@article {pmid41707424,
year = {2026},
author = {Zhao, L and Liu, Z and Ding, G and Zhu, Y and Wang, H and Liu, R and Qu, F and Ao, Q and Zhu, X and Zhang, Y and Yang, G and Wang, Z},
title = {Dual-readout aptasensor based on CRISPR/Cas12a and nanozyme for accurate detection of KIM-1 and its application in kidney transplant prognosis.},
journal = {Biosensors & bioelectronics},
volume = {302},
number = {},
pages = {118533},
doi = {10.1016/j.bios.2026.118533},
pmid = {41707424},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; Humans ; *Hepatitis A Virus Cellular Receptor 1/isolation & purification ; *Aptamers, Nucleotide/chemistry ; CRISPR-Cas Systems ; *Kidney Transplantation ; Limit of Detection ; Prognosis ; Metal Nanoparticles/chemistry ; Silver/chemistry ; Colorimetry/methods ; Metal-Organic Frameworks/chemistry ; SELEX Aptamer Technique ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Kidney injury molecule-1 (KIM-1) has emerged as a pivotal prognostic biomarker for renal allograft function. However, its detection remains challenging due to sensitivity and accuracy limitations. An innovative biosensing platform synergizing aptamer recognition, CRISPR trans-cleavage and nanozyme amplification for dual-readout KIM-1 detection has been presented in this work. This platform employs a meticulously selected high-affinity aptamer with capillary electrophoresis SELEX for specific target recognition, leverages the CRISPR/Cas12a system for signal transduction and cascade amplification, and utilizes engineered FeNi MOF@AgNPs nanozyme for dual-signal output. With the presence of KIM-1, whose binding with aptamer, effectively inhibits the trans-cleavage activity of the CRISPR/Cas12a system, and uninhibited Cas12a subsequently cleaves nanozyme-conjugated magnetic probes, releasing FeNi MOF@AgNPs nanozymes that catalyze a TMB-based reaction to generate intense colorimetric and fluorescent dual-readout signals. The as developed aptasensor demonstrates satisfied sensitivity achieving detection limits of 58.7 pg/mL (colorimetric) and 34.4 pg/mL (fluorometric), and dependable accuracy achieving average relative deviation of -2.7% (colorimetric) and 3.2% (fluorometric) with commercial ELISA kit in urine samples from patients with acute kidney injury. Moreover, longitudinally track the dynamic changes in urinary KIM-1 concentrations over the first 8 days following renal transplantation was successfully realized. This work not only provides a robust analytical tool for KIM-1 detection but also establishes a generic research approach for extending CRISPR-based systems to the precise detection of proteins.},
}

*Biosensing Techniques/methods
Humans
*Hepatitis A Virus Cellular Receptor 1/isolation & purification
*Aptamers, Nucleotide/chemistry
CRISPR-Cas Systems
*Kidney Transplantation
Limit of Detection
Prognosis
Metal Nanoparticles/chemistry
Silver/chemistry
Colorimetry/methods
Metal-Organic Frameworks/chemistry
SELEX Aptamer Technique
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41707427,
year = {2026},
author = {Tu, Z and Wang, Y and Qian, X and Chen, J and Li, L and Wang, T and Chen, H and Wei, H and Yang, P and Pan, J and Wang, S and Rong, Z},
title = {Plasmonic magnetic nanoparticles-enabled universal enrichment, photothermal lysis, and duplex CRISPR detection of bacteria in urine samples.},
journal = {Biosensors & bioelectronics},
volume = {302},
number = {},
pages = {118518},
doi = {10.1016/j.bios.2026.118518},
pmid = {41707427},
issn = {1873-4235},
mesh = {Humans ; *Escherichia coli/isolation & purification/genetics ; *Magnetite Nanoparticles/chemistry ; *Enterococcus faecalis/isolation & purification/genetics ; *Biosensing Techniques/methods ; *Urinary Tract Infections/microbiology/urine/diagnosis ; CRISPR-Cas Systems ; Surface Plasmon Resonance/methods ; *Escherichia coli Infections/urine/microbiology/diagnosis ; Limit of Detection ; Boronic Acids/chemistry ; },
abstract = {Urinary tract infections (UTIs), exacerbated by antibiotic resistance and evolving pathogen diversity, demand rapid and sensitive diagnostics. This study introduces an integrated platform combining magnetic enrichment, photothermal lysis, and CRISPR-based detection (ME-CRISPR) for simultaneous identification of Escherichia coli (E. coli) and Enterococcus faecalis (E. faecalis) in UTIs. We engineered plasmonic magnetic nanoparticles functionalized with 4-mercaptophenylboronic acid for broad-spectrum bacterial capture within 10 min. Subsequent near-infrared laser irradiation leveraged localized surface plasmon resonance to lyse captured bacteria in situ. The released nucleic acids were directly analyzed in a single-step duplex RPA-CRISPR/Cas12a-Cas13a assay. This 40-min workflow achieved a limit of detection of 10 CFU/mL for both pathogens. Validation with 90 clinical samples (39 E. coli, 26 E. faecalis, and 25 negative controls) demonstrated 100% sensitivity and specificity, matching qPCR performance while significantly reducing turnaround time. The platform overcomes limitations of centrifugation and Gram-class-dependent lysis, offering a rapid and ultrasensitive point-of-care testing tool to curb antibiotic misuse.},
}

Humans
*Escherichia coli/isolation & purification/genetics
*Magnetite Nanoparticles/chemistry
*Enterococcus faecalis/isolation & purification/genetics
*Biosensing Techniques/methods
*Urinary Tract Infections/microbiology/urine/diagnosis
CRISPR-Cas Systems
Surface Plasmon Resonance/methods
*Escherichia coli Infections/urine/microbiology/diagnosis
Limit of Detection
Boronic Acids/chemistry

@article {pmid41707429,
year = {2026},
author = {Nong, J and Pan, Z and Li, Y and Wei, J and Gong, Y and Li, J and Zhang, K and Liao, X},
title = {COF-confined CsPbBr3 nanocomposite with CRISPR/Cas12a-driven DNA walking for ultrasensitive electrochemiluminescent detection of circulating tumor DNA.},
journal = {Biosensors & bioelectronics},
volume = {302},
number = {},
pages = {118536},
doi = {10.1016/j.bios.2026.118536},
pmid = {41707429},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; Humans ; *Nanocomposites/chemistry ; Luminescent Measurements/methods ; CRISPR-Cas Systems/genetics ; Electrochemical Techniques/methods ; Limit of Detection ; *Circulating Tumor DNA/blood/isolation & purification/genetics ; Endodeoxyribonucleases/chemistry ; DNA Probes/chemistry ; Metal-Organic Frameworks/chemistry ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {A highly sensitive electrochemiluminescence biosensor was developed for circulating tumor DNA detection by integrating a covalent organic framework-confined CsPbBr3 nanocomposite with a CRISPR/Cas12a-driven amplification strategy. The covalent organic framework not only stabilizes CsPbBr3 nanocrystals in aqueous environments but also regulates interfacial charge transfer and enables programmable immobilization of DNA probes. Upon recognition of the target sequence, Cas12a is activated and progressively cleaves surface-tethered quencher-modified DNA strands, leading to stepwise restoration of electrochemiluminescence emission. This surface-confined signal amplification eliminates the need for polymerase chain reaction or isothermal preamplification. Under optimized conditions, the biosensor exhibited a linear response over a concentration range from 10 fM to 10 nM, with a detection limit of 5.4 fM. The method demonstrated good selectivity toward single-base mismatches and satisfactory performance in diluted serum and clinical plasma samples. These results highlight a synergistic material-enzyme strategy for sensitive and robust nucleic acid detection and provide a proof-of-concept platform for electrochemiluminescence-based circulating tumor DNA analysis.},
}

*Biosensing Techniques/methods
Humans
*Nanocomposites/chemistry
Luminescent Measurements/methods
CRISPR-Cas Systems/genetics
Electrochemical Techniques/methods
Limit of Detection
*Circulating Tumor DNA/blood/isolation & purification/genetics
Endodeoxyribonucleases/chemistry
DNA Probes/chemistry
Metal-Organic Frameworks/chemistry
Bacterial Proteins
CRISPR-Associated Proteins

@article {pmid41708610,
year = {2026},
author = {Klann, M and Miura, S and Lee, SH and Vianello, SD and Ross, R and Watanabe, M and Gairin, E and Liang, Y and Hutto, HW and McCluskey, BM and Herrera, M and Solnica-Krezel, L and Besseau, L and Pigolotti, S and Parichy, DM and Kinoshita, M and Laudet, V},
title = {Cell-cell communication as underlying principle governing color pattern formation in teleost fishes.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41708610},
issn = {2041-1723},
mesh = {Animals ; *Cell Communication/genetics/physiology ; Zebrafish/genetics ; Gap Junctions/metabolism ; *Pigmentation/genetics/physiology ; Mutation, Missense ; Connexins/genetics/metabolism ; Fish Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Genome-Wide Association Study ; Phenotype ; Gene Editing ; *Perciformes/genetics ; },
abstract = {The diverse pigmentation patterns of animals are crucial for predation avoidance and behavioral display. This diversity arises from interactions among distinct pigment cell types, yet mechanisms generating pattern variation across teleost fishes remain incompletely understood. In zebrafish, Turing models have been proposed to explain stripe patterns, but it is unclear if they apply to other fishes. Here, we investigate the Snowflake mutant of the anemonefish Amphiprion ocellaris, which displays enlarged white bars with irregular boundaries. Using genome-wide association mapping and targeted sequencing, we identify a missense mutation (E42K) in gja5b, encoding the gap junction protein Connexin 41.8. CRISPR/Cas9-mediated genome editing recapitulates the Snowflake phenotype, while pharmacological inhibition of gap junctions phenocopies the boundary defects, supporting a causal role for impaired intercellular communication. Expression analyses reveal that, unlike zebrafish, anemonefish gja5b is predominantly expressed in iridophores. With functional in vitro assays we demonstrate that the E42K mutation acts as a dominant negative, strongly reducing gap junctional coupling. Introducing the same mutation in zebrafish reveals context-dependent effects on pigment patterning. Taken together our findings highlighting gap junction-mediated communication as a conserved but flexible mechanism controlling pigment boundary positioning and pattern diversification.},
}

Animals
*Cell Communication/genetics/physiology
Zebrafish/genetics
Gap Junctions/metabolism
*Pigmentation/genetics/physiology
Mutation, Missense
Connexins/genetics/metabolism
Fish Proteins/genetics/metabolism
CRISPR-Cas Systems
Genome-Wide Association Study
Phenotype
Gene Editing
*Perciformes/genetics

@article {pmid41708664,
year = {2026},
author = {Huo, Y and Mei, J and Zhang, D and Yan, B and Zhang, D and Dong, C and Yin, S and Liu, M and Wang, X and Chen, D and Guan, Y and Song, G and Du, B and Wang, Y and Zheng, Z and Liu, H and Li, D and Yang, L and Wang, L},
title = {Engineered Un1Cas12f1 for multiplex genome editing with enhanced activity and targeting scope.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41708664},
issn = {2041-1723},
support = {U24A20677//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32025023//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32230064//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32311530111//National Natural Science Foundation of China (National Science Foundation of China)/ ; 24J22800400//Science and Technology Commission of Shanghai Municipality (Shanghai Municipal Science and Technology Commission)/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Animals ; Humans ; Mice ; HEK293 Cells ; Genome, Human ; Mutation ; Genetic Therapy/methods ; *CRISPR-Associated Proteins/genetics/metabolism ; },
abstract = {The compact CRISPR-Cas12f system is promising for AAV-delivered gene therapy, but its application has been constrained by restrictive PAM recognition (e.g., TTTR) and suboptimal editing efficiency. Through bacterial library screening and mammalian cell validation, we engineer evoCas12f, an optimized variant incorporating five key mutations, that dramatically expands PAM recognition to NTNR/NYTR. This advancement reduces median distance between two neighbouring PAM sites to 2 nucleotides in the human genome. It also demonstrates 1.4-fold enhanced activity at TTTR sites compared to wild-type Un1Cas12f1, achieving up to 91% editing efficiency. Remarkably, evoCas12f enables efficient generation of homozygous mutations in F0 generation mice, even at non-canonical PAM sites. We further adapt this system for robust transcriptional activation and precise base editing with a well-defined editing window. As a compact yet highly efficient platform, evoCas12f represents a significant advance in CRISPR technology, enabling multiplexed editing for high-resolution targeting applications and expanding possibilities for therapeutic genome engineering.},
}

*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Animals
Humans
Mice
HEK293 Cells
Genome, Human
Mutation
Genetic Therapy/methods
*CRISPR-Associated Proteins/genetics/metabolism

@article {pmid41709648,
year = {2026},
author = {Doghish, AS and Ghaiad, HR and Elfar, N and El Said, NH and Radwan, AF and Abd-Elmawla, MA and Mohamed, HH and Mohammed, OA and Rizk, HA},
title = {Unraveling the Function of lncRNAs in Gliomas: Interaction With Signaling Pathways and Therapeutic Opportunities.},
journal = {Journal of biochemical and molecular toxicology},
volume = {40},
number = {3},
pages = {e70756},
doi = {10.1002/jbt.70756},
pmid = {41709648},
issn = {1099-0461},
mesh = {Humans ; *RNA, Long Noncoding/genetics/metabolism ; *Glioma/genetics/metabolism/therapy/pathology ; *Signal Transduction ; *Brain Neoplasms/metabolism/genetics/therapy/pathology ; Animals ; *Gene Expression Regulation, Neoplastic ; *RNA, Neoplasm/metabolism/genetics ; },
abstract = {Brain tumors represent some of the most formidable challenges in neuro-oncology due to their aggressive clinical course, resistance to therapy, and profound molecular heterogeneity. Among the emerging regulatory elements reshaping our understanding of tumor biology are long non-coding RNAs (lncRNAs), a diverse class of RNA transcripts that modulate gene expression and cellular behavior without encoding proteins. This review provides an in-depth and integrative examination of the biogenesis, regulatory mechanisms, and functional roles of lncRNAs in brain tumor development and progression. We systematically explore both canonical and non-canonical pathways of lncRNA biogenesis, detailing how these influence structural specificity and molecular interactions. This review synthesized evidence retrieved from PubMed/MEDLINE, Scopus, and Web of Science, covering publications from January 2010 to June 2025. This analysis highlights key gaps, such as context-dependent therapeutic effects that limit translational applicability. A major focus is placed on the interplay between lncRNAs and core oncogenic signaling pathways, including Phosphoinositide 3-kinase (PI3K)/serine/threonine kinase (AKT), Signal Transducer and Activator of Transcription 3 (STAT3), Wingless/Int-1 (Wnt)/β-catenin, and Transforming Growth Factor-Beta (TGF-β), which drive malignant transformation, invasion, stemness, and therapeutic resistance in gliomas. Furthermore, we dissect the molecular functions of lncRNAs as epigenetic regulators, competitive endogenous RNAs (ceRNAs), and structural scaffolds, and discuss their contribution to the dynamic tumor microenvironment. By synthesizing the latest findings, this review underscores the academic and translational importance of targeting lncRNA-associated networks. It also highlights emerging therapeutic approaches, such as antisense oligonucleotides, RNA interference, CRISPR-Cas systems, and natural lncRNA-modulating compounds, which collectively represent a promising frontier in precision medicine for brain tumors. This work offers a critical framework for future research and therapeutic innovation in the lncRNA landscape of neuro-oncology.},
}

Humans
*RNA, Long Noncoding/genetics/metabolism
*Glioma/genetics/metabolism/therapy/pathology
*Signal Transduction
*Brain Neoplasms/metabolism/genetics/therapy/pathology
Animals
*Gene Expression Regulation, Neoplastic
*RNA, Neoplasm/metabolism/genetics

@article {pmid41709870,
year = {2026},
author = {Feng, L and Yu, P and He, N and Zhang, Q and Tang, Y and Geng, J and Lu, Q and Song, C and Chen, F},
title = {Label-Free MicroRNA Diagnostics: From CRISPR Nucleases to Nanomaterial-Enhanced Transducers.},
journal = {ACS synthetic biology},
volume = {15},
number = {3},
pages = {915-954},
doi = {10.1021/acssynbio.5c00868},
pmid = {41709870},
issn = {2161-5063},
mesh = {*MicroRNAs/genetics/analysis ; *Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *Nanostructures/chemistry ; },
abstract = {Label-free detection of microRNAs (miRNAs) has emerged as a pivotal approach in molecular diagnostics, integrating the programmability of CRISPR systems with the high sensitivity of nanomaterial-based transduction. miRNAs are short, noncoding RNAs that play central roles in gene regulation and disease pathogenesis, serving as valuable biomarkers for early diagnosis and prognosis. Conventional miRNA detection methods rely on labeling and multistep amplification, which hinder their adaptability for rapid and point-of-care applications. In contrast, label-free biosensing translates molecular recognition into intrinsic optical, electrochemical, or mechanical signals, enabling real-time, amplification-free analysis. This review summarizes recent advances in label-free miRNA biosensing, with emphasis on CRISPR/Cas12a, Cas13a, and Cas14a systems that couple target recognition with signal transduction, and nanomaterial-assisted platforms including gold and silver nanoparticles, carbon nanotubes, quantum dots, silica nanostructures, and magnetic composites. Particular attention is given to innovations that achieve attomolar-level sensitivity, single-nucleotide discrimination, and multiplex detection. We also discuss integration into microfluidic and wearable platforms, addressing persistent challenges in repeatability and stability, antifouling performance, and clinical translation. Emerging trends in artificial intelligence-assisted data processing, molecular logic circuits, and digital single-molecule biosensing are highlighted. These advances collectively outline the pathway toward intelligent, amplification-free, and portable miRNA diagnostics, bridging molecular biology and synthetic bioengineering for next-generation healthcare applications.},
}

*MicroRNAs/genetics/analysis
*Biosensing Techniques/methods
Humans
*CRISPR-Cas Systems/genetics
*Nanostructures/chemistry

@article {pmid41710881,
year = {2026},
author = {Karaoglu, IC and Odabas, A and Önder, T and Kizilel, S},
title = {Single-gene knockout of RNLS or HIVEP2 are insufficient to protect β-cell spheroids from allo- and xeno-rejection.},
journal = {Frontiers in immunology},
volume = {17},
number = {},
pages = {1759835},
pmid = {41710881},
issn = {1664-3224},
mesh = {Animals ; Mice ; *Insulin-Secreting Cells/transplantation/immunology/metabolism ; Humans ; *Graft Rejection/genetics/immunology/prevention & control ; Gene Knockout Techniques ; *Spheroids, Cellular/immunology/transplantation/metabolism ; CRISPR-Cas Systems ; *Islets of Langerhans Transplantation ; Cell Line ; Gene Editing ; Transplantation, Heterologous ; },
abstract = {INTRODUCTION: β-Cell replacement therapy offers a potential cure for type 1 diabetes, but its success is limited by rapid graft rejection. While genome-wide CRISPR screens have recently identified RNLS and HIVEP2 as candidate genes capable of protecting β-cells from autoimmune destruction, their efficacy against the distinct mechanisms of allogeneic and xenogeneic rejection remains unknown. This study aimed to test the hypothesis that single-gene ablation of RNLS or HIVEP2 protects β-cell spheroids from allo- and xenorejection in immunocompetent hosts.

METHODS: Murine β-TC-6 and human EndoC-βH1 β-cell lines were genetically edited using CRISPR-Cas9 to knockout RNLS or HIVEP2. Editing efficiencies were confirmed via T7 endonuclease I assay and Tracking of Indels by Decomposition (TIDE) analysis. Cells were aggregated into uniform, size-controlled spheroids using an optimized agarose suspension culture. Functional integrity was assessed via glucose-stimulated insulin secretion (GSIS). To evaluate immune evasion in vivo, luciferase-labeled spheroids were transplanted subcutaneously into immunocompetent CD-1 mice, modelling allogeneic (murine-to-murine) and xenogeneic (human-to-murine) rejection, with graft survival monitored longitudinally by bioluminescence imaging.

RESULTS: Robust editing efficiencies were achieved for both targets. Functional characterization indicated that Rnls deletion modestly impaired GSIS in murine cells, whereas HIVEP2 deletion showed no functional alterations in either cell line. In vivo assessment revealed no protective effects of RNLS or HIVEP2 deletion; grafts from both knockout groups displayed rejection kinetics indistinguishable from non-targeting controls. While allogeneic grafts survived longer than xenogeneic grafts, both were ultimately cleared by the host immune system regardless of genotype.

DISCUSSION: These data indicate that single-gene deletions of RNLS or HIVEP2 are insufficient to protect β-cell grafts from the barriers of allo- or xenorejection. By defining the limitations of these targets in isolation, our findings highlight the necessity for combinatorial genome editing strategies or complementary integration with immunomodulatory biomaterials to achieve effective and sustained β-cell graft survival.},
}

Animals
Mice
*Insulin-Secreting Cells/transplantation/immunology/metabolism
Humans
*Graft Rejection/genetics/immunology/prevention & control
Gene Knockout Techniques
*Spheroids, Cellular/immunology/transplantation/metabolism
CRISPR-Cas Systems
*Islets of Langerhans Transplantation
Cell Line
Gene Editing
Transplantation, Heterologous

@article {pmid41710969,
year = {2026},
author = {Xu, Z and Wei, M and Jiang, M and Wang, Y and He, K},
title = {Single-step duplex CRISPR coupled with lateral flow assay for point-of-care detection of human immunodeficiency virus and Treponema pallidum.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {10},
pages = {1996-2004},
doi = {10.1039/d5ay02168e},
pmid = {41710969},
issn = {1759-9679},
mesh = {Humans ; *Treponema pallidum/genetics/isolation & purification ; *Point-of-Care Systems ; *HIV Infections/diagnosis/virology ; *Syphilis/diagnosis/microbiology ; *CRISPR-Cas Systems/genetics ; *HIV/genetics/isolation & purification ; Point-of-Care Testing ; Sensitivity and Specificity ; },
abstract = {Continued acquired immunodeficiency syndrome (AIDS) caused by the human immunodeficiency virus (HIV) and syphilis caused by Treponema pallidum (TP) pose great challenges for global health, highlighting the need for rapid and sensitive diagnostics. Here, we introduced DIAL (Duplex Integrated All-in-one CRISPR Lateral flow assay), a simplified CRISPR-based diagnostic system for simultaneous detection of HIV and TP. We developed a single-step duplex CRISPR assay for dual-target recognition, which incorporated reverse transcription, multiplex recombinase polymerase amplification, transcription, CRISPR/Cas12a detection, and CRISPR/Cas13a detection in one pot. Then, we combined CRISPR detection assay with a "line-elimination" dual-line lateral flow assay for low-cost and equipment-free visual readout. We further developed reagent lyophilization and extraction-free sample lysis techniques to meet the point-of-care testing. The optimized DIAL system detected as low as 100 copies per µL of both HIV RNA and TP DNA within 45 minutes. In a clinical evaluation of 46 samples, it demonstrated 100% sensitivity and 97.5% specificity for HIV and 90% sensitivity and 100% specificity for TP, relative to quantitative PCR. The DIAL provided a scalable, accessible approach for decentralized screening of sexually transmitted infections or other pathogens in resource-limited settings.},
}

Humans
*Treponema pallidum/genetics/isolation & purification
*Point-of-Care Systems
*HIV Infections/diagnosis/virology
*Syphilis/diagnosis/microbiology
*CRISPR-Cas Systems/genetics
*HIV/genetics/isolation & purification
Point-of-Care Testing
Sensitivity and Specificity

@article {pmid41711690,
year = {2026},
author = {Shi, H and Chi, H},
title = {Next-generation CRISPR screens enable causal systems immunology.},
journal = {The Journal of experimental medicine},
volume = {223},
number = {3},
pages = {},
doi = {10.1084/jem.20241266},
pmid = {41711690},
issn = {1540-9538},
support = {//American Lebanese Syrian Associated Charities/ ; CA253188/NH/NIH HHS/United States ; CA281868/NH/NIH HHS/United States ; AI105887/NH/NIH HHS/United States ; AI131703/NH/NIH HHS/United States ; AI140761/NH/NIH HHS/United States ; AI150241/NH/NIH HHS/United States ; AI150514/NH/NIH HHS/United States ; //Lupus Research Alliance/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Systems Biology/methods ; Animals ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Single-Cell Analysis ; Immunoinformatics ; },
abstract = {Mapping the causal circuits that shape the phenotypic and functional landscape of immune cells remains a formidable challenge. Recent advances in pooled CRISPR-based screens, coupled with multiplexed single-cell profiling and imaging-based spatial readouts, make this goal increasingly attainable. In this Perspective, we discuss how CRISPR-based genetic screens will fundamentally transform our understanding of immunobiology. We highlight the applications of state-of-the-art, high-throughput pooled perturbation approaches, including emerging methodologies for bulk, single-cell, and spatial CRISPR screens, to advance our understanding of immunity and in vivo biology. Additionally, we summarize new strategies to address the complexity of combinatorial perturbations to uncover genetic interactions and mechanistic drivers of immunity at unprecedented scale and resolution. By integrating CRISPR screening data with experimental insights, we advocate a new framework in immunology research that leverages perturbation-driven regulatory effects and networks to discover new therapeutic targets and establish causal systems biology and immunology for advancing immunological knowledge and therapeutic application.},
}

Humans
*CRISPR-Cas Systems/genetics
*Systems Biology/methods
Animals
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Single-Cell Analysis
Immunoinformatics

@article {pmid41711853,
year = {2026},
author = {Guo, Y and Zhao, J and Li, X and Zheng, X and Yuan, Y and Deng, J and Ren, Y and Xu, K},
title = {Detection of alkaline phosphatase activity based on ATP hydrolysis and CRISPR/Cas12a.},
journal = {Analytical and bioanalytical chemistry},
volume = {418},
number = {8},
pages = {2337-2344},
pmid = {41711853},
issn = {1618-2650},
support = {2023JJ30430//Natural Science Foundation of Hunan Province/ ; 23A0075//Education Department of Hunan Province/ ; 82173572//National Natural Science Foundation of China/ ; 82574159//National Natural Science Foundation of China/ ; },
mesh = {*Alkaline Phosphatase/metabolism/analysis ; *Adenosine Triphosphate/metabolism/chemistry ; *CRISPR-Cas Systems ; Hydrolysis ; Animals ; Limit of Detection ; Milk/enzymology ; Aptamers, Nucleotide/chemistry ; *CRISPR-Associated Proteins/metabolism ; Biosensing Techniques/methods ; Cattle ; *Endodeoxyribonucleases/metabolism ; Bacterial Proteins ; },
abstract = {Alkaline phosphatase (ALP) activity serves as a crucial biomarker for livestock disease diagnosis, nutritional management, and the evaluation of milk pasteurization efficacy, holding significant implications for food safety and food science. In this study, we developed a novel method for detecting ALP activity in food samples, based on ATP hydrolysis coupled with a CRISPR/Cas12a system. The assay employs a DNA molecular recognition lock probe, comprising an ATP-specific aptamer and an activator strand designed to trigger the CRISPR/Cas12a trans-cleavage activity. In the absence of ALP, ATP acts as a trans-hydrolysis substrate to open the lock structure, releasing the activator strand. This subsequently activates the Cas12a protein, leading to the generation of a fluorescent signal. Conversely, when the target ALP is present, it catalyzes the hydrolysis of ATP via a dephosphorylation reaction. This prevents the opening of the "Aptamer-Activator" molecular lock, thereby inhibiting Cas12a activation and resulting in a corresponding decrease in fluorescence intensity. Under optimized conditions, the assay demonstrated a detection limit of 2.52 mU/mL for ALP activity, with a linear range of 0-18.75 mU/mL. The total detection time was 70 min. The method was successfully applied to detect ALP activity in samples from various livestock (chickens, pigs, sheep, cattle) and in milk, achieving recovery rates between 92 and 99%. In conclusion, we have developed a sensitive, cost-effective, and rapid method for ALP detection. This work provides a promising strategy for the development of point-of-care testing (POCT) devices in food safety monitoring.},
}

*Alkaline Phosphatase/metabolism/analysis
*Adenosine Triphosphate/metabolism/chemistry
*CRISPR-Cas Systems
Hydrolysis
Animals
Limit of Detection
Milk/enzymology
Aptamers, Nucleotide/chemistry
*CRISPR-Associated Proteins/metabolism
Biosensing Techniques/methods
Cattle
*Endodeoxyribonucleases/metabolism
Bacterial Proteins

@article {pmid41712102,
year = {2026},
author = {Lin, J and Hazaisi, H and Guan, Y and Bai, M},
title = {Multiplex gene editing drives revolution in crop breeding: overlaid editing of multiple genes and customization of complex traits.},
journal = {Advanced biotechnology},
volume = {4},
number = {1},
pages = {5},
pmid = {41712102},
issn = {2948-2801},
support = {2023ZD040360104//Chinese Academy of Agricultural Sciences from Institute of Crop Science/ ; },
abstract = {Modern agriculture currently demands higher standards for the simultaneous improvement of crop yield, quality and stress resistance. However, traditional crop breeding methods can no longer meet the needs of modern agricultural development. Improving a single trait is no longer sufficient to meet the multifaceted demands of modern agricultural production and consumer expectations. Multiple traits breeding has increasingly become a key objective in current crop breeding. Over the past decade, CRISPR/Cas9-based multiplex genome editing (MGE) has enabled efficient pyramiding and precise regulation of multiple traits via targeted editing of multiple gene loci, revolutionizing crop breeding. In this review, we briefly describe the core CRISPR/Cas-based MGE strategies and technical workflows, and thoroughly discuss the practical outcomes of MGE applications in various fields, such as enhancing crop stress resistance, increasing yield and improving quality. This review aims to provide a summary and theoretical reference for crop breeding, as well as open up new ideas for achieving different breeding goals.},
}

@article {pmid41712409,
year = {2026},
author = {Nenad, WC and Kuhlers, PC and Sturgill, IR and Biju, I and Bucklan, M and Hernandez, L and Zhu, LC and Hoadley, KA and Raab, JR},
title = {Hepatocyte-targeted Bap1 reduction in the liver primes an inflammatory transcriptional response.},
journal = {G3 (Bethesda, Md.)},
volume = {16},
number = {5},
pages = {},
pmid = {41712409},
issn = {2160-1836},
support = {/GM/NIGMS NIH HHS/United States ; },
mesh = {*Ubiquitin Thiolesterase/genetics/metabolism ; Animals ; *Hepatocytes/metabolism ; *Tumor Suppressor Proteins/genetics/metabolism ; Mice ; *Liver/metabolism/pathology ; *Inflammation/genetics/metabolism/pathology ; Mice, Knockout ; Transcriptome ; CRISPR-Cas Systems ; *Transcription, Genetic ; Gene Expression Profiling ; },
abstract = {BRCA1-associated protein 1 (BAP1) is a deubiquitinase, frequently altered in cancers including hepatocellular carcinoma and cholangiocarcinoma. While Bap1 has been shown to play key roles in metabolism, maintenance of tissue homeostasis, and immune cell development, little is known about its normal functions in the liver in vivo. Using AAV8-mediated CRISPR/CAS9 genome editing, we generated a mouse hepatocyte-specific model of Bap1 knockout to define the changes that occur in liver biology in an in vivo system and characterize how loss of Bap1 alters the liver's response to injury. Single-cell resolution spatial transcriptomics were performed in conjunction with immunohistochemistry to analyze cell-type composition and immune cell recruitment changes. Bulk RNA-sequencing was performed to further assess the impact of Bap1 loss on transcription. Hepatocyte-specific depletion of Bap1-induced transcriptional changes shared with acute injury. We observed a strong dysregulation of inflammatory pathways associated with Bap1 loss. Moreover, the transcriptional response of Bap1 depletion in hepatocytes to damage was markedly different than in control liver, with Bap1-depleted livers showing a decreased hepatocyte identity based on gene expression. Spatial transcriptomics and quantitative texture analysis of immunohistochemistry revealed an altered immune environment prior to damage and an impaired recruitment of immune cells in Bap1-depleted livers after damage. Our data suggest Bap1 is a critical modulator in the liver's immune cell response and its loss leads to an inflammatory environment prior to damage and disrupts the recruitment immune cells. Our quantitative spatial analysis highlights the power of such approaches to characterize the spatial distribution of different cell types in a tissue.},
}

*Ubiquitin Thiolesterase/genetics/metabolism
Animals
*Hepatocytes/metabolism
*Tumor Suppressor Proteins/genetics/metabolism
Mice
*Liver/metabolism/pathology
*Inflammation/genetics/metabolism/pathology
Mice, Knockout
Transcriptome
CRISPR-Cas Systems
*Transcription, Genetic
Gene Expression Profiling

@article {pmid41712528,
year = {2026},
author = {Magdy, M and Tinker-Kulberg, R and Josephs, EA},
title = {Polyvalent Guide RNAs Enhance the CRISPR-Mediated Suppression of a Human Coronavirus.},
journal = {ACS synthetic biology},
volume = {15},
number = {5},
pages = {1738-1750},
doi = {10.1021/acssynbio.5c00574},
pmid = {41712528},
issn = {2161-5063},
support = {R35GM133483/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Coronavirus 229E, Human/genetics ; Gene Editing/methods ; RNA, Viral/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; HEK293 Cells ; },
abstract = {While CRISPR enzymes have become important tools for targeted gene editing in mammalian cells, they can also be used to specifically target and deplete viral nucleic acids to treat infections; this can be accomplished by delivering an RNA-targeting CRISPR effector like Cas13 along with a guide RNA (gRNA) that recognizes sequences from the genomes of single-stranded RNA (ssRNA) viruses. Previously, we hypothesized that by designing individual gRNAs able to target multiple, similar-but-not-identical viral sequences simultaneously ("polyvalent" guide RNAs or pgRNAs), gRNA's polyvalency would overcome any deficits caused by mispairing between the gRNA and the viral targets and, hence, still increase Cas13's antiviral potency and prevent mutagenic escape. We subsequently demonstrated this was the case using a model of viral infection in plants; however, it was not determined whether this strategy would also work against a human virus. Here, pgRNAs were designed to target multiple RNA sequences within human coronavirus 229E (hCoV-229E) and delivered along with Cas13 into a human lung epithelial cell line infected by hCoV-229E. CRISPR antiviral treatments using pgRNAs exhibited significant viral suppression in a CRISPR-dependent manner─more so than their single-target gRNA counterparts, even when multiple single-target gRNAs were used simultaneously. This improvement was also observed even as Cas13 with those same pgRNAs exhibited less "collateral" or nonspecific RNase activity relative to their single-target counterparts, which could imply that they may have greater specificity and safety profiles as therapeutic agents. Our findings demonstrate a computational and experimental pipeline by which pgRNAs, created using an unconventional gRNA design strategy, can be generated and validated to target human viruses using CRISPR antiviral biotechnologies more effectively.},
}

Humans
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Coronavirus 229E, Human/genetics
Gene Editing/methods
RNA, Viral/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
HEK293 Cells

@article {pmid41712626,
year = {2026},
author = {Sünderhauf, D and Ringger, JR and Payne, LJ and Pinilla-Redondo, R and Gaze, WH and Brown, SP and van Houte, S},
title = {CRISPR-Cas is beneficial in plasmid competition, but limited by competitor toxin-antitoxin activity when horizontally transferred.},
journal = {PLoS biology},
volume = {24},
number = {2},
pages = {e3003658},
pmid = {41712626},
issn = {1545-7885},
mesh = {*Plasmids/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Transfer, Horizontal ; Escherichia coli/genetics ; *Toxin-Antitoxin Systems/genetics ; },
abstract = {Bacteria can encode dozens of different immune systems that protect them from infection by mobile genetic elements (MGEs). MGEs themselves may also carry immune systems, such as CRISPR-Cas, to target competitor MGEs. It is unclear when this is favored by natural selection, and whether toxin-antitoxin (TA) systems-common competitive mechanisms carried by plasmids-can alter their efficacy. Here, we develop and test novel theory to analyze the outcome of competition between plasmids when one carries a CRISPR-Cas system that targets the other plasmid. Our mathematical model and experiments using Escherichia coli and competing IncP plasmids reveal that plasmid-borne CRISPR-Cas is beneficial to the plasmid carrying it when the plasmid has not recently transferred to a new host. However, CRISPR-Cas is selected against when the plasmid carrying it transfers horizontally, if a resident competitor plasmid encodes a TA system that elicits post-segregational killing. Consistent with a TA barrier to plasmid-borne CRISPR-Cas, a bioinformatic analysis reveals that naturally occurring CRISPR-Cas-bearing plasmids avoid targeting other plasmids with TA systems across bacterial genera. Our work shows how the benefit of plasmid-borne CRISPR-Cas is severely reduced against TA-encoding competitor plasmids, but only when plasmid-borne CRISPR-Cas is horizontally transferred. These findings have key implications for the distribution of prokaryotic defenses and our understanding of their role in competition between MGEs, and the utility of CRISPR-Cas as a tool to remove plasmids from pathogenic bacteria.},
}

*Plasmids/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Gene Transfer, Horizontal
Escherichia coli/genetics
*Toxin-Antitoxin Systems/genetics

@article {pmid41713039,
year = {2026},
author = {Zhang, H and Cui, C and Wang, X and Liu, S and Wang, X and Wang, Y and Ge, S and Cai, Y and Bao, J and Wang, Z},
title = {Development of a one-pot RT-RAA/CRISPR-Cas13a assay for rapid genotyping of Nipah virus in pigs.},
journal = {Diagnostic microbiology and infectious disease},
volume = {115},
number = {2},
pages = {117316},
doi = {10.1016/j.diagmicrobio.2026.117316},
pmid = {41713039},
issn = {1879-0070},
mesh = {Animals ; Swine ; *Nipah Virus/genetics/isolation & purification/classification ; Sensitivity and Specificity ; *Swine Diseases/virology/diagnosis ; Genotype ; *CRISPR-Cas Systems ; *Genotyping Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; *Henipavirus Infections/veterinary/diagnosis/virology ; *Molecular Diagnostic Techniques/methods ; Polymorphism, Single Nucleotide ; },
abstract = {INTRODUCTION: Nipah virus (NiV) is a highly pathogenic zoonotic virus transmitted from bats to humans through pigs as a key intermediate host. Given the existence of two distinct NiV genotypes, which differ in clinical manifestations and transmission patterns in both humans and pigs, rapid and sensitive method for detection and genotyping is crucial for effective disease control. Isothermal amplification combined with CRISPR/Cas-based assay provides a promising approach to meet this need.

METHODS: Conserved regions were identified by aligning the N gene sequences from 67 NiV strains. Specific primers and probes were designed for reverse transcription recombinase-aided amplification (RT-RAA) to detect NiV. Subsequently, single nucleotide polymorphisms within the conserved region were analyzed, and corresponding crRNAs were designed to establish a one-pot RT-RAA/CRISPR-Cas13a assay for NiV genotyping. The assays were evaluated using simulated pig serums spiked with NiV pseudovirus.

RESULTS: The RT-RAA assay exhibited a detection sensitivity of 10[-2] Infection Unit/mL (IU/mL) for NiV pseudovirus, outperforming conventional qRT-PCR in simulated pig serum samples. No cross-reactivity was observed with viral RNA or DNA of PCV2, PEDV, PRRSV, PRV and SVA, confirming high specificity. The entire one-pot RT-RAA/CRISPR-Cas13a assay could be completed within 1 hour and clearly discriminated between the two NiV genotypes without requiring sophisticated instruments. Evaluation with simulated samples showed a sensitivity of 100% (95% CI, 92.87-100%) and a specificity of 94% (95% CI, 83.78-98.36%), with a detection limit of 10[-1] IU/mL for NiV pseudovirus.

CONCLUSION: The one-pot RT-RAA/CRISPR-Cas13a assay provides a rapid and sensitive platform for NiV genotyping.},
}

Animals
Swine
*Nipah Virus/genetics/isolation & purification/classification
Sensitivity and Specificity
*Swine Diseases/virology/diagnosis
Genotype
*CRISPR-Cas Systems
*Genotyping Techniques/methods
*Nucleic Acid Amplification Techniques/methods
*Henipavirus Infections/veterinary/diagnosis/virology
*Molecular Diagnostic Techniques/methods
Polymorphism, Single Nucleotide

@article {pmid41713565,
year = {2026},
author = {Shashikala, T and Yogi, D and Akshay, K and Nagesh, SN and Manamohan, M and Venkataravanappa, V and Jha, GK and Ashok, K and Asokan, R},
title = {First report of CRISPR/Cas13a-based rapid detection of groundnut bud necrosis virus without amplification.},
journal = {Methods (San Diego, Calif.)},
volume = {249},
number = {},
pages = {9-22},
doi = {10.1016/j.ymeth.2026.02.010},
pmid = {41713565},
issn = {1095-9130},
mesh = {*CRISPR-Cas Systems/genetics ; *Plant Diseases/virology ; RNA, Viral/genetics/isolation & purification ; *Tospovirus/genetics/isolation & purification ; Vigna/virology ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Globally, the groundnut bud necrosis virus (GBNV) (Bunyaviridae), pose a serious threat to solanaceous and leguminous crops causing serious crop loss. This requires a rapid and sensitive diagnostics for initially identifying at the earliest stage and further to initiate disease management. This study presents the first report of a CRISPR/Cas13a-based diagnostic assay for GBNV detection without amplification. GBNV was maintained in cowpea cv. C152 through mechanical inoculation, further viral RNA was isolated to clone the two target genes viz. nucleocapsid (NP) and movement protein (MP) genes. These genes were ligated to pTZ57R/T vector and sequenced. Similarly, the LshCas13a gene was cloned from pUC19 into pET28a, expressed in E. coli BL21, and purified using Ni-NTA affinity chromatography. Guide RNAs targeting conserved regions of NP and MP genes were synthesized by in vitro transcription and mixed with Cas13a protein to form ribonucleoprotein (RNP) complex. Target RNA, obtained either by in vitro-transcription or crude extract of infected tomato was used to detect GBNV, using a fluorescence-based reporter assay. This method found to be highly sensitive that could detect GBNV at as low as 0.01 ng. From the field perspective, GBNV could be detected from the crude extract of the GBNV infected tomato leaves using an alkaline PEG buffer. Thus CRISPR/Cas13a-based assay provides a rapid, amplification-free, and field-deployable diagnostic platform for GBNV. This lays the groundwork for a field adoptable CRISPR diagnostics for other plant RNA viruses also.},
}

*CRISPR-Cas Systems/genetics
*Plant Diseases/virology
RNA, Viral/genetics/isolation & purification
*Tospovirus/genetics/isolation & purification
Vigna/virology
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid41713574,
year = {2026},
author = {Hu, T and Hou, Z and Zhang, Y and Jing, P and Dai, X and Wang, H},
title = {Development of a one-pot integrated rapid detection method for white spot syndrome virus based on RAA-CRISPR/Cas12a technology.},
journal = {Journal of invertebrate pathology},
volume = {216},
number = {},
pages = {108569},
doi = {10.1016/j.jip.2026.108569},
pmid = {41713574},
issn = {1096-0805},
mesh = {*White spot syndrome virus 1/isolation & purification ; Animals ; *Penaeidae/virology ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; Aquaculture ; Sensitivity and Specificity ; },
abstract = {Pathogenic microorganisms, particularly white spot syndrome virus (WSSV), pose a major threat to global shrimp aquaculture, causing mass mortalities and substantial economic losses. To address the urgent need for rapid, accurate, and field-deployable detection methods, this study developed an innovative one-pot RAA-CRISPR/Cas12a assay. The platform integrates recombinase-aided amplification (RAA) with CRISPR/Cas12a technology using sucrose-mediated density gradient phase separation in a closed-tube format: sucrose acts as a density modifier to form distinct layers, spatially isolating RAA amplification reagents from CRISPR/Cas12a detection components to avoid cross-interference and enable sequential reactions without manual intervention. Under isothermal conditions at 37℃ for 60 min, the optimized assay achieves a limit of detection as low as 1 copy/μL, validated by both fluorescence and lateral flow dipstick (LFD) readouts. High specificity was confirmed by the absence of cross-reactivity with four other prevalent shrimp pathogens: infectious hypodermal and hematopoietic necrosis virus (IHHNV), Decapod iridescent virus 1 (DIV1), Enterocytozoon hepatopenaei (EHP), and Vibrio parahaemolyticus associated with acute hepatopancreatic necrosis disease (VpAHPND). Clinical validation with 30 field samples showed concordant results with the chinese national detection standard (GB/T 28630.2-2012). This novel nucleic acid detection platform combines highly sensitive, excellent specificity, and user-friendly visual interpretation, making it highly suitable for point-of-care testing and large-scale disease surveillance in shrimp aquaculture.},
}

*White spot syndrome virus 1/isolation & purification
Animals
*Penaeidae/virology
*Nucleic Acid Amplification Techniques/methods
*CRISPR-Cas Systems
Aquaculture
Sensitivity and Specificity

@article {pmid41714766,
year = {2026},
author = {Taha, BA and Addie, AJ and Haider, AJ and Jasim, AJ and Ahmed, NM and Arsad, N},
title = {Perspective of smart nanocapsule swallowable laser-guided for integrated sensing and crispr-mediated cancer gene editing.},
journal = {Cancer gene therapy},
volume = {33},
number = {3},
pages = {351-365},
pmid = {41714766},
issn = {1476-5500},
mesh = {Humans ; *Gene Editing/methods ; *Nanocapsules/chemistry/administration & dosage ; *Neoplasms/genetics/therapy ; *CRISPR-Cas Systems ; Lasers ; Biosensing Techniques/methods ; Animals ; *Genetic Therapy/methods ; },
abstract = {Current therapeutic techniques for cancer often lack specificity. They also cause systemic toxicity and lack genetic control. Thus, cancer ranks among the most complex and crucial global health issues. The novel concept of smart nanocapsules is discussed in this Perspective. These oral medications modify genes using CRISPR technology and integrate biosensing and laser-guided activation to enable more personalized cancer therapies. The creation of these versatile nanocapsules is driven by three objectives. First, they aim to enable controlled gene editing in the gastrointestinal tract. Second, they deliver treatments to specific target areas. Third, they detect tumors in real time. Nanocapsules equipped with biosensing components provide microenvironmental input. An external laser can trigger the release of light-absorbing agents. Moreover, these features reduce off-target effects and allow spatiotemporal precision, thhe enteric-coated architecture ensures oral stability. Surface functionalization enhances selective tumor accumulation. AI-guided control algorithms can manage diagnostic interpretation and activation. The CRISPR-based cancer medicines offer the potential for improved safety, specificity, and translational use in the future. Combining advanced nanotechnology, gene editing, and AI-guided control could create innovative solutions.},
}

Humans
*Gene Editing/methods
*Nanocapsules/chemistry/administration & dosage
*Neoplasms/genetics/therapy
*CRISPR-Cas Systems
Lasers
Biosensing Techniques/methods
Animals
*Genetic Therapy/methods

@article {pmid41715004,
year = {2026},
author = {Metz, P and Alves-Vasconcelos, S and Wallbank, R and Riepsaame, J and Brown, S and Hassan, AB},
title = {Variation in guide RNA library representation results in gene effect score bias in genome-wide CRISPR screens.},
journal = {BMC genomics},
volume = {27},
number = {1},
pages = {},
pmid = {41715004},
issn = {1471-2164},
support = {OxPOS//F. Hoffmann-La Roche/ ; GEO//John Fell Fund, University of Oxford/ ; MPNST//Grenfell Shaw Charity/ ; Oxford Clinical Fellowships/CRUK_/Cancer Research UK/United Kingdom ; },
mesh = {Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems ; *Gene Library ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Genome, Human ; },
abstract = {Genome wide CRISPR-based perturbation screens are powerful discovery tools enabling the identification of novel gene dependencies through either gain or loss of function. While genome wide guide RNA (gRNA) libraries have advantages when using enAsCas12a, such as multiplex single gRNAs per gene, they may be subject to similar confounding factors that can affect the interpretation of large genome-wide datasets. Here, we examine the impact of these variables in over twenty enAsCas12a multiple gRNA based perturbation screens performed using Humagne C, Humagne D and Inzolia libraries in human cells, as well as external datasets containing Cas9-based CRISPR library screens, including from DepMap. We demonstrate that the choice of CRISPR library is often the most significant factor that influences genetic perturbation results, outweighing other variables such as either target cell lines or culture media conditions. A potential contributor to this effect is gRNA representation within a given CRISPR library, where lower gRNA representation can lead to variable and more pronounced gene effect scores using either log fold change or Chronos analysis. These effects may be mitigated by using either multiple gRNA constructs per gene, by optimisation of CRISPR library production processes or by targeting with multiple independent gRNA libraries. Importantly, we also propose strategies for addressing gRNA representation bias during CRISPR screen hit prioritisation. CRISPR library gRNA representation dependent bias remains a major challenge in the interpretation of gene essentiality in perturbation screens.},
}

Humans
*RNA, Guide, CRISPR-Cas Systems/genetics
*CRISPR-Cas Systems
*Gene Library
*Clustered Regularly Interspaced Short Palindromic Repeats
Genome, Human

@article {pmid41715150,
year = {2026},
author = {Zhao, Y and Li, X and Du, Y},
title = {AI-driven CRISPR screening: optimizing gene editing through automation and intelligent decision support.},
journal = {Journal of translational medicine},
volume = {24},
number = {1},
pages = {},
pmid = {41715150},
issn = {1479-5876},
support = {No. Z04J2024E107-B-12//Bethune Charitable Foundation/ ; },
mesh = {Humans ; *Artificial Intelligence ; *Gene Editing ; *Automation ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Decision Support Techniques ; *CRISPR-Cas Systems/genetics ; Deep Learning ; *Genetic Testing/methods ; },
abstract = {BACKGROUND: CRISPR-based genetic screening has become a central methodology in functional genomics, enabling systematic interrogation of gene function, genetic interactions and context-dependent vulnerabilities at scale. However, the rapid expansion of screening modalities-including multi-condition designs, combinatorial perturbations, in vivo applications and single-cell readouts-has exposed fundamental limitations of heuristic-driven experimental design and post hoc statistical analysis.

MAIN BODY: This Review synthesizes how artificial intelligence is reshaping CRISPR screening by introducing predictive, adaptive and system-level intelligence across the experimental lifecycle. We organize recent advances into two tightly coupled modules. First, machine learning and deep learning (ML/DL) methods optimize experimental design by learning context-dependent perturbation behavior, anticipating confounding effects and enabling iterative, information-efficient screening strategies. Second, large language model-agent (LLM-agent) systems complement these advances by externalizing scientific reasoning, integrating biological knowledge at scale and coordinating analysis and decision-making in human-in-the-loop workflows.

CONCLUSIONS: Together, ML/DL and LLM-agent approaches reframe CRISPR screening from a static analytical pipeline into an intelligent experimental system, with important implications for robustness, scalability and biological discovery.},
}

Humans
*Artificial Intelligence
*Gene Editing
*Automation
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*Decision Support Techniques
*CRISPR-Cas Systems/genetics
Deep Learning
*Genetic Testing/methods

@article {pmid41715936,
year = {2026},
author = {Shimizu, Y and Kataoka, K},
title = {DIPA-CRISPR Mediated Knockout of Vermilion Generates a Visible Eye Color Marker for The Band-Legged Ground Cricket Dianemobius nigrofasciatus.},
journal = {Archives of insect biochemistry and physiology},
volume = {121},
number = {2},
pages = {e70135},
doi = {10.1002/arch.70135},
pmid = {41715936},
issn = {1520-6327},
support = {JPJ009237//Bio-oriented Technology Research Advancement Institution, BRAIN/ ; 21K05614//JSPS KAKENHI Grant-in-Aid for Scientific Research (C)/ ; 21J23478/22KJ2609//Grant-in-Aid for JSPS Fellows/ ; },
mesh = {Animals ; Female ; *Gryllidae/genetics/growth & development/physiology ; CRISPR-Cas Systems ; *Insect Proteins/genetics/metabolism ; *Eye Color/genetics ; Photoperiod ; Gene Knockout Techniques ; Gene Editing ; },
abstract = {The molecular basis of photoperiodism, by which insects use photoperiodic cues to anticipate seasonal changes and regulate key life-history events such as development, diapause, and reproduction, remains poorly understood. Studies on the molecular mechanisms of photoperiodism in hemimetabolous insects are limited compared with those in holometabolous insects, largely due to the lack of appropriate model organisms. The band-legged ground cricket Dianemobius nigrofasciatus represents a valuable model system because it exhibits clear photoperiodic responses in the maternal induction of embryonic diapause, the wing morph, and the rate of nymphal development. With the recent availability of the D. nigrofasciatus genome sequence, the establishment of effective genome-editing methods and reliable marker genes is expected to promote functional genomic analyses. In this study, we aimed to establish a direct parental (DIPA)-CRISPR genome-editing approach and evaluate the utility of vermilion (Dn-v), a gene involved in ommochrome synthesis, as a visible eye color marker for mutant screening. Cas9 ribonucleoprotein complexes were injected into females 3-5 days after adult emergence, during the vitellogenic stage, successfully yielding Dn-v knockout mutants. These mutants had white compound eyes throughout development, with pigmentation reaching a vermilion color about 20 days after adult emergence. We further examined the photoperiodic response associated with maternal diapause induction in knockout mutants. Similar to the wild-type, knockout mutants exhibited low and high diapause incidence under long-day and short-day conditions, respectively. Our results demonstrate that DIPA-CRISPR is an effective genome-editing method in D. nigrofasciatus and that Dn-v serves as a practical and reliable marker gene. The establishment of these genomic tools provides a foundation for future functional analyses aimed at elucidating the molecular basis of photoperiodism in hemimetabolous insects.},
}

Animals
Female
*Gryllidae/genetics/growth & development/physiology
CRISPR-Cas Systems
*Insect Proteins/genetics/metabolism
*Eye Color/genetics
Photoperiod
Gene Knockout Techniques
Gene Editing

@article {pmid41717108,
year = {2026},
author = {Balasubramani, S and Li, Z and Gayathri, E and Gurusamy, D and Singh, A},
title = {Editorial: Advancing plant defense: genome editing, RNAi, and synthetic biology for sustainable pest control.},
journal = {Frontiers in plant science},
volume = {17},
number = {},
pages = {1785705},
pmid = {41717108},
issn = {1664-462X},
}

@article {pmid41717201,
year = {2026},
author = {Mittal, A and Manna, S and Nelson, V and Ladha, N},
title = {In Silico Design of gRNA for CRISPR System for Detection of Multidrug Resistant Tuberculosis Using Indian Mycobacterium tuberculosis Genomes: A Computational Study.},
journal = {Cureus},
volume = {18},
number = {1},
pages = {e101851},
pmid = {41717201},
issn = {2168-8184},
abstract = {Background Multidrug-resistant tuberculosis (MDR-TB) continues to pose a major challenge to TB elimination in India, where drug resistance and delayed diagnosis contribute significantly to ongoing transmission. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) based diagnostics have emerged as versatile tools, compared to GeneXpert, capable of detecting resistance-associated mutations with rapid turnaround and high accuracy. This study aimed to design and in silico validate Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated protein (CRISPR-Cas)-based guide RNAs (gRNAs) targeting major drug-resistance mutations in Indian Mycobacterium tuberculosis (M. tuberculosis) isolates. Methods Whole-genome mutation profiles were analyzed using TBProfiler, and gRNAs were designed using CHOPCHOP. Off-target evaluation was performed using Cas-OFFinder and Basic Local Alignment Search Tool (BLAST). High-confidence mutations in gyrA, rpoB, katG, rpsL, embB, and ethA were selected based on prevalence in Indian isolates and WHO-defined resistance markers. Results Numerous drug resistance-associated mutations were identified in the drug-resistant tuberculosis genome isolates. The study identified six key genetic mutations identified in MTB isolates that are associated with phenotypic drug resistance, including gyrA (Asp94Gly), rpoB (Ser450Leu), and katG (Ser315Thr). For each of the six genes, the chromosome position, locus ID, mutation type, and affected amino acids were identified, and tailored guide RNAs were designed in silico. Top-ranked gRNAs demonstrated optimal GC content, high predicted cleavage efficiency, and zero off-target activity. Each resistance locus yielded multiple candidate gRNAs suitable for CRISPR-based assays. Conclusions This computational in silico analysis provides a robust panel of mutation-targeted gRNAs tailored to Indian MDR-TB genomic profiles. These findings lay a strong foundation for developing rapid, affordable CRISPR diagnostics for point-of-care detection of drug resistance. Future laboratory validation and clinical testing are essential for translation into diagnostic practice.},
}

@article {pmid41717698,
year = {2026},
author = {Tadokoro, T and Li, H and Gan, P and Xu, Z and Tan, W and Alzhanov, D and Sánchez-Ortiz, E and McAnally, JR and Guo, L and Xu, L and Ruan, P and Liu, N and Olson, EN},
title = {Ablation of PKCα Phosphorylation by CRISPR-Cas9 Base Editing Rescues Heart Failure.},
journal = {Circulation research},
volume = {138},
number = {7},
pages = {e326738},
pmid = {41717698},
issn = {1524-4571},
support = {P01 HL160488/HL/NHLBI NIH HHS/United States ; P50 HD087351/HD/NICHD NIH HHS/United States ; R01 HL157281/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; *Protein Kinase C-alpha/genetics/metabolism ; *Heart Failure/genetics/therapy/enzymology/physiopathology/pathology/metabolism ; Phosphorylation ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Mice ; Humans ; Myocytes, Cardiac/metabolism/enzymology ; Mice, Inbred C57BL ; Male ; Disease Models, Animal ; Induced Pluripotent Stem Cells ; },
abstract = {BACKGROUND: The prevalence of heart failure is increasing globally, with poor prognosis, highlighting the need for novel therapeutic strategies. PKCα (protein kinase C alpha), encoded by PRKCA, plays a central role in heart failure pathogenesis. Phosphorylation of PKCα at threonine 497 (T497) triggers a series of intramolecular phosphorylation events, leading to its activation. Ablation of T497 phosphorylation leads to reduced stability and activity of PKCα.

METHODS: We generated mice harboring a phospho-resistant PKCα (T497A) mutation in the germline using CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9)-mediated homology-directed repair. To assess the clinical feasibility of postnatal genome editing, we used CRISPR-Cas9 adenine base editing delivered by adeno-associated virus 9 to introduce the T497A substitution into the Prkca gene (Prkca[T497A]) in wild-type mice. Mice underwent transverse aortic constriction to model heart failure. Cardiac function, hypertrophy, fibrosis, and transcriptional changes were evaluated by echocardiography, wheat germ agglutinin staining, Masson's trichrome staining, and RNA-sequencing. The editing efficiency of Prkca[T497A] was assessed using Sanger sequencing and deep amplicon sequencing. To further explore its clinical potential, we introduced the PRKCA[T497A] mutation into human induced pluripotent stem cells by nucleofection-mediated adenine base editing. Ca[2+] homeostasis was analyzed in Fura-2-loaded human induced pluripotent stem cell-derived cardiomyocytes with PRKCA[T497A] under chronic AngII (angiotensin II) stimulation.

RESULTS: The T497A mutation in PKCα prevented its subsequent phosphorylation and led to PKCα protein degradation. Four weeks after transverse aortic constriction surgery, wild-type mice showed impaired cardiac function, cardiac remodeling, and increased lung weight. In contrast, PKCα phospho-resistant mice showed protection against heart failure-related aberrant changes in cardiac hypertrophy, fibrosis, and cardiac gene expression. Mice administered with adeno-associated virus 9 base editors to prevent T497 phosphorylation exhibited similar cardioprotective effects. In vitro, PKCα-edited induced pluripotent stem cell-derived cardiomyocytes were protected from AngII-induced impairments in contractility and Ca[2+] transients.

CONCLUSIONS: The editing of PRKCA[T497A] through adenine base editing represents a potential therapeutic approach for human cardiac diseases.},
}

Animals
*Protein Kinase C-alpha/genetics/metabolism
*Heart Failure/genetics/therapy/enzymology/physiopathology/pathology/metabolism
Phosphorylation
*CRISPR-Cas Systems
*Gene Editing/methods
Mice
Humans
Myocytes, Cardiac/metabolism/enzymology
Mice, Inbred C57BL
Male
Disease Models, Animal
Induced Pluripotent Stem Cells

@article {pmid41718462,
year = {2026},
author = {Ren, Y and Wu, R and Yang, X and Yao, B and Wang, Y},
title = {Membrane-Confined CRISPR-Cas12a Activation via Split Activator for Wash-Free Detection of Tumor-Specific Extracellular Vesicles.},
journal = {ACS sensors},
volume = {11},
number = {3},
pages = {2356-2365},
doi = {10.1021/acssensors.5c04166},
pmid = {41718462},
issn = {2379-3694},
mesh = {*Extracellular Vesicles/metabolism/chemistry ; *CRISPR-Cas Systems ; Humans ; Aptamers, Nucleotide/chemistry/metabolism ; *Biosensing Techniques/methods ; *CRISPR-Associated Proteins/metabolism ; *Endodeoxyribonucleases/metabolism/genetics ; *Bacterial Proteins/metabolism ; *Neoplasms/diagnosis ; },
abstract = {Current CRISPR-Cas12a biosensing systems for protein analysis rely on the release of a blocked activator from an aptamer through a competitive mechanism. However, the design of the complementary length between the activator and the aptamer involves a critical trade-off: excessively long sequences impede aptamer-target binding, whereas overly short sequences fail to activate Cas12a effectively. To overcome this limitation, we split the full-length Cas12a activator into two short single-stranded DNA fragments (S1 and S2; 10 nt each). S1 was specifically sequestered within the hairpin structure of an allosteric aptamer. The binding of the target protein to the aptamer triggered a conformational change, exposing S1 and thereby enabling its assembly with S2 to activate Cas12a. The strategy successfully preserved the binding affinity of the aptamer without compromising the efficiency of Cas12a activation. Furthermore, we anchored the Cas12a substrate to the membrane surface of extracellular vesicles (EVs) via hydrophobic interactions, localizing the cleavage reaction to the EVs interface and thereby significantly enhancing local catalytic efficiency. Finally, the strategy provided highly sensitive detection of tumor-specific EVs, not only accurately distinguishing cancer patients from healthy individuals (77.5% accuracy for early-stage and 100% for advanced-stage) but also holding potential for monitoring dynamic postoperative changes. Overall, our study provided a wash-free, one-pot detection platform for EVs analysis and established a new paradigm for CRISPR-based clinical diagnostics.},
}

*Extracellular Vesicles/metabolism/chemistry
*CRISPR-Cas Systems
Humans
Aptamers, Nucleotide/chemistry/metabolism
*Biosensing Techniques/methods
*CRISPR-Associated Proteins/metabolism
*Endodeoxyribonucleases/metabolism/genetics
*Bacterial Proteins/metabolism
*Neoplasms/diagnosis

@article {pmid41718886,
year = {2026},
author = {Cao, X and He, Y and Guo, H and Cao, X and Zhang, D and Lai, Y and Yang, W and Ma, Z and Yu, X and Wang, L and Li, D and Zeng, Z},
title = {Optimization of RNP-CRISPR for high-efficiency gene editing in mouse intestinal organoids.},
journal = {Molecular and cellular biochemistry},
volume = {481},
number = {4},
pages = {1817-1827},
pmid = {41718886},
issn = {1573-4919},
support = {2019YFA0110802//National Key Research and Development Program of China/ ; No.32025023//National Natural Science Foundation of China/ ; No.32200732//National Natural Science Foundation of China/ ; 20MC1920400//Shanghai Municipal Commission for Science and Technology/ ; 22YF1437700//Shanghai Municipal Commission for Science and Technology/ ; 2019-01-07-00-05-E00054//Innovation Program of Shanghai Municipal Education Commission/ ; },
mesh = {Animals ; *Organoids/metabolism/cytology ; *Gene Editing/methods ; Mice ; *CRISPR-Cas Systems ; *Ribonucleoproteins/genetics/metabolism ; *Intestines/cytology ; Wnt Signaling Pathway ; Mice, Knockout ; beta Catenin/metabolism ; },
abstract = {Mouse intestinal organoids are ideal models for investigating intestinal development and diseases. The full potential of these models hinges on the ability to precisely engineer their genome, yet traditional methods for CRISPR-based editing in 3D cultures often surfer from low efficiency, high cytotoxicity, and inconsistent post-editing differentiation, which limits their applications. Here, we developed an electroporation approach mediated by ribonucleoprotein (RNP)-CRISPR that achieves over 90% gene editing efficiency in mouse intestinal organoids. Using this optimized method, we generated APC-knockout organoids that exhibit Wnt pathway hyperactivation, demonstrated by R-spondin1-independent growth, increased nuclear β-catenin, and enhanced proliferation. Our method addresses a critical technical gap in murine organoid research, offering a scalable platform for intestinal disease modeling.},
}

Animals
*Organoids/metabolism/cytology
*Gene Editing/methods
Mice
*CRISPR-Cas Systems
*Ribonucleoproteins/genetics/metabolism
*Intestines/cytology
Wnt Signaling Pathway
Mice, Knockout
beta Catenin/metabolism

@article {pmid41719905,
year = {2026},
author = {Yu, F and Yue, D and Wang, F and Fu, B and Qin, G and Wei, S and Zang, W and Zhang, Q and Cui, L and Wang, T},
title = {Structure-initiated CHA variant coordinating SDA for cascade amplification in CRISPR/Cas12a-based miRNA analysis.},
journal = {Talanta},
volume = {304},
number = {},
pages = {129558},
doi = {10.1016/j.talanta.2026.129558},
pmid = {41719905},
issn = {1873-3573},
mesh = {*MicroRNAs/genetics/analysis/blood ; *CRISPR-Cas Systems/genetics ; Humans ; *Nucleic Acid Amplification Techniques/methods ; *Biosensing Techniques/methods ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {MicroRNAs (miRNAs) are well-established biomarkers for tumor diagnosis and monitoring. Herein, we report a novel biosensing platform by engineering a structure-initiated variant of catalytic hairpin assembly (VCHA) that coordinates with strand displacement amplification (SDA) to drive cascade amplification for CRISPR/Cas12a-based detection. This system employs three hairpin probes which, upon recognizing the target miRNA, self-assemble into a key 5'-end dangling three-way conjugate (5'-DTC) structure. This structure serves as the exclusive trigger, simultaneously propagating the VCHA cycle and priming the SDA process through the coordinated action of polymerase and nicking enzyme. Consequently, VCHA and SDA operate synergistically within a unified circuit, generating abundant single-stranded activator DNA (acDNA) products. These acDNA molecules then activate the trans-cleavage activity of CRISPR/Cas12a, yielding a significantly amplified fluorescence readout. The VCHA-SDA/Cas12a platform demonstrated excellent performance for miRNA-155 detection, achieving a broad dynamic range from 1 pmol/L to 10 nmol/L with an ultra-low detection limit of 0.166 pmol/L. Furthermore, the platform successfully quantified miRNA levels in clinical plasma specimens and various cell lines, confirming its considerable potential as a robust tool for molecular diagnostics and clinical translation.},
}

*MicroRNAs/genetics/analysis/blood
*CRISPR-Cas Systems/genetics
Humans
*Nucleic Acid Amplification Techniques/methods
*Biosensing Techniques/methods
Limit of Detection
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41719925,
year = {2026},
author = {Park, YJ and Song, DY and Jeon, HB and Kim, DM},
title = {Nucleic acid detection via protein readout through Cas-controlled gating of cell-free protein synthesis.},
journal = {Biosensors & bioelectronics},
volume = {301},
number = {},
pages = {118514},
doi = {10.1016/j.bios.2026.118514},
pmid = {41719925},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; Cell-Free System ; *Protein Biosynthesis/genetics ; *Escherichia coli O157/genetics ; Bacillus anthracis/genetics ; *CRISPR-Cas Systems/genetics ; *RNA, Ribosomal, 16S/genetics/analysis ; *Nucleic Acids/analysis/genetics ; Bacterial Proteins/genetics ; },
abstract = {We present a modular platform that converts CRISPR target recognition into programmable protein outputs for nucleic acid detection. This system couples Cas-mediated collateral cleavage with cell-free protein synthesis. In the presence of a target, Cas-mediated collateral cleavage releases an extension-competent trigger DNA that gates reporter expression. Although collateral cleavage is inherently indiscriminate, we achieve deterministic fragment generation by employing chemically programmed precursors bearing backbone modifications-exemplified here by phosphorothioate linkages-that constrain cleavage to predefined sites. Using Bacillus anthracis and E. coli O157:H7 as a model, the developed CRIVER assay enables concurrent readouts of 16S rRNA together with the species-specific capB or ecf1 loci by integrating Cas13a-mediated RNA recognition and Cas12a-mediated DNA recognition into a dual-channel workflow. Taken together, proposed method establishes addressable signaling at the protein layer, supports protein-based outputs in a modular workflow, and provides a general route to sensitive, dual-channel nucleic acid detection.},
}

*Biosensing Techniques/methods
Cell-Free System
*Protein Biosynthesis/genetics
*Escherichia coli O157/genetics
Bacillus anthracis/genetics
*CRISPR-Cas Systems/genetics
*RNA, Ribosomal, 16S/genetics/analysis
*Nucleic Acids/analysis/genetics
Bacterial Proteins/genetics

@article {pmid41720308,
year = {2026},
author = {Shi, Q and Huang, W and Hu, D and Zhang, P and Chen, X and Hu, H and Wang, Y and Zhou, J and Weng, R and Quan, J and Zhao, D and Du, X and Yu, Y and Jiang, Y},
title = {The nationwide genomic characteristics and phylogenetic evolution of ST23-K1 hypervirulent Klebsiella pneumoniae in relation to virulence and antimicrobial resistance acquisition.},
journal = {The Journal of infection},
volume = {92},
number = {3},
pages = {106709},
doi = {10.1016/j.jinf.2026.106709},
pmid = {41720308},
issn = {1532-2742},
mesh = {*Klebsiella pneumoniae/genetics/pathogenicity/drug effects/classification ; *Klebsiella Infections/microbiology/epidemiology ; Virulence/genetics ; Phylogeny ; Animals ; China/epidemiology ; Mice ; Humans ; Plasmids/genetics ; Whole Genome Sequencing ; Genome, Bacterial ; Evolution, Molecular ; Anti-Bacterial Agents/pharmacology ; *Drug Resistance, Bacterial/genetics ; Bacterial Proteins/genetics ; Virulence Factors/genetics ; beta-Lactamases/genetics ; Drug Resistance, Multiple, Bacterial/genetics ; },
abstract = {OBJECTIVES: Hypervirulent Klebsiella pneumoniae (hvKp) ST23-K1 poses a global health threat due to its high virulence and increasing antimicrobial resistance. This study aimed to characterise the genomic feature and phylogenetic evolution of ST23-K1 in China.

METHODS: K1 isolates from a nationwide epidemiological surveillance project underwent whole-genome sequencing. Virulence was assessed using hypermucoviscosity phenotyping and a murine infection model. For ST23-K1 carrying acquired antimicrobial resistance genes (ARGs), the CRISPR/Cas system, protospacers, anti-CRISPR (Acr) genes, and plasmidome were characterised. Time-resolved phylogenetic analysis was performed using integrated locally generated and publicly available data.

RESULTS: Among 400 K1 isolates, ST23 was the most prevalent sequence type, and its effective population size increased following CG23-I divergence. The CG23-I sub-lineage was widely distributed nationwide with limited evidence of clonal transmission. Isolates with an incomplete cps locus exhibited significantly reduced virulence compared with those carrying an intact locus. The prevalence of extended-spectrum β-lactamase-positive ST23-K1 isolates increased over time, whereas carbapenemase-producing isolates remained stable. Among acquired ARGs-positive ST23-K1 isolates, a conserved protospacer corresponding to a prevalent spacer was identified. This protospacer, together with AcrIE genes, was frequently co-located on IncFII-type plasmids.

CONCLUSION: ST23-K1 remains a hypervirulent lineage undergoing ongoing evolutionary expansion. The presence of acquired ARGs in ST23-K1 may be associated with AcrIE-harbouring IncFII plasmids, and functional validation is required to clarify the underlying mechanisms. Continuous genomic surveillance is essential to monitor the evolution and antimicrobial resistance trends of ST23-K1.},
}

*Klebsiella pneumoniae/genetics/pathogenicity/drug effects/classification
*Klebsiella Infections/microbiology/epidemiology
Virulence/genetics
Phylogeny
Animals
China/epidemiology
Mice
Humans
Plasmids/genetics
Whole Genome Sequencing
Genome, Bacterial
Evolution, Molecular
Anti-Bacterial Agents/pharmacology
*Drug Resistance, Bacterial/genetics
Bacterial Proteins/genetics
Virulence Factors/genetics
beta-Lactamases/genetics
Drug Resistance, Multiple, Bacterial/genetics

@article {pmid41720778,
year = {2026},
author = {Lee, YJ and Zhang, D and Stolze, SC and Saridis, G and Ebert, MK and Nakagami, H and Doehlemann, G},
title = {Ustilago maydis disrupts carbohydrate signaling networks to induce hypertrophy in host cells.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41720778},
issn = {2041-1723},
support = {DO1421/3-3//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; //European Union’s Horizon 2020 research and innovation program/ ; },
mesh = {*Zea mays/microbiology/metabolism/genetics ; Signal Transduction ; Plant Proteins/metabolism/genetics ; *Plant Diseases/microbiology ; Starch/metabolism/biosynthesis ; Gene Expression Regulation, Plant ; Host-Pathogen Interactions ; Fungal Proteins/metabolism/genetics ; Plant Leaves/microbiology/metabolism ; Mesophyll Cells/metabolism/microbiology ; *Carbohydrate Metabolism ; Endoreduplication ; Virulence Factors/metabolism/genetics ; Cell Cycle/genetics ; CRISPR-Cas Systems ; Basidiomycota ; },
abstract = {Ustilago maydis infection in maize causes hypertrophic leaf tumors; however, the underlying mechanisms driving this excessive cell growth are unknown. In this study, we identify Hap1 (hypertrophy-associated protein 1) as an effector and virulence factor that regulates mesophyll cell hypertrophy. Using CRISPR-Cas9 mutagenesis, we demonstrate that Hap1 contributes to endoreduplication and starch accumulation in infected tissues. Transcriptomics revealed Hap1-dependent upregulation of starch biosynthesis and cell cycle genes, as well as suppression of plant defense. This links Hap1 to metabolic and cell cycle reprogramming, and immune suppression. To identify the target of Hap1 that drives metabolic reprogramming, we investigated its interaction with ZmSnRK1α in maize. We found that Hap1 interferes with the phosphorylation of SnRK1 substrates and that two Hap1-interacting effectors, Hip1 and Hip2, enhance its protein stability. We conclude that Hap1 contributes to the reprogramming of maize metabolism and cell cycle, as well as mesophyll cell hypertrophy, by modulating the SnRK1 signaling pathway to regulate starch biosynthesis and host defense responses.},
}

*Zea mays/microbiology/metabolism/genetics
Signal Transduction
Plant Proteins/metabolism/genetics
*Plant Diseases/microbiology
Starch/metabolism/biosynthesis
Gene Expression Regulation, Plant
Host-Pathogen Interactions
Fungal Proteins/metabolism/genetics
Plant Leaves/microbiology/metabolism
Mesophyll Cells/metabolism/microbiology
*Carbohydrate Metabolism
Endoreduplication
Virulence Factors/metabolism/genetics
Cell Cycle/genetics
CRISPR-Cas Systems
Basidiomycota

@article {pmid41720881,
year = {2026},
author = {Ong, JK and Bhunia, S and Hilbert, B and Kirschner, V and Duglosz, S and Zimmermann, F and Freichel, M and Cornean, A},
title = {ABE9 fused to SpRY Cas9 nickase enables precise generation of bystander free mouse models.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {41720881},
issn = {2045-2322},
mesh = {Animals ; *Gene Editing/methods ; Mice ; *CRISPR-Cas Systems ; *Deoxyribonuclease I/genetics/metabolism ; Humans ; Disease Models, Animal ; *CRISPR-Associated Protein 9/genetics/metabolism ; Point Mutation ; },
abstract = {Point mutations cause many genetic disorders, but modelling them in organisms is technically challenging. Creating mouse models that mimic these mutations is crucial for establishing a causal relationship between mutations and disease phenotype, thereby supporting the development of therapeutic strategies. Adenine base editors (ABEs) can correct single-nucleotide variants (SNVs) in disease modelling without double-stranded breaks (DSBs) or donor DNA, achieving higher product purity than traditional Cas9 methods. Earlier ABE techniques faced issues like limited targetability, bystander editing, and off-target effects. By combining two editor advancements, we introduced and tested ABE9-SpRY, an improved ABE variant fused with a PAM-flexible SpRY-Cas9 nickase. Our results show that ABE9-SpRY effectively generates three out of four targeted A-to-G mutations in mouse embryos, achieving desired editing efficiencies of up to 96% in individual adult founder mice. Furthermore, we observe fewer off-target events at predicted DNA sites in mouse embryos and in an orthogonal R-loop assay compared with ABE8e-SpRY. ABE9-SpRY also enhances product purity in mouse embryos under pooled sgRNA injections and, as a proof-of-concept, at a single endogenous locus in human induced pluripotent stem cells (hiPSCs), relative to ABE8e-SpRY. Our findings support ABE9-SpRY's precision at the loci tested and PAM-flexible versatility. Although performance remains sequence-dependent, these data support ABE9-SpRY as a PAM-flexible tool for generating precise point-mutation models where bystander editing is a concern.},
}

Animals
*Gene Editing/methods
Mice
*CRISPR-Cas Systems
*Deoxyribonuclease I/genetics/metabolism
Humans
Disease Models, Animal
*CRISPR-Associated Protein 9/genetics/metabolism
Point Mutation

@article {pmid41720886,
year = {2026},
author = {Nagalakshmi, U and Rodriguez, JE and Nguyen, T and Weissman, RF and Thornton, BW and Terrace, CI and Savage, DF and Dinesh-Kumar, SP},
title = {High-efficiency, transgene-free plant genome editing by viral delivery of an engineered TnpB.},
journal = {Nature plants},
volume = {12},
number = {3},
pages = {503-511},
pmid = {41720886},
issn = {2055-0278},
support = {IOS-2303522//National Science Foundation (NSF)/ ; DGE-2146752//National Science Foundation (NSF)/ ; },
mesh = {*Gene Editing/methods ; Plants, Genetically Modified/genetics ; *Genome, Plant/genetics ; *Plant Viruses/genetics ; CRISPR-Cas Systems ; Transgenes ; *Nicotiana/genetics ; Genetic Vectors ; *Arabidopsis/genetics ; },
abstract = {Genome editing has revolutionized plant biology research[1], yet the efficient delivery of editing reagents remains a challenge. Current methods are labour intensive, involving lengthy tissue culture and complex transformation and regeneration steps. Viral delivery can mitigate these issues[2] but CRISPR-Cas nucleases exceed viral cargo limits, restricting guide RNA (gRNA) delivery into Cas9-expressing transgenic plants[2-11]. This requires generating an initial Cas9 transgenic line. Furthermore, gRNAs delivered by plant viral vectors can induce somatic edits, although only a few produce heritable edits[3-7,9-12]. Some engineered plant negative-strand rhabdoviruses can deliver both Cas9 and gRNA, but they face other challenges, including the need for tissue regeneration or pruning infected plants, and some rhabdoviruses can be delivered only through vector transmission[13-16]. Recently, smaller editors such as TnpBs were discovered, but they are significantly less active than Cas9[17-19]. Here we optimized a tobacco rattle virus-based system to deliver recently engineered, highly active ISDra2 TnpB variants. The eTnpBc variant enables effective somatic editing in systemic leaves and achieves up to 90% editing efficiency at target loci. In addition, up to 89% of offspring exhibit a mutant phenotype, with editing efficiencies reaching 100%. The design principles outlined here could promote wider use of eTnpBc for efficient, transformation- and transgene-free plant genome editing.},
}

*Gene Editing/methods
Plants, Genetically Modified/genetics
*Genome, Plant/genetics
*Plant Viruses/genetics
CRISPR-Cas Systems
Transgenes
*Nicotiana/genetics
Genetic Vectors
*Arabidopsis/genetics

@article {pmid41721525,
year = {2026},
author = {Chen, N and Gao, M and Bai, Y and Wang, M and Liu, M and Xiong, W and Mo, B},
title = {5'UTR Editing of the ribosomal protein UL3Z gene unveils its critical roles in pre-rRNA processing and global mRNA translation dynamics.},
journal = {Plant physiology},
volume = {200},
number = {3},
pages = {},
doi = {10.1093/plphys/kiag073},
pmid = {41721525},
issn = {1532-2548},
support = {32470592//National Natural Science Foundation of China/ ; 32270595//National Natural Science Foundation of China/ ; 2023B001//SZU 2035 Excellence Research Program/ ; },
mesh = {*5' Untranslated Regions/genetics ; *Ribosomal Proteins/genetics/metabolism ; *Arabidopsis/genetics/metabolism ; *RNA Precursors/metabolism/genetics ; *Arabidopsis Proteins/genetics/metabolism ; *Protein Biosynthesis/genetics ; RNA, Messenger/metabolism/genetics ; Gene Expression Regulation, Plant ; *RNA Processing, Post-Transcriptional/genetics ; Gene Editing ; CRISPR-Cas Systems ; Plants, Genetically Modified ; },
abstract = {The EMBRYO DEFECTIVE 2207 (EMB2207) gene, encoding ribosomal protein UL3Z, is critical for embryonic development in Arabidopsis, with loss of function resulting in embryo lethality. Despite its importance, the role of UL3Z in the complicated protein translation machinery in plants remains poorly understood due to the lack of viable hypomorphic alleles. In this study, we utilized CRISPR/Cas9 to edit the 5' untranslated region (5'UTR) of UL3Z, generating 5 ul3z mutants with varying degrees of reduced expression levels of UL3Z proteins. The mutant with the lowest expression exhibited the most severe developmental defects. In contrast, null mutants of its paralog UL3Y displayed no observable phenotypes. Interestingly, expression of UL3Y driven by the UL3Z/EMB2207 promoter successfully rescued the phenotypes of ul3z, demonstrating that these 2 paralogous ribosomal proteins actually possess functionally interchangeable roles. GUS staining results showed that UL3Z was constitutively expressed in all examined tissues, while UL3Y was only appreciably expressed in specific tissues. Molecular analysis further revealed the accumulation of ribosomal RNA (rRNA) maturation intermediates and increased polysome levels in ul3z mutants, indicating compromised pre-rRNA processing and disturbed global mRNA translation. Interestingly, 3' ends of many rRNA precursors in ul3z had higher frequency of non-encoded tails compared with Col-0. This study demonstrates that CRISPR/Cas9-mediated 5'UTR editing is an effective tool for generating viable hypomorphic alleles of lethal genes and uncovers the critical roles of UL3Z/EMB2207 in pre-rRNA processing and the maintenance of appropriate mRNA translation on ribosomes, underscoring its importance in plant development.},
}

*5' Untranslated Regions/genetics
*Ribosomal Proteins/genetics/metabolism
*Arabidopsis/genetics/metabolism
*RNA Precursors/metabolism/genetics
*Arabidopsis Proteins/genetics/metabolism
*Protein Biosynthesis/genetics
RNA, Messenger/metabolism/genetics
Gene Expression Regulation, Plant
*RNA Processing, Post-Transcriptional/genetics
Gene Editing
CRISPR-Cas Systems
Plants, Genetically Modified

@article {pmid41721881,
year = {2026},
author = {Jin, Z and Liu, M and Liu, M and Qi, X and Zhao, L and Yu, X and Guo, Y and Wen, Y},
title = {CRISPR/Cas9-mediated dual editing of VviGAI and VviFLC generates a novel early-flowering grapevine germplasm.},
journal = {Planta},
volume = {263},
number = {3},
pages = {},
pmid = {41721881},
issn = {1432-2048},
support = {No.32272670//Natural Science Foundation for Young Scientists of Shanxi Province/ ; No.31972986//the National Natural Science Foundation of China/ ; },
mesh = {*Vitis/genetics/growth & development/physiology ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Flowers/genetics/growth & development/physiology ; *Plant Proteins/genetics/metabolism ; Phenotype ; Plants, Genetically Modified ; Gene Expression Regulation, Plant ; Mutation ; },
abstract = {CRISPR/Cas9-mediated dual knockout of VviGAI1 and VviFLC in grapevine promotes early flowering and induces distinctive morphological changes, offering novel genetic resources for breeding. CRISPR/Cas9-mediated genome editing offers a transformative approach for grapevine improvement. In this study, we achieved simultaneous knockout of two central flowering regulators VviGAI1, a DELLA protein ortholog, and VviFLC, a floral repressor in Vitis vinifera 'Cabernet Sauvignon' using a dual-sgRNA vector system. Remarkably, all 15 independent edited lines exhibited biallelic mutations in both genes, primarily consisting of frameshifts that led to premature termination. The dual-mutant plants displayed a range of distinctive phenotypic alterations, including dwarfism, shortened internodes, modified leaf morphology, and disrupted tendril development. Notably, one line (EL-43) showed precocious flowering under greenhouse conditions, underscoring the synergistic role of VviGAI1 and VviFLC in repressing floral transition. Comparative analysis with previously reported gai mutants revealed both conserved and novel traits, suggesting that structural variation within the DELLA domain contributes to phenotypic diversity. Collectively, our findings establish that dual editing of VviGAI1 and VviFLC not only accelerates flowering but also introduces unique vegetative and reproductive characteristics, providing a valuable genetic resource for future grapevine domestication and precision breeding efforts.},
}

*Vitis/genetics/growth & development/physiology
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Flowers/genetics/growth & development/physiology
*Plant Proteins/genetics/metabolism
Phenotype
Plants, Genetically Modified
Gene Expression Regulation, Plant
Mutation

@article {pmid41722369,
year = {2026},
author = {Boumpoureka, I and Gorgogietas, V and Petkovski, E and Massart, F and Mellick, GD and Krüger, R},
title = {Generation of two isogenic control iPSC lines (LCSBi001-A-2 and LCSBi001-A-3) from a Parkinson's disease patient line (LCSBi001-A) carrying the pathogenic VPS35 p.D620N mutation.},
journal = {Stem cell research},
volume = {92},
number = {},
pages = {103944},
doi = {10.1016/j.scr.2026.103944},
pmid = {41722369},
issn = {1876-7753},
mesh = {Humans ; *Parkinson Disease/genetics/pathology/metabolism ; *Induced Pluripotent Stem Cells/metabolism/cytology/pathology ; *Vesicular Transport Proteins/genetics ; *Mutation/genetics ; Cell Line ; CRISPR-Cas Systems ; Cell Differentiation ; },
abstract = {The pathogenic mutation VPS35 p.D620N has been identified to cause autosomal dominant, late-onset Parkinson's disease (PD) in multiple individuals and families worldwide. Here, we describe the generation of two new isogenic control lines (LCSBi001-A-2 and LCSBi001-A-3) from an already established patient-derived line (LCSBi001-A) carrying the heterozygous VPS35 p.D620N mutation. The control lines were generated with CRISPR/Cas9 technology, and the correction of the mutation was verified with Sanger sequencing. Both lines express pluripotency markers, are capable of in vitro differentiation into the three germ layers, and have a normal karyotype. The mutant and control iPSC lines are available for research purposes.},
}

Humans
*Parkinson Disease/genetics/pathology/metabolism
*Induced Pluripotent Stem Cells/metabolism/cytology/pathology
*Vesicular Transport Proteins/genetics
*Mutation/genetics
Cell Line
CRISPR-Cas Systems
Cell Differentiation

@article {pmid41722572,
year = {2026},
author = {Abadie, FMC and Suiter, CC and Smith, NT and Daza, RM and Rominger, MC and Parrish, P and McDiarmid, TA and Lalanne, JB and Martin, B and Calderon, D and Ellison, A and Berger, AH and Shendure, J and Starita, LM},
title = {A multiplex, prime editing framework for identifying drug resistance variants at scale.},
journal = {Cell genomics},
volume = {6},
number = {5},
pages = {101167},
pmid = {41722572},
issn = {2666-979X},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Point Mutation/genetics ; *Drug Resistance, Neoplasm/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Protein Kinase Inhibitors/pharmacology ; Cell Line, Tumor ; },
abstract = {CRISPR-based genome editing has revolutionized functional genomics, enabling thousands of perturbations to be concurrently assayed in single experiments. However, for methods such as saturation genome editing (SGE), which aims to generate and assay libraries of point mutations, a challenge is that only one region (e.g., one exon) is studied per experiment. Here, we describe prime-SGE, a prime editing-based framework in which libraries of specific point mutations are installed into genes throughout the genome and then functionally assessed by sequencing of prime editing guide RNAs (pegRNAs) rather than the mutations themselves. We apply prime-SGE in two cell lines to assay thousands of point mutations in eight oncogenes for their ability to confer drug resistance to four tyrosine kinase inhibitors. Our prime-SGE strategy, combined with ongoing improvements in prime editing efficiency, opens the door to efficient positive selection screens of large numbers of point mutations at locations throughout the genome.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Point Mutation/genetics
*Drug Resistance, Neoplasm/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Protein Kinase Inhibitors/pharmacology
Cell Line, Tumor

@article {pmid41723140,
year = {2026},
author = {Shen, K and Seow, WY and Keng, CT and Lim, MGL and Lim, DS and Guo, K and Meliani, A and Irfan Bin Hajis, M and Wang, B and Prabhakar, S and Chen, KH and Chew, WL},
title = {Spatial perturb-seq: single-cell functional genomics within intact tissue architecture.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41723140},
issn = {2041-1723},
support = {PREPARE-OC-VT-2024-008//Ministry of Health -Singapore (MOH)/ ; SC18/21-1089UI//Agency for Science, Technology and Research (A*STAR)/ ; OFIRG24jul-0096//MOH | National Medical Research Council (NMRC)/ ; OFYIRG23jul-0050//MOH | National Medical Research Council (NMRC)/ ; NRF-CRP25-2020-0001//National Research Foundation Singapore (National Research Foundation-Prime Minister's office, Republic of Singapore)/ ; },
mesh = {Animals ; *Single-Cell Analysis/methods ; Mice ; *Genomics/methods ; Brain/metabolism/cytology ; CRISPR-Cas Systems ; Neurodegenerative Diseases/genetics ; Mice, Knockout ; Neurons/metabolism ; Cell Communication/genetics ; Gene Knockout Techniques ; },
abstract = {We develop Spatial Perturb-Seq, an in vivo CRISPR technology that interrogates multiple genes within single cells of intact tissues, compatible with both sequencing-based and probe-based spatial technologies. We apply Spatial Perturb-Seq to knock out risk genes for neurodegenerative diseases in the mouse brain, uncovering cell autonomous and cell-cell microenvironmental effects within the spatially intact tissue. Spatial Perturb-Seq functionally screens multiple genes in situ and in vivo, bypasses cell processing steps that skew cell type representation, identifies intracellular and intercellular effects of knockouts, and identifies candidate genes underlying dysregulated neuronal intercellular communication pathways.},
}

Animals
*Single-Cell Analysis/methods
Mice
*Genomics/methods
Brain/metabolism/cytology
CRISPR-Cas Systems
Neurodegenerative Diseases/genetics
Mice, Knockout
Neurons/metabolism
Cell Communication/genetics
Gene Knockout Techniques

@article {pmid41723978,
year = {2026},
author = {Zhen, Z and Yu, L and Zhu, Y and Lu, Z and Huang, Z},
title = {Molecular mechanism of CRISPR-SpyCas9 inhibition by AcrIIA26.},
journal = {Biochemical and biophysical research communications},
volume = {809},
number = {},
pages = {153491},
doi = {10.1016/j.bbrc.2026.153491},
pmid = {41723978},
issn = {1090-2104},
mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/chemistry/metabolism/genetics ; *Bacterial Proteins/chemistry/metabolism/genetics ; Gene Editing ; Cryoelectron Microscopy ; *Streptococcus/metabolism/genetics ; RNA, Guide, CRISPR-Cas Systems/metabolism/genetics/chemistry ; Protein Binding ; Protein Domains ; Models, Molecular ; },
abstract = {In the ongoing arms race between bacteria and phages, CRISPR-Cas systems and anti-CRISPR proteins (Acrs) have evolved to counteract one another. Recently, AcrIIA26 from Streptococcus sp. was identified as a modulator of SpyCas9 activity, although its molecular mechanism remains unclear. Here, we present the cryo-EM structure of the SpyCas9-sgRNA-AcrIIA26 complex, revealing a two-domain architecture. The 5A domain of AcrIIA26 binds the PI and WED domains of Cas9, while the 4A domain contacts the REC2 domain. This dual interaction sterically blocks target DNA binding and prevents the conformational changes required for cleavage. Notably, AcrIIA26's sgRNA-independent binding to Cas9 allows the modulation of gene editing across an extended temporal window. Our study elucidates the molecular mechanism of AcrIIA26 and provides novel strategies for precise SpyCas9 regulation.},
}

*CRISPR-Cas Systems
*CRISPR-Associated Protein 9/chemistry/metabolism/genetics
*Bacterial Proteins/chemistry/metabolism/genetics
Gene Editing
Cryoelectron Microscopy
*Streptococcus/metabolism/genetics
RNA, Guide, CRISPR-Cas Systems/metabolism/genetics/chemistry
Protein Binding
Protein Domains
Models, Molecular

@article {pmid41724384,
year = {2026},
author = {Burnham, CM and Kurilung, A and Wanchai, V and Regenberg, B and Delgado-Calle, J and Basnakian, AG and Nookaew, I},
title = {An enhancement of extrachromosomal circular DNA enrichment and amplification to address the extremely low overlap between replicates.},
journal = {The Journal of biological chemistry},
volume = {302},
number = {4},
pages = {111302},
pmid = {41724384},
issn = {1083-351X},
mesh = {*DNA, Circular/genetics ; Humans ; Animals ; Mice ; *Nucleic Acid Amplification Techniques/methods ; CRISPR-Cas Systems ; Female ; Cell Line, Tumor ; },
abstract = {Extrachromosomal circular DNA (eccDNA) of chromosomal origin is present in all eukaryotic organisms and tissues that have been tested. Populations of eccDNA exhibit immense diversity and a characteristically low degree of overlap between samples, suggesting low inheritance of eccDNA between cells or a deficiency in the methods by which eccDNA is detected. This study revisits the Circle-Seq approach for enrichment of eccDNA to address these limitations, hypothesizing that experimental procedures significantly contribute to the observed low eccDNA overlap. We optimized the protocol by reducing the time needed to complete the procedure. Linear DNA is digested by increasing Exonuclease V activity. We employed CRISPR-Cas9 for mitochondrial linearization, which proved superior to using restriction enzymes. A key finding is the critical role of random hexamer primer concentration and genomic DNA input in rolling circle amplification (RCA) for generating high-quality long concatemeric tandem copy amplicons from eccDNA, essential for confident de novo eccDNA construction from long-read sequencing data. Lower primer concentrations substantially increased the percentage of concatemer-derived eccDNA and improved the overlap of identified eccDNAs in technical replicates. Applying this revised approach to human myeloma and breast cancer cell lines, as well as xenograft models, demonstrated >50% overlap in detected eccDNA, a substantial improvement over the <1% overlap observed in previous studies. Additionally, the oncogenic signature of eccDNAs can be identified across all replicates. These findings provide guidelines for developing standardized procedures for eccDNA profiling, advancing our understanding of eccDNA biology, and its potential clinical applications.},
}

*DNA, Circular/genetics
Humans
Animals
Mice
*Nucleic Acid Amplification Techniques/methods
CRISPR-Cas Systems
Female
Cell Line, Tumor

@article {pmid41724454,
year = {2026},
author = {Ghasemian, A and Al-Marzoqi, AH and Ali, ZA and Nouruzi, F and Abdollahi, A and Montaseri, Z and Memariani, M and Zarenezhad, E},
title = {Engineered Bacteria as living detectors of tumor DNA: A new diagnostic frontier.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {586},
number = {},
pages = {120914},
doi = {10.1016/j.cca.2026.120914},
pmid = {41724454},
issn = {1873-3492},
mesh = {Humans ; Animals ; *Biosensing Techniques/methods ; *Neoplasms/diagnosis/genetics ; *DNA, Neoplasm/genetics/analysis ; *Bacteria/genetics ; *Genetic Engineering ; },
abstract = {The identification of tumor-generated DNA must be accurate, minimally invasive, and precise, as it forms a fundamental aspect of effective cancer diagnosis, prognosis, and customized treatment plans. Recent advances in synthetic biology have pioneered the creation of genetically engineered bacteria as innovative biosensors capable of detecting tumor-derived DNA directly in situ. This review explores key developments in designing these microbial sentinels to pinpoint oncogenic DNA alterations, particularly emphasizing KRAS mutations that drive many cancers. By leveraging natural competence and horizontal gene transfer, in combination with CRISPR-Cas tools for selective targeting and integration of mutant DNA sequences, engineered bacteria can distinguish between tumor and wild-type DNA and produce observable reporter outputs. We further elaborate on various molecular engineering strategies using unique genetic circuits, homologous recombination, multiplexed CRISPR systems and safety circuits to improve specificity, sensitivity and biosafety. An additional perspective in the discussion incorporates diverse bacterial species and various cancer types, with a specific emphasis on colorectal and gastrointestinal cancers, while also considering possible applications to other solid tumors. Detection modalities encompass in vitro assays, organoid models, in vivo mouse models, and non-invasive stool sampling, offering an impressive range of platforms for validating biosensors. The positive aspects of these approaches, such as real-time detection, affordability, programmability, and reduced invasiveness, need to be balanced with their negative aspects concerning biosafety, colonization efficiency, and detection sensitivity limitations. Looking forward, this review delves into the translational potential of engineered bacterial biosensors for clinical cancer diagnostics, their integration with therapeutic delivery systems, and future directions that involve multiplexed detection and the incorporation of digital health. Indubitably, engineered bacterial tumor DNA biosensors represent a key fusion of microbiology, synthetic biology, and oncology, aimed at revolutionizing the diagnosis and management of cancers.},
}

Humans
Animals
*Biosensing Techniques/methods
*Neoplasms/diagnosis/genetics
*DNA, Neoplasm/genetics/analysis
*Bacteria/genetics
*Genetic Engineering

@article {pmid41724570,
year = {2026},
author = {Vizoso, M},
title = {DiLiCre2.0 mouse model: An advanced genome-editing tool to induce mutagenesis in vivo with high spatio-temporal resolution.},
journal = {Methods in cell biology},
volume = {203},
number = {},
pages = {41-58},
doi = {10.1016/bs.mcb.2025.12.003},
pmid = {41724570},
issn = {0091-679X},
mesh = {Animals ; Mice ; *Mutagenesis/genetics ; Mice, Transgenic ; *Gene Editing/methods ; Disease Models, Animal ; Humans ; *Carcinogenesis/genetics ; Neoplasms/genetics/pathology ; CRISPR-Cas Systems/genetics ; },
abstract = {For decades, transgenic mouse models have been developed and utilized to study tumorigenesis in vivo, offering the ability to manipulate oncogene and tumor suppression gene expression systemically or within entire organs and tissue compartments. However, the induction of tumorigenesis in many of these experimental models contrasts sharply with the development of most human cancers, where mutations affecting gene expression occur in a spatially restricted manner and lesions generally originates from the clonal expansion of one single mutated cell. This discrepancy raises critical questions regarding the relevance of the existing transgenic mouse models in accurately replicating the mechanisms of tumor initiation observed in humans. To overcome this limitation and study tumor initiation in vivo, I developed an innovative mouse model to induce early tumorigenesis through light-targeted mutagenesis of single cells, achieving unprecedented spatio-temporal resolution. This model provides a more accurate representation of tumor initiation processes, thus enhancing our understanding of cancer mechanisms at its inception.},
}

Animals
Mice
*Mutagenesis/genetics
Mice, Transgenic
*Gene Editing/methods
Disease Models, Animal
Humans
*Carcinogenesis/genetics
Neoplasms/genetics/pathology
CRISPR-Cas Systems/genetics

@article {pmid41724722,
year = {2026},
author = {Limia, CG and Steffey, V and Cheng, HC and Machado, D and Hart, T and McHargue, MC and Brizzee, C and Crawford, J},
title = {Sequential, chromosome-specific glutamine synthetase double knockout with Cas-CLOVER establishes enhanced CHO platforms for cell line development.},
journal = {Biotechnology progress},
volume = {42},
number = {2},
pages = {e70113},
pmid = {41724722},
issn = {1520-6033},
mesh = {CHO Cells ; Animals ; *Glutamate-Ammonia Ligase/genetics/metabolism ; Cricetulus ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Gene Knockout Techniques/methods ; },
abstract = {Cas-CLOVER is an emerging high-fidelity genome editing system that enables precise and efficient cell engineering. In this study, we applied Cas-CLOVER to establish a robust, gene-edited platform in suspension-adapted CHO-K1 cells supporting cell line development (CLD) for biopharmaceutical production. An attractive strategy for high-yield clone selection is the use of glutamine synthetase (GS) knockout CHO cells. The primary GS gene resides on chromosome 5 (GS5), while a recently identified GS pseudogene is located on chromosome 1 (GS1). To compare editing efficiency, we evaluated Cas-CLOVER and Cas9 at both GS loci using the Neon™ Transfection System. Cas-CLOVER achieved 84% editing at GS5 and 74% at GS1, markedly higher than Cas9. Leveraging Cas-CLOVER's dual-guide RNA design, we generated a GS5 single knockout (GS5-SKO) and subsequently a double knockout (GS-DKO) line at both the GS5 and GS1 loci, both with none detected off-target mutations analyzed in 40 predictably off-target sites. For functional validation, these cell lines were engineered with the proprietary Harbor-IN transposase system to stably express trastuzumab. Using an optimized protocol, the resulting GS-DKO platform, termed CleanCut GS CHO, enabled stringent selection and yielded high-producing clones with cell-specific productivity exceeding 100 pg/cell/day and antibody titers greater than 5 g/L in 24 deep well-plate fed-batch cultures after 14 days. The antibody titer stability analysis showed consistency over 60 generations. Collectively, these findings establish Cas-CLOVER as a versatile genome editing tool for developing high-yield CHO host platforms in CLD.},
}

CHO Cells
Animals
*Glutamate-Ammonia Ligase/genetics/metabolism
Cricetulus
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Gene Knockout Techniques/methods

@article {pmid41724841,
year = {2026},
author = {Ibrahim, R and Aranjani, JM},
title = {Bacterial defense mechanisms against bacteriophages: an evolutionary arms race.},
journal = {Archives of microbiology},
volume = {208},
number = {5},
pages = {},
pmid = {41724841},
issn = {1432-072X},
mesh = {*Bacteriophages/physiology/genetics ; *Bacteria/virology/genetics/immunology ; Biological Evolution ; CRISPR-Cas Systems ; Biofilms/growth & development ; },
abstract = {Bacteria and bacteriophages are in a co-evolutionary arms race, developing intricate bacterial defense mechanisms that enable phage resistance and counterstrategies. Bacteria evolve diverse defense mechanisms to inhibit each stage of the phage infection cycle.Surface-based defenses prevent phage adsorption and infection, including receptor modifications, capsule production, and biofilm formation. Intracellular systems such as restriction-modification (R-M) and abortive infection (Abi) mechanisms degrade phage DNA or sacrifice infected cells to protect the population. Adaptive immunity, particularly through CRISPR-Cas systems, enables bacteria to recognize and neutralize recurring phage attacks. Phages counter these defenses through anti-CRISPR proteins, receptor mimicry, and depolymerization, which degrade capsules and biofilm matrices. These dynamic interactions shape microbial ecosystems, offering insights for the development of novel antimicrobial strategies. Emerging approaches, including engineered phages and combination therapies, hold promise for addressing bacterial resistance. Understanding these bacterial-phage dynamics is critical for advancing phage therapy as a powerful tool against multidrug-resistant bacterial infections. This review aims to systematically examine and integrate current knowledge on bacterial antiphage defense systems and the evolutionary adaptations employed by bacteriophages to overcome these barriers.},
}

*Bacteriophages/physiology/genetics
*Bacteria/virology/genetics/immunology
Biological Evolution
CRISPR-Cas Systems
Biofilms/growth & development

@article {pmid41724842,
year = {2026},
author = {Jana, UK and Bawankar, P and Gupta, P and Kango, N},
title = {CRISPR/Cas systems in fungal biotechnology: advancing high-value metabolite synthesis for industrial and food security applications.},
journal = {Archives of microbiology},
volume = {208},
number = {5},
pages = {},
pmid = {41724842},
issn = {1432-072X},
}

@article {pmid41725298,
year = {2026},
author = {Chew, YP and Ferenczi, A and Dannay, M and Ponce-Lilly, C and Kovac, A and Tóth, D and Tóth, SZ and Molnar, A},
title = {Enhancing CRISPR/Cas-Mediated Gene Knockout With Short Non-Homologous Oligonucleotides.},
journal = {Plant biotechnology journal},
volume = {24},
number = {6},
pages = {3742-3753},
pmid = {41725298},
issn = {1467-7652},
support = {//Darwin Trust of Edinburgh/ ; //UK Research and Innovation/ ; BB/W003538/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Knockout Techniques/methods ; *Chlamydomonas reinhardtii/genetics ; Gene Editing/methods ; *Oligonucleotides/genetics ; DNA End-Joining Repair ; },
abstract = {Chlamydomonas reinhardtii is a model green microalga that has great industrial potential as a sustainable bio-factory for recombinant protein and high-value chemical production. Efficient genome editing tools are required to redesign this organism for synthetic biology applications. CRISPR-Cas editing technologies have already been adapted for gene knockout, transgene knock-in, and precise gene editing in C. reinhardtii. However, the efficacy of CRISPR/Cas-mediated gene knockout (KO) is low, which hampers pathway engineering and functional genomic studies. Here we report that co-delivery of CRISPR-Cas gene editing reagents with short double-stranded non-homologous oligodeoxynucleotides (dsNHO) increases gene knockout efficacy up to 100-fold in C. reinhardtii. This phenomenon, referred to as non-homologous oligonucleotide enhancement (NOE), is heavily affected by the length, structure, and chemical modifications of dsNHO, and is largely mediated by the DNA double-stranded break sensor KU70/80 (KU) heterodimer in a Cas nuclease-, locus-, and strain-independent manner. Our data suggest that dsNHOs disrupt the cell's double-stranded break (DSB) sensing pathways, consequently shifting the balance of DNA repair from canonical non-homologous end joining (c-NHEJ) towards the more error-prone, microhomology-mediated end joining (MMEJ), which could be harnessed as a strategy for improving gene KO efficiency in Chlamydomonas and beyond.},
}

*CRISPR-Cas Systems/genetics
*Gene Knockout Techniques/methods
*Chlamydomonas reinhardtii/genetics
Gene Editing/methods
*Oligonucleotides/genetics
DNA End-Joining Repair

@article {pmid41727253,
year = {2026},
author = {Meenakshi, and Komal, and Sharma, A and Prabhu, S and Awasthi, S},
title = {Modern plant stress adaptation: integrating defense, nanotechnology and genetics.},
journal = {3 Biotech},
volume = {16},
number = {3},
pages = {105},
pmid = {41727253},
issn = {2190-572X},
abstract = {This review critically analyses plant adaptive responses to biotic and abiotic stress, with a focus on recent advancements in molecular defense pathways, emerging nanotechnology approaches and CRISPR/Cas-based genome editing strategies. We critically reviewed structural, physiological, biochemical and genetic adaptations. Key regulatory processes include phytohormonal regulation, antioxidants, reactive oxygen species (ROS) signaling and stress-response gene networks are explored along with advances in nanotechnology-based strategies and CRISPR/Cas genome editing. A comparative evaluation of conventional breeding, molecular breeding, and genome-editing approaches highlights the advantages of CRISPR/Cas systems, particularly their precision, efficiency and ability to generate targeted phenotypic changes. In parallel, nanomaterials have shown promise in improved nutrient delivery, protecting cellular structures and enhancing genome-editing efficiency under stress conditions. By integrating nanotechnology and genome-editing approaches with traditional agricultural practices, it may be possible to enhance plant resilience, sustain crop productivity and reduce reliance on chemical inputs. Overall, this review provides a cohesive perspective on how these technologies can be combined to support future crop improvement efforts to tackle climate-induced agricultural challenges.},
}

@article {pmid41727334,
year = {2025},
author = {Ramezani, R and Behbahani, M and Mohabatkar, H and Sarraf Mamouri, K and Hejazi, F},
title = {Comparison of CRISPR Sequences in Archaea and Bacteria with Eukaryotic microRNAs.},
journal = {Avicenna journal of medical biotechnology},
volume = {17},
number = {4},
pages = {258-276},
pmid = {41727334},
issn = {2008-2835},
abstract = {BACKGROUND: This study explores repetitive Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) sequences from the archaea Acidianus sp. and Acidianus ambivalens (A. ambivalens), as well as from the bacterium Yersinia ruckeri (Y. ruckeri). These sequences are compared with human microRNA (miRNA) sequences to investigate potential genetic similarities and disease associations.

METHODS: CRISPR sequences were retrieved from the CRISPR/Cas[++] database, and human miRNA sequences were obtained from miRBase. Sequence alignments were performed using BLASTn with an E-value threshold of 1e-5 to identify significant similarities. Genes associated with matched human miRNAs were identified through the HGNC and GeneCards databases. Further analyses included comparison with disease-associated miRNAs reported in human and mouse datasets.

RESULTS: In Y. ruckeri, alignments revealed similarities to miRNAs linked with genes such as FOXO1, PTEN, PAX7, and DOCK3, which are associated with lung cancer and muscular dystrophies. In A. ambivalens, aligned miRNAs corresponded to loci including CHM13 and GRCh38, potentially linked to periembolic adenocarcinoma and mild pre-eclampsia. For Acidianus sp., matches were observed with miRNAs associated with genes like Irak2, NOS2, STAT1, and Numb, which have been implicated in Psoriatic arthritis, Alzheimer's disease, Hepatocellular carcinoma, and Coronary artery disease.

CONCLUSION: CRISPR sequences from these prokaryotes show notable similarities with human miRNAs, suggesting possible indirect links to genes involved in major diseases. These preliminary findings emphasize the need for further investigation into shared sequence motifs and their functional roles in host-pathogen interactions or evolutionary biology.},
}

@article {pmid41728195,
year = {2025},
author = {Zhu, F and Liu, Z and Zheng, Z},
title = {An AI-driven framework for enhancing regulatory precision and efficiency in CRISPR-Cas gene-edited crops: challenges, opportunities, and global harmonization.},
journal = {Frontiers in plant science},
volume = {16},
number = {},
pages = {1693105},
pmid = {41728195},
issn = {1664-462X},
abstract = {INTRODUCTION: The rapid advancement and adoption of CRISPR-Cas technologies in crop improvement has significantly outpaced existing regulatory frameworks, leading to inconsistencies in the global oversight of gene-edited organisms. As governments and international bodies struggle to reconcile scientific innovation with policy governance, a pressing need has emerged for methodologies that can translate biological edits into regulatory-compliant representations across jurisdictions. Traditional approaches often compartmentalize genomic and legal domains, lacking the formalism to bridge biological intent and compliance precision. These methods are typically static, unable to adapt to jurisdictional policy drift or incorporate real-time exemption logic, thereby undermining both regulatory interpretability and technical fidelity.

METHODS: To address this gap, I propose a unified computational framework built around the novel GeneRegAlignNet model and the Constraint-Aware Policy Induction (CAPI) strategy. This framework embeds regulatory semantics directly into the learning architecture, enabling the alignment of gene-editing features with heterogeneous policy descriptors in a shared latent space. GeneRegAlignNet employs symbolic gating, contrastive manifold learning, and exemption-aware vectorization to predict alignment likelihoods between edits and legal categories with high precision. CAPI extends this model with a risk-calibrated policy optimization pipeline that accounts for policy evolution, regulatory variance, and jurisdictional priorities.

RESULTS AND DISCUSSION: Empirical validation demonstrates improved performance in regulatory alignment accuracy and resilience to policy drift across a diverse set of gene-editing scenarios. By tightly integrating formal representations of molecular edits with dynamic, multi-jurisdictional policy inference, our framework offers a scalable and interpretable path forward in enhancing regulatory precision and global harmonization in the oversight of CRISPR-Cas-edited crops.},
}

@article {pmid41728946,
year = {2026},
author = {Dang, QT and Chang, CW and Chen, PY and Truong, VA and Huang, PY and Nguyen, MTT and Hu, YC},
title = {CRISPR-associated transposon for programmable viral vector engineering and prime editing.},
journal = {Nucleic acids research},
volume = {54},
number = {4},
pages = {},
pmid = {41728946},
issn = {1362-4962},
support = {NSTC 114-2223-E-007-013//National Science and Technology Council/ ; 113-2321-B-007-004//National Science and Technology Council/ ; 113-2223-E-007-010//National Science and Technology Council/ ; 113-2622-E-007-012//National Science and Technology Council/ ; 112-2622-E-007-030//National Science and Technology Council/ ; 112-2223-E-007-002//National Science and Technology Council/ ; //Veterans General Hospitals/ ; VGHUST115-G6-1-1//University System of Taiwan/ ; //National Science and Technology Council/ ; VGHUST115-G6-1-1//University System of Taiwan Joint Research Program/ ; },
mesh = {*Gene Editing/methods ; Humans ; HEK293 Cells ; *Baculoviridae/genetics ; *Genetic Vectors/genetics ; *DNA Transposable Elements/genetics ; Animals ; *CRISPR-Cas Systems ; Escherichia coli/genetics ; Transgenes ; *Genetic Engineering/methods ; },
abstract = {Baculovirus, an insect virus commonly used for recombinant protein expression in insect cells and gene delivery in mammalian systems, is often generated through bacmid-based engineering. To enable flexible and programmable bacmid engineering, we developed SHOT 2.0, an optimized CRISPR-associated transposon platform that mediates RNA-guided and customized bacmid editing in Escherichia coli. The edited bacmid can be transfected into insect cells to produce recombinant baculoviruses. SHOT 2.0 supported site-specific integration of large DNA cargos (at least 14 kb) into defined loci such as v-cath and ODVe56, with integration at ODVe56 markedly improving transgene stability during serial virus passaging. The system is fully compatible with the Bac-to-Bac® workflow, enabling dual-gene insertion into the bacmid and derived baculovirus. Leveraging this platform, we constructed an all-in-one baculovirus encoding the PE5max prime editor. This vector-mediated prime editing achieves efficiencies up to 85.6% in HEK293T cells and achieves robust prime editing in hard-to-transfect cell types, including iPSCs and liver cancer cells, with efficiencies up to 37.1%. These results demonstrate that SHOT 2.0 substantially expands the baculovirus engineering toolbox, providing a flexible platform for genome editing and future gene delivery.},
}

*Gene Editing/methods
Humans
HEK293 Cells
*Baculoviridae/genetics
*Genetic Vectors/genetics
*DNA Transposable Elements/genetics
Animals
*CRISPR-Cas Systems
Escherichia coli/genetics
Transgenes
*Genetic Engineering/methods

@article {pmid41728950,
year = {2026},
author = {King, HE and O'Connell, S and Kavanagh, D and Mason, S and McCool, C and Fernandez-Chamorro, J and Chaffer, CL and Clark, SJ and Vieira, HGS and Sterne-Weiler, T and Weatheritt, RJ},
title = {Isoform-specific single-cell perturb-seq reveals distinct functions of alternative promoters in drug response.},
journal = {Nucleic acids research},
volume = {54},
number = {4},
pages = {},
pmid = {41728950},
issn = {1362-4962},
support = {DP250103133//Australian Research Council Discovery/ ; FT210100355//Future Fellowship/ ; //Scrimshaw Foundation/ ; },
mesh = {*Promoter Regions, Genetic ; Humans ; *Single-Cell Analysis/methods ; Tamoxifen/pharmacology/therapeutic use ; *Estrogen Receptor alpha/genetics ; *Breast Neoplasms/genetics/drug therapy/pathology ; CRISPR-Cas Systems ; Female ; Protein Isoforms/genetics ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic/drug effects ; },
abstract = {CRISPR interference (CRISPRi) screens have emerged as powerful tools for dissecting gene function, yet their application to genes with multiple promoters, which comprise over 60% of human genes, remains poorly understood. Here, we demonstrate that CRISPR-dCas9-based screens exhibit widespread promoter specificity, with untargeted promoters often showing compensatory upregulation to maintain gene expression. Leveraging this selective targeting of individual promoters within the same gene, we developed Isoform-Specific single-cell Perturb-Seq to systematically analyse alternative promoter function. Our analysis revealed that alternative promoters in 51.6% of targeted genes drive distinct transcriptional programs. This suggests that promoter selection represents a fundamental mechanism for generating cellular diversity rather than mere transcriptional redundancy. In breast cancer models, this promoter-specific targeting revealed differential effects on drug sensitivity, where distinct estrogