Wang, Z and Li, Y and Jiao, X and Li, Q},
title = {Prevalence and genomic insights into type III-A CRISPR-Cas system acquisition in global Staphylococcus argenteus strains.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1644286},
pmid = {40792102},
issn = {2235-2988},
mesh = {*CRISPR-Cas Systems/genetics ; *Staphylococcus/genetics/classification/isolation & purification ; Humans ; *Genome, Bacterial ; *Staphylococcal Infections/microbiology/epidemiology/veterinary ; Animals ; Genomics ; DNA Transposable Elements ; Prevalence ; Food Microbiology ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {INTRODUCTION: The CRISPR-Cas system serves as a defense mechanism in bacteria and archaea, protecting them against the invasion of mobile genetic elements. Staphylococcus argenteus, a Gram-positive bacterium that diverged from Staphylococcus aureus, is characterized by the rare presence of the CRISPR-Cas system in only a few isolates.

METHODS: In this study, we analyzed the prevalence of the type III-A CRISPR-Cas system in 368 S. argenteus genome sequences from animals, food sources, and humans across 26 countries, available in public database.

RESULTS: Our findings revealed that 44.0% of these strains carry this immune system, with 98.1% of them belonging to the sequence type 2250 (ST2250). Genomic localization analysis indicated that the CRISPR-Cas is closely associated with SCCmec (mecA-ΔmecR1-IS1272-ccrB2-ccrA2) or Insertion sequence 1272 (IS1272) transposase. Further analysis identified a common IS1272 target inverted repeats (IR) sequence in ST2250 strains, providing insights into why these strains are more likely to acquire the CRISPR-Cas system. CRISPR typing identified 41 sequences types, classifying these strains into two clusters, with Cluster II being the predominant one. Homology analysis of spacers revealed that all the identified 15 spacers exhibited homology to sequences from plasmids, lytic phages, or prophages.

CONCLUSION: This study suggests that the acquisition of the CRISPR-Cas system in S. argenteus enhances its resistance to phage attacks and plasmid invasions in environmental settings, potentially posing significant challenges for clinical treatment of infections caused by these strains and hindering efforts to control their spread in food products using phage-based interventions.},
}

*CRISPR-Cas Systems/genetics
*Staphylococcus/genetics/classification/isolation & purification
Humans
*Genome, Bacterial
*Staphylococcal Infections/microbiology/epidemiology/veterinary
Animals
Genomics
DNA Transposable Elements
Prevalence
Food Microbiology
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40790872,
year = {2025},
author = {Kale, SM and Paina, C and Füchtbauer, WS and Bjarup, P and Sarup, PM and Schatz-Jakobsen, JA and Orabi, J and Borum, F and Jahoor, A and Brinch-Pedersen, H},
title = {An introgression from Triticum timopheevii reduces grain protein content in winter wheat populations.},
journal = {The plant genome},
volume = {18},
number = {3},
pages = {e70090},
doi = {10.1002/tpg2.70090},
pmid = {40790872},
issn = {1940-3372},
support = {Halm til det hele//Promilleafgiftsfonden for Landbrugs/ ; },
mesh = {*Triticum/genetics/metabolism ; Genome-Wide Association Study ; *Grain Proteins/metabolism ; Phenotype ; Quantitative Trait Loci ; Plant Breeding ; *Genetic Introgression ; Edible Grain/genetics ; Plant Proteins/genetics/metabolism ; Chromosomes, Plant/genetics ; },
abstract = {Improving grain protein content (GPC) in wheat (Triticum aestivum L.) is crucial for enhancing end-use quality and ensuring efficient nitrogen (N) utilization, thereby reducing environmental damage caused by excess N. However, progress in increasing GPC has been limited because of the strong negative correlation between GPC and grain yield (GY), as well as the scarcity of multi-location, multi-year phenotypic data. In this study, we analyzed the variation in GPC, GY, and grain protein deviation (GPD) using multi-location, multi-year phenotypic data from winter wheat varieties in Scandinavian regions. As reported previously, we observed a negative correlation between GY and GPC, with recent cultivars showing higher GY but lower GPC. Additionally, a genome-wide association study (GWAS) in two independent populations identified significant marker-trait associations (MTAs) for GPC and GPD, with key MTAs located on chromosome 2B (chr2B), highlighting its central role in the regulation of these traits. Interestingly, the MTA for GPD on chr2B coincided with an introgression from Triticum timopheevii, which was associated with reduced GPC and GPD in elite lines carrying this region. This introgression, which contains a powdery mildew resistance gene (Pm6), appears to negatively affect GPC, likely due to linkage drag. These findings emphasize the importance of chr2B in wheat breeding and suggest that advanced genomic techniques, such as mutagenesis and CRISPR-Cas, could be employed to mitigate negative pleiotropic effects and improve GPC and GPD. Overall, this study provides valuable insights into the genetic architecture underlying GPC in wheat and offers directions for future breeding strategies aimed at enhancing protein content.},
}

*Triticum/genetics/metabolism
Genome-Wide Association Study
*Grain Proteins/metabolism
Phenotype
Quantitative Trait Loci
Plant Breeding
*Genetic Introgression
Edible Grain/genetics
Plant Proteins/genetics/metabolism
Chromosomes, Plant/genetics

@article {pmid40790091,
year = {2025},
author = {Kocsy, K and Wilkinson, H and Felix-Ilemhenbhio, F and Bax, B and Van Agtmael, T and Azzouz, M and Majid, A},
title = {Gene editing for collagen disorders: current advances and future perspectives.},
journal = {Gene therapy},
volume = {},
number = {},
pages = {},
pmid = {40790091},
issn = {1476-5462},
abstract = {Collagen disorders encompass a wide range of genetic conditions caused by pathogenic variants in collagen genes for which there is an unmet need for treatments. They present various clinical features, ranging from localised tissue abnormalities to severe systemic complications. Symptoms differ significantly and depend on the pathogenic variant, which can affect various systems, including the musculoskeletal, cardiovascular, and respiratory systems, highlighting the complex implications of collagen gene pathogenic variants and the wide range of expression patterns among different collagen types. Gene-editing technologies, particularly Clustered Regularly Interspaced Palindromic Repeats (CRISPR)-Cas systems, have emerged as promising therapeutic options for these disorders, representing a putative one-for-all treatment strategy. This review provides an overview of current gene-editing strategies aimed at collagen-related diseases, including osteogenesis imperfecta, Alport syndrome, and dystrophic epidermolysis bullosa. We explore the application of CRISPR-Cas9, which facilitates targeted DNA modifications, base editing (BE), and prime editing (PE), enabling precise single-nucleotide alterations without double-strand breaks (DSB). Preclinical and clinical studies have shown the potential of gene therapy to enhance collagen production, restore tissue integrity, and alleviate symptoms. However, challenges persist, including the lack of recurring mutations, the need for improved delivery methods, the reduction of off-target effects, and the development of novel therapies. Despite these challenges, advancements in gene editing techniques appear promising in enhancing editing efficiency while minimising unintended mutations, paving the way for more precise and safer genetic interventions for collagen disorders. Gene editing is fundamentally transforming medicine and biotechnology. Its applications encompass advanced diagnostics, tailored therapeutic strategies, and solutions for rare genetic disorders. By enabling precise genetic modifications, gene editing is paving the way for treatments of previously untreatable diseases, including those linked to collagen pathogenic variants. This review discusses the latest advancements in gene therapy techniques targeting collagen-related disorders. It explores innovative approaches like CRISPR-Cas9-mediated gene editing and highlights emerging strategies, such as allele-specific inactivation and base editing (BE). By examining these cutting-edge therapies and their potential clinical applications, this review highlights the transformative impact of gene editing in treating collagen-related conditions, while also identifying critical challenges and future directions for research.},
}

@article {pmid40696172,
year = {2025},
author = {Xu, Z and Wang, G and Zhu, X and Wang, R and Zhu, L and Tu, L and Liu, Y and Peng, R and Lindsey, K and Wang, M and Zhang, X and Jin, S},
title = {Genome assembly of two allotetraploid cotton germplasms reveals mechanisms of somatic embryogenesis and enables precise genome editing.},
journal = {Nature genetics},
volume = {57},
number = {8},
pages = {2028-2039},
pmid = {40696172},
issn = {1546-1718},
support = {32325039//China National Funds for Distinguished Young Scientists/ ; 32201856//National Science Foundation of China | Young Scientists Fund/ ; },
mesh = {*Gossypium/genetics ; *Genome, Plant/genetics ; *Gene Editing/methods ; Tetraploidy ; *Plant Somatic Embryogenesis Techniques/methods ; Retroelements/genetics ; CRISPR-Cas Systems ; },
abstract = {Somatic embryogenesis is crucial for plant genetic engineering, yet the underlying mechanisms in cotton remain poorly understood. Here we present a telomere-to-telomere assembly of Jin668 and a high-quality assembly of YZ1, two highly regenerative allotetraploid cotton germplasms. The completion of the Jin668 genome enables characterization of ~30.1 Mb of centromeric regions invaded by centromeric retrotransposon of maize and Tekay retrotransposons, an ~8.1 Mb 5S rDNA array containing 25,190 copies and a ~75.1 Mb major 45S rDNA array with 8,131 copies. Comparative analyses of regenerative and recalcitrant genotypes reveal dynamic transcriptional patterns and chromatin accessibility during the initial regeneration process. A hierarchical gene regulatory network identifies AGL15 as a contributor to regeneration. Additionally, we demonstrate that genetic variation affects sgRNA target sites, while the Jin668 genome assembly reduces the risk of off-target effects in CRISPR-based genome editing. Together, the complete Jin668 genome reveals the complexity of genomic regions and cotton regeneration, and improves the precision of genome editing.},
}

*Gossypium/genetics
*Genome, Plant/genetics
*Gene Editing/methods
Tetraploidy
*Plant Somatic Embryogenesis Techniques/methods
Retroelements/genetics
CRISPR-Cas Systems

@article {pmid40607979,
year = {2025},
author = {Cohen, O and Maru, P and Liang, Q and Saeij, JPJ},
title = {Surface antigen SAG1 mediates Toxoplasma gondii fitness and host cell attachment in IFNγ-stimulated cells.},
journal = {Infection and immunity},
volume = {93},
number = {8},
pages = {e0001025},
doi = {10.1128/iai.00010-25},
pmid = {40607979},
issn = {1098-5522},
support = {//Societe de Pathologie Infectieuse de Langue Francaise/ ; R01AI173803//National Institute of Allergy and Infectious Diseases/ ; },
mesh = {*Toxoplasma/physiology/genetics/immunology ; *Interferon-gamma/immunology/pharmacology ; *Antigens, Protozoan/genetics/metabolism/immunology ; *Protozoan Proteins/genetics/metabolism/immunology ; *Host-Parasite Interactions ; Animals ; Humans ; Toxoplasmosis/parasitology/immunology ; Cell Adhesion ; CRISPR-Cas Systems ; Mice ; },
abstract = {Toxoplasma gondii is an obligate intracellular protozoan parasite that can establish lifelong infections and cause severe disease in immunocompromised individuals. Interferon gamma (IFNγ) is a key host defense cytokine that induces a variety of toxoplasmacidal mechanisms. Recent CRISPR/Cas9 loss-of-function screens identified multiple Toxoplasma genes important for fitness in IFNγ-stimulated cells. One consistent hit in several screens was the parasite surface antigen, SAG1. Here, we used CRISPR/Cas9 to generate a SAG1 knockout strain and found that SAG1 is important for parasite fitness specifically in IFNγ-stimulated cells. Mechanistic studies revealed that host surface sialic acids are important for parasite attachment, especially in IFNγ-stimulated cells. SAG1-deficient parasites had reduced attachment efficiency, which was exacerbated in IFNγ-treated cells. These findings highlight the role of SAG1 in mediating robust parasite attachment, especially in the context of immune pressure.},
}

*Toxoplasma/physiology/genetics/immunology
*Interferon-gamma/immunology/pharmacology
*Antigens, Protozoan/genetics/metabolism/immunology
*Protozoan Proteins/genetics/metabolism/immunology
*Host-Parasite Interactions
Animals
Humans
Toxoplasmosis/parasitology/immunology
Cell Adhesion
CRISPR-Cas Systems
Mice

@article {pmid40579538,
year = {2025},
author = {Hanlon, VCT and Cagan, A and Eves-van den Akker, S},
title = {How and when organisms edit their own genomes.},
journal = {Nature genetics},
volume = {57},
number = {8},
pages = {1823-1834},
pmid = {40579538},
issn = {1546-1718},
support = {BB/S006397/1//RCUK | Biotechnology and Biological Sciences Research Council (BBSRC)/ ; BB/X006352/1//RCUK | Biotechnology and Biological Sciences Research Council (BBSRC)/ ; BB/Y513246/1//RCUK | Biotechnology and Biological Sciences Research Council (BBSRC)/ ; RPG-2023-001//Leverhulme Trust/ ; R01AG087974//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Animals ; Humans ; *Genome/genetics ; Bacteria/genetics ; Host-Pathogen Interactions/genetics ; },
abstract = {Mutations are often thought of as untargeted and non-adaptive, but in rare cases, organisms perform programmed, targeted and adaptive rearrangements of their own DNA sequences. Notable examples include the somatic diversification of immunoglobulin genes, which is the foundation of the vertebrate immune system, and natural CRISPR spacer arrays in bacteria, which recognize and cleave foreign DNA. These systems, along with a dozen known analogs scattered across the tree of life, often underlie critical biological functions, particularly in host-pathogen conflicts. In this Review, we compare the mechanisms by which organisms edit their own genomes. We show that superficially dissimilar editing systems often rely on surprisingly similar genetic mechanisms, regardless of function or taxon. Finally, we argue that the recurrence of editing in host-pathogen conflicts and the bias to a handful of well-studied organisms strongly suggest that new editing systems will be found in understudied pathogens and their hosts.},
}

*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Animals
Humans
*Genome/genetics
Bacteria/genetics
Host-Pathogen Interactions/genetics

@article {pmid40519079,
year = {2025},
author = {Geronazzo, J and Heimerl, A and Lindell, L and McCrimmon, S and Stormer, C and Horvai, B and Johnson, IP and Peterson, TM and Zuckerman, J and Scott, AI and Course, MM},
title = {Characterizing fatty acid oxidation genes in Drosophila.},
journal = {G3 (Bethesda, Md.)},
volume = {15},
number = {8},
pages = {},
doi = {10.1093/g3journal/jkaf139},
pmid = {40519079},
issn = {2160-1836},
support = {//CC Natural Sciences Executive Committee Research and Development/ ; //CC Student Collaborative Research/ ; //Seattle Children's Research Institute Center for Clinical and Translational Research/ ; //AIS/ ; },
mesh = {Animals ; *Fatty Acids/metabolism ; Oxidation-Reduction ; *Drosophila Proteins/genetics/metabolism ; Phenotype ; *Drosophila/genetics/metabolism ; Humans ; CRISPR-Cas Systems ; Mutation ; *Drosophila melanogaster/genetics/metabolism ; },
abstract = {In this study, we leverage the power and tractability of Drosophila genetics to better understand the molecular mechanisms underlying a group of rare genetic diseases known as fatty acid oxidation disorders. We use CRISPR-Cas9 to generate mutations in 6 putative fatty acid oxidation genes in Drosophila, then analyze the phenotypes and acylcarnitine profiles of these flies. We find that while Arc42 and CG4860 are both predicted orthologs of human ACADS, only Arc42 loss of function mirrors the acylcarnitine profile of ACADS loss of function. Acylcarnitine profiles also support our previous identification of Mcad as the likely ACADM ortholog, and reveal the deleterious effects of a single codon deletion in Mtpα (the predicted human HADHA ortholog). Finally, we observe that loss of function in Etf-QO and in CG7834-predicted orthologs of human ETFDH and ETFB, respectively-is homozygous lethal in flies. Producing animal models like these will enable new approaches to studying fatty acid oxidation disease progression, symptomatic variability, and therapeutic intervention.},
}

Animals
*Fatty Acids/metabolism
Oxidation-Reduction
*Drosophila Proteins/genetics/metabolism
Phenotype
*Drosophila/genetics/metabolism
Humans
CRISPR-Cas Systems
Mutation
*Drosophila melanogaster/genetics/metabolism

@article {pmid40476353,
year = {2025},
author = {Weisman, AS and Fisher, NM and Hunter, CP},
title = {Efficient iterative CRISPR/Cas9 editing using sid-1 co-conversion and feeding RNAi in Caenorhabditis elegans.},
journal = {G3 (Bethesda, Md.)},
volume = {15},
number = {8},
pages = {},
doi = {10.1093/g3journal/jkaf128},
pmid = {40476353},
issn = {2160-1836},
support = {GM089795/NH/NIH HHS/United States ; 62579/NH/NIH HHS/United States ; },
mesh = {Animals ; *Caenorhabditis elegans/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Caenorhabditis elegans Proteins/genetics ; *RNA Interference ; Phenotype ; Membrane Proteins ; },
abstract = {We present a sid-1 loss-of-function and restoration-of-function CRISPR/Cas9 co-conversion protocol in Caenorhabditis elegans. Introducing CRISPR reagents that induce sid-1 loss-of-function can produce survivors on lethal RNAi foods while reagents that induce sid-1 restoration-of-function can be screened for restoration of visible RNAi phenotypes. Both methods efficiently reduce the pool of candidates from hundreds or thousands of F1 progeny to tens with minimal experimenter effort. Furthermore, our optimized sid-1 CRISPR design allows a high ratio of CRISPR reagents targeting the gene of interest, maximizing successful co-conversion events. The interconvertibility of the sid-1 locus readily enables this strategy to be leveraged to iteratively create complex strains with multiple gene edits.},
}

Animals
*Caenorhabditis elegans/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
*Caenorhabditis elegans Proteins/genetics
*RNA Interference
Phenotype
Membrane Proteins

@article {pmid40460280,
year = {2025},
author = {Nalley, MJ and Banerjee, S and Huang, MY and Madhani, HD},
title = {Near 100% efficient homology-dependent genome engineering in the human fungal pathogen Cryptococcus neoformans.},
journal = {G3 (Bethesda, Md.)},
volume = {15},
number = {8},
pages = {},
doi = {10.1093/g3journal/jkaf118},
pmid = {40460280},
issn = {2160-1836},
support = {//R01 AI000272/ ; },
mesh = {*Cryptococcus neoformans/genetics ; *Genome, Fungal ; CRISPR-Cas Systems ; *Gene Editing/methods ; Humans ; DNA End-Joining Repair ; *Genetic Engineering/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {We recently described CRISPR/Cas9-based short homology-dependent genome engineering in the human fungal pathogen Cryptococcus neoformans, a haploid budding yeast that is the most common cause of fungal meningitis and an emerging model organism. This was achieved by electroporation of strains stably expressing a codon-optimized Cas9 with 2 separate DNA molecules, one encoding a selectable marker flanked by short homology arms and a second encoding a sgRNA under the control of the U6 snRNA promoter. However, the efficiency of desired homology-dependent repair relative to undesired non-homologous end-joining (NHEJ) events can be low and variable. Here, we describe methods and strains enabling extremely efficient (∼99%) homology-dependent genome editing in C. neoformans. This high-efficiency method requires 2 manipulations. First, we placed the sgRNA-expressing segment into the marker-containing DNA flanked by targeting homology; thus, only a single DNA molecule is introduced into cells. Second, we used a strain mutant for the non-homologous end-joining factor Ku80 (encoded by YKU80). We also report the engineering of a yku80::amdS mutant strain harboring an insertion mutation that can be removed scarlessly via recombination between direct repeats. This enables the functional restoration of YKU80 after homology-dependent genome editing after selection against the amdS marker using fluoroacetamide. This approach minimizes documented drawbacks of using Ku-defective strains in downstream experiments. Finally, we describe a plasmid series that enables rapid cloning of sgRNA-marker constructs for genomic manipulation of C. neoformans, including gene deletion and C-terminal tagging. These methods, strains, and plasmids accelerate the genomic manipulation of C. neoformans.},
}

*Cryptococcus neoformans/genetics
*Genome, Fungal
CRISPR-Cas Systems
*Gene Editing/methods
Humans
DNA End-Joining Repair
*Genetic Engineering/methods
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid40188316,
year = {2025},
author = {Teter, OM and McQuade, A and Hagan, V and Liang, W and Dräger, NM and Sattler, SM and Holmes, BB and Castillo, VC and Papakis, V and Leng, K and Boggess, S and Nowakowski, TJ and Wells, J and Kampmann, M},
title = {CRISPRi-based screen of autism spectrum disorder risk genes in microglia uncovers roles of ADNP in microglia endocytosis and synaptic pruning.},
journal = {Molecular psychiatry},
volume = {30},
number = {9},
pages = {4176-4193},
pmid = {40188316},
issn = {1476-5578},
support = {U01 MH115747/MH/NIMH NIH HHS/United States ; R01 MH125516/MH/NIMH NIH HHS/United States ; 2034836//National Science Foundation (NSF)/ ; AARF-22-973222//Alzheimer's Association/ ; 2022-A-016-FEL//Larry L. Hillblom Foundation (Larry L. Hillblom Foundation, Inc.)/ ; U01 MH115747/MH/NIMH NIH HHS/United States ; R01 MH125516/MH/NIMH NIH HHS/United States ; },
mesh = {*Microglia/metabolism ; *Autism Spectrum Disorder/genetics/metabolism ; Humans ; Endocytosis/genetics/physiology ; Phagocytosis/genetics ; Neurons/metabolism ; Induced Pluripotent Stem Cells/metabolism ; *Neuronal Plasticity/genetics/physiology ; CRISPR-Cas Systems/genetics ; Coculture Techniques ; *Nerve Tissue Proteins/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Synapses/metabolism ; Genetic Predisposition to Disease/genetics ; Brain/metabolism ; },
abstract = {Autism Spectrum Disorders (ASD) are a set of neurodevelopmental disorders with complex biology. The identification of ASD risk genes from exome-wide association studies and de novo variation analyses has enabled mechanistic investigations into how ASD-risk genes alter development. Most functional genomics studies have focused on the role of these genes in neurons and neural progenitor cells. However, roles for ASD risk genes in other cell types are largely uncharacterized. There is evidence from postmortem tissue that microglia, the resident immune cells of the brain, appear activated in ASD. Here, we used CRISPRi-based functional genomics to systematically assess the impact of ASD risk gene knockdown on microglia activation and phagocytosis. We developed an iPSC-derived microglia-neuron coculture system and high-throughput flow cytometry readout for synaptic pruning to enable parallel CRISPRi-based screening of phagocytosis of beads, synaptosomes, and synaptic pruning. Our screen identified ADNP, a high-confidence ASD risk genes, as a modifier of microglial synaptic pruning. We found that microglia with ADNP loss have altered endocytic trafficking, remodeled proteomes, and increased motility in coculture.},
}

*Microglia/metabolism
*Autism Spectrum Disorder/genetics/metabolism
Humans
Endocytosis/genetics/physiology
Phagocytosis/genetics
Neurons/metabolism
Induced Pluripotent Stem Cells/metabolism
*Neuronal Plasticity/genetics/physiology
CRISPR-Cas Systems/genetics
Coculture Techniques
*Nerve Tissue Proteins/genetics/metabolism
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Synapses/metabolism
Genetic Predisposition to Disease/genetics
Brain/metabolism

@article {pmid39394482,
year = {2025},
author = {Mantena, S and Pillai, PP and Petros, BA and Welch, NL and Myhrvold, C and Sabeti, PC and Metsky, HC},
title = {Model-directed generation of artificial CRISPR-Cas13a guide RNA sequences improves nucleic acid detection.},
journal = {Nature biotechnology},
volume = {43},
number = {8},
pages = {1266-1273},
pmid = {39394482},
issn = {1546-1696},
support = {U01AI151812//Division of Intramural Research, National Institute of Allergy and Infectious Diseases (Division of Intramural Research of the NIAID)/ ; T32 GM144273/GM/NIGMS NIH HHS/United States ; T32GM007753//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; T32GM144273//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; U19AI110818//Division of Intramural Research, National Institute of Allergy and Infectious Diseases (Division of Intramural Research of the NIAID)/ ; D18AC00006//United States Department of Defense | Defense Advanced Research Projects Agency (DARPA)/ ; 75D30122C15113//U.S. Department of Health & Human Services | Centers for Disease Control and Prevention (CDC)/ ; K01AI163498//U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; NU50CK000629//U.S. Department of Health & Human Services | Centers for Disease Control and Prevention (CDC)/ ; INV-034761/GATES/Gates Foundation/United States ; INV-034761/GATES/Gates Foundation/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Algorithms ; Base Sequence ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {CRISPR guide RNA sequences deriving exactly from natural sequences may not perform optimally in every application. Here we implement and evaluate algorithms for designing maximally fit, artificial CRISPR-Cas13a guides with multiple mismatches to natural sequences that are tailored for diagnostic applications. These guides offer more sensitive detection of diverse pathogens and discrimination of pathogen variants compared with guides derived directly from natural sequences and illuminate design principles that broaden Cas13a targeting.},
}

*CRISPR-Cas Systems/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics
Algorithms
Base Sequence
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid40790054,
year = {2025},
author = {Patel, MA and Boribong, BP and Sinha, H and Xiao, B and Xie, K and Vo, PQN and Chin, AB and Ellouzi, A and Little, SR and Shih, S and Wu, H and Muller, WJ and Hirukawa, A},
title = {Miniaturized scalable arrayed CRISPR screening in primary cells enables discovery at the single donor resolution.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {29350},
pmid = {40790054},
issn = {2045-2322},
mesh = {Humans ; *CRISPR-Cas Systems ; Electroporation/methods ; *Gene Editing/methods ; Microfluidics/methods ; CD4-Positive T-Lymphocytes/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Miniaturization ; },
abstract = {High-efficiency gene editing in primary human cells is critical for advancing therapeutic development and functional genomics, yet conventional electroporation platforms often require high cell input and are poorly suited to parallelized experiments. Here we introduce a next-generation digital microfluidics (DMF) electroporation platform that enables high-throughput, low-input genome engineering using discrete droplets manipulated on a planar electrode array. The system supports 48 independently programmable reaction sites and integrates seamlessly with laboratory automation, allowing efficient delivery of CRISPR-Cas9 RNPs and mRNA cargo into as few as 3,000 primary human cells per condition. The platform was validated across diverse primary human cell types and cargo modalities, demonstrating efficient delivery of various cargo, with high rates of transfection, gene knockout via non-homologous end joining, and precise knock-in through homology-directed repair. To showcase its utility in functional genomics, we applied the platform to an arrayed CRISPR-Cas9 screen in chronically stimulated human CD4[+] T cells, identifying novel regulators of exhaustion, including epigenetic and transcriptional modulators. These findings establish our DMF-based electroporation platform as a powerful tool for miniaturized genome engineering in rare or precious cell populations and provide a scalable framework for high-content genetic screening in primary human cells.},
}

Humans
*CRISPR-Cas Systems
Electroporation/methods
*Gene Editing/methods
Microfluidics/methods
CD4-Positive T-Lymphocytes/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats
Miniaturization

@article {pmid40790042,
year = {2025},
author = {Li, H and He, Y and Jiang, J and Liu, Z and Liu, Y and Shi, Q and Ding, J and Li, H and Sun, W and Hu, X and Chen, Z and He, X},
title = {CRISPR screening reveals that RNA helicase DDX41 triggers ribosome biogenesis and cancer progression through R-loop-mediated RPL/RPS transcription.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {7409},
pmid = {40790042},
issn = {2041-1723},
support = {82121004//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82472626//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82472641//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82172937//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*DEAD-box RNA Helicases/genetics/metabolism ; Humans ; *Ribosomes/metabolism/genetics ; Animals ; Mice ; *Liver Neoplasms/genetics/pathology/metabolism/drug therapy ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; *Ribosomal Proteins/genetics/metabolism ; Transcription, Genetic ; Disease Progression ; Histone Acetyltransferases/metabolism ; Promoter Regions, Genetic ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Neoplasms/genetics/pathology ; Protein Biosynthesis ; },
abstract = {The RNA helicase DDX41 is a DEAD-box helicase that is well known as a virus sensor in dendritic cells and a tumor suppressor that is frequently mutated in myeloid neoplasms. However, the functions and relevance of DDX41 in solid tumors remain largely unexplored. In this study, through in vivo CRISPR screening, we demonstrate that DDX41 is highly expressed in various solid tumor types and promotes tumorigenicity in liver cancer. Mechanistically, DDX41 facilitates R-loop processing and accelerates the transcription of RPL/RPS genes, thereby promoting ribosome biogenesis and protein synthesis. Additionally, we show that the acetyltransferase KAT8 is required for H3K9ac modification of the DDX41 promoter and that NR2C1/NR2C2 are responsible for DDX41 expression. Moreover, elevated DDX41 levels increase liver cancer cell sensitivity to protein synthesis inhibitors; treatment with homoharringtonine (HHT), an approved drug, significantly inhibits tumor growth in DDX41-overexpressing liver cancer models. Taken together, the results of this study highlight that DDX41 acts as an oncogene in liver cancer and suggest that protein synthesis inhibition may be a promising therapy for liver cancers with high DDX41 expression.},
}

*DEAD-box RNA Helicases/genetics/metabolism
Humans
*Ribosomes/metabolism/genetics
Animals
Mice
*Liver Neoplasms/genetics/pathology/metabolism/drug therapy
Cell Line, Tumor
Gene Expression Regulation, Neoplastic
*Ribosomal Proteins/genetics/metabolism
Transcription, Genetic
Disease Progression
Histone Acetyltransferases/metabolism
Promoter Regions, Genetic
CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
Neoplasms/genetics/pathology
Protein Biosynthesis

@article {pmid40790018,
year = {2025},
author = {Wang, K and Wang, J and Yang, X and Sun, W and Sheng, G and Wang, Y},
title = {Structural insights into Type II-D Cas9 and its robust cleavage activity.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {7396},
pmid = {40790018},
issn = {2041-1723},
support = {Z231100007223004//Beijing Municipal Science and Technology Commission/ ; 32330055, 31930065, 22121003, 32071198, 32071444//National Natural Science Foundation of China (National Science Foundation of China)/ ; 5232022//Natural Science Foundation of Beijing Municipality (Beijing Natural Science Foundation)/ ; XDB0570000, XDB37010202//Chinese Academy of Sciences (CAS)/ ; },
mesh = {*CRISPR-Associated Protein 9/metabolism/chemistry/genetics/ultrastructure ; Cryoelectron Microscopy ; DNA/metabolism/chemistry/genetics ; RNA, Guide, CRISPR-Cas Systems/metabolism/genetics/chemistry ; DNA Cleavage ; CRISPR-Cas Systems ; Gene Editing ; *Bacterial Proteins/metabolism/genetics/chemistry ; Catalytic Domain ; },
abstract = {Type II-D Cas9 proteins (Cas9d) are more compact than typical Type II-A/B/C Cas9s. Here, we demonstrate that NsCas9d from Nitrospirae bacterium RBG_13_39_12 derived from a metagenomic assembly exhibits robust dsDNA cleavage activity comparable to SpCas9 in vitro. Unlike typical Cas9 enzymes that generate blunt ends, NsCas9d produces 3-nucleotide staggered overhangs. Our high-resolution cryo-EM structure of the NsCas9d-sgRNA-dsDNA complex in its catalytic state reveals the target and non-target DNA strands positioned within the HNH and RuvC catalytic pockets, respectively. NsCas9d recognizes the 5'-NRG-3' protospacer adjacent motif (PAM), with 5'-NGG-3' showing the highest cleavage efficiency. Its sgRNA structure, resembling the 5' end of IscB ωRNA, along with structural features shared with other Cas9 variants, suggests that Cas9d are hypothesized to resemble evolutionary intermediates between other Cas9 sub-types and IscB. These findings deepen our understanding of Cas9 evolution and mechanisms, highlighting NsCas9d as a promising genome-editing tool due to its compact size, DNA cleavage pattern, and efficient PAM recognition.},
}

*CRISPR-Associated Protein 9/metabolism/chemistry/genetics/ultrastructure
Cryoelectron Microscopy
DNA/metabolism/chemistry/genetics
RNA, Guide, CRISPR-Cas Systems/metabolism/genetics/chemistry
DNA Cleavage
CRISPR-Cas Systems
Gene Editing
*Bacterial Proteins/metabolism/genetics/chemistry
Catalytic Domain

@article {pmid40789840,
year = {2025},
author = {Goyal, H and Kaur, J},
title = {Long non-coding RNAs and autophagy: dual drivers of Hepatocellular carcinoma progression.},
journal = {Cell death discovery},
volume = {11},
number = {1},
pages = {376},
pmid = {40789840},
issn = {2058-7716},
abstract = {Hepatocellular carcinoma (HCC), a leading cause of cancer-related mortality worldwide, is characterized by poor prognosis, high recurrence rates, and limited responsiveness to current therapies. Autophagy, a conserved catabolic pathway essential for cellular homeostasis, plays a paradoxical role in HCC, acting as a tumor suppressor during initiation but promoting survival and progression in advanced stages. Long non-coding RNAs (lncRNAs) have emerged as critical regulators of autophagy, influencing tumorigenesis, metastasis, and therapy resistance through mechanisms such as miRNA sponging, chromatin remodeling, and protein interactions. This review describes how autophagy contributes to HCC at different stages, outlines the dual functions of lncRNAs as oncogenic drivers or tumor suppressors, and illustrates their integration into key signaling networks of autophagy (e.g., PI3K/AKT/mTOR, AMPK, Beclin-1). LncRNAs have been shown to modulate drug resistance, including resistance to first-line agents, by altering autophagic flux and associated molecular pathways. We also explored emerging strategies for targeting the lncRNA-autophagy axis, such as siRNAs, antisense oligonucleotides, and CRISPR/Cas systems, that have shown promise in preclinical studies and may be adapted for HCC. Furthermore, autophagy-related lncRNAs hold potential as non-invasive diagnostic and prognostic biomarkers and as predictors of recurrence. Integrating multi-omics approaches to validate these candidates will be critical for translation into clinical practice. Collectively, this review highlights the lncRNA-autophagy network as a promising frontier of biomarker discovery for precision diagnostics and targets for innovative therapeutics. The regulatory role of lncRNAs in autophagy presents a paradigm shift, heralding new strategies for targeted treatment.},
}

@article {pmid40789802,
year = {2025},
author = {Noormohamadi, H and Soleimani Samarkhazan, H and Kargar, M and Maroufi, F and Servatian, N and Davami, F},
title = {CRISPR/Cas technologies in pancreatic cancer research and therapeutics: recent advances and future outlook.},
journal = {Discover oncology},
volume = {16},
number = {1},
pages = {1530},
pmid = {40789802},
issn = {2730-6011},
abstract = {Pancreatic cancer is marked by a poor prognosis and an exceptionally high mortality rate, with its aggressive nature contributing to its classification as a highly malignant disease. For effective therapeutic strategies, the development of sophisticated and regulated DNA manipulation methods is essential. Originally part of the prokaryotic immune system, CRISPR/Cas has emerged as a pivotal genome-editing tool with promising applications in pancreatic cancer research and therapy. This gene editing method is known for simplicity, rapid advancement, and superior precision compared to earlier techniques. Its adaptability allows precise gene editing for therapeutic purposes, including oncogene silencing and correction of pathogenic mutations. Additionally, CRISPR-driven gene editing has facilitated the development of pancreatic cancer models, which serve as valuable platforms for drug discovery and personalized treatment strategies, offering deeper insights into the genetic landscape of pancreatic tumors. This article provides an overview of the current applications of CRISPR technology in gene therapy and cancer research, particularly in the context of pancreatic cancer, and lays the foundation for future studies.},
}

@article {pmid40789027,
year = {2025},
author = {Huang, Y and Li, C and Desingu Rajan, AR and Bronner, ME},
title = {Sox11 genes affect neuronal differentiation in the developing zebrafish enteric nervous system.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {33},
pages = {e2510548122},
doi = {10.1073/pnas.2510548122},
pmid = {40789027},
issn = {1091-6490},
support = {HD105604//HHS | NIH | Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)/ ; DK133480//HHS | NIH | National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)/ ; },
mesh = {Animals ; *Zebrafish/genetics/embryology ; *Enteric Nervous System/embryology/metabolism/cytology ; *Zebrafish Proteins/genetics/metabolism ; *SOXC Transcription Factors/genetics/metabolism ; *Cell Differentiation/genetics ; Gene Expression Regulation, Developmental ; Neural Crest/metabolism/cytology ; *Neurons/metabolism/cytology ; CRISPR-Cas Systems ; Neurogenesis/genetics ; },
abstract = {The vertebrate enteric nervous system (ENS) is derived from vagal neural crest cells, which enter the foregut as progenitors that migrate from rostral to caudal to populate the entire length of the gut. Here, we show that transcription factors sox11a and sox11b, zebrafish orthologs of the human SOX11 gene, are highly expressed in neural crest cells transitioning from progenitors to differentiating neuronal subtypes. Accordingly, CRISPR-Cas9 depletion shows that loss of sox11 paralogs reduces the number of neurons that express the inhibitory motor neuron marker adcyap1b without affecting cell proliferation or death. Transcription factor footprinting analysis of open chromatin regions identified by ATAC-seq reveals Sox11 binding sites in the adcyap1b enhancer. Furthermore, mutational analysis shows these binding sites are required for mediating enhancer-driven reporter expression. Taken together, our results demonstrate an important and previously unrecognized role for sox11a and sox11b in neuronal subtype specification in the developing zebrafish ENS.},
}

Animals
*Zebrafish/genetics/embryology
*Enteric Nervous System/embryology/metabolism/cytology
*Zebrafish Proteins/genetics/metabolism
*SOXC Transcription Factors/genetics/metabolism
*Cell Differentiation/genetics
Gene Expression Regulation, Developmental
Neural Crest/metabolism/cytology
*Neurons/metabolism/cytology
CRISPR-Cas Systems
Neurogenesis/genetics

@article {pmid40788907,
year = {2025},
author = {Zhang, Y and Fu, Z and Yang, B and Wang, J and Lu, T and Shi, DL and Shao, M},
title = {Generation of Maternal Mutants Using zpc:cas9 Knock-in Zebrafish.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {221},
pages = {},
doi = {10.3791/68642},
pmid = {40788907},
issn = {1940-087X},
mesh = {Animals ; *Zebrafish/genetics ; Female ; *Gene Knock-In Techniques/methods ; *CRISPR-Cas Systems ; Animals, Genetically Modified ; *RNA-Binding Proteins/genetics ; *Zebrafish Proteins/genetics ; },
abstract = {Oogenesis and early embryonic development are critically dependent on maternally derived mRNAs and proteins. Eliminating these maternal factors necessitates homozygous mutations in female zebrafish, often resulting in lethal or infertile phenotypes, which prevent the acquisition of maternal mutant embryos. Our previous work introduced a rapid approach to bypass zygotic lethality through oocyte-specific genome editing. However, the previously reported cas9 transgene exhibits instability and undergoes gradual silencing over successive generations. Furthermore, the presence of Tol2 transposable elements flanking the zpc:cas9 cassette in this line hinders the potential for further sgRNA transgenesis using Tol2 system, which is currently the most efficient transgenic system in zebrafish. Consequently, there is a critical need for a Tol2-free zebrafish line that ensures stable and robust oocyte-specific Cas9 expression. Here, we present a line with zpccas9 knock-in at the rbm24a locus that addresses this requirement. Using this enhanced tool, we provide a pipeline for the rapid generation of maternal mutants of genes with zygotically lethal mutant phenotypes within the zebrafish model.},
}

Animals
*Zebrafish/genetics
Female
*Gene Knock-In Techniques/methods
*CRISPR-Cas Systems
Animals, Genetically Modified
*RNA-Binding Proteins/genetics
*Zebrafish Proteins/genetics

@article {pmid40788566,
year = {2025},
author = {Risse, J and Pietrek, L and Cantz, T and Krzemien, M and Schnalke, J and Eggenschwiler, R and Heinemann, T and Dederer, HG},
title = {"Snip, snip, cure"? Philosophical, legal and biomedical perspectives on novel somatic genomic therapies.},
journal = {Medicine, health care, and philosophy},
volume = {},
number = {},
pages = {},
pmid = {40788566},
issn = {1572-8633},
abstract = {The advent of innovative techniques, such as the CRISPR/Cas system, has opened up a new range of possibilities for modifying the genome, with the potential to address previously unmet therapeutic needs of patients with genetic diseases. These new possibilities have not only raised ethical concerns but also challenged existing classifications of genome modification techniques. While the legal status of some of these new therapies remains uncertain, there is an ongoing debate within philosophy of biology about the information-related metaphors adopted by scientists to describe and classify the genome and its therapeutic modification. Given the continuing advance of new genomic therapies, we show, employing an interdisciplinary approach, that a comprehensive framework for the classification of these technologies is needed to resolve legal and philosophical issues. The first section provides an analysis of the current state of novel genome-modifying techniques in medical genetics. In the second section, we assess the regulatory status of these techniques within the European regulatory framework for advanced therapy medicinal products (ATMPs). Drawing on these results, we argue in the third section from a philosophical perspective that metaphors, such as 'editing' the genome, which are based on a conception of the genome as linear information, cannot adequately capture the breadth of advanced genomic technologies. To accurately categorise these techniques in a manner that meets their diverse applications, we propose introducing the umbrella term 'somatic genomic therapies' (SGTs). Urging an integrative approach to defining and classifying new technologies in medical genetics, we advocate for the development of an integrative concept of SGTs.},
}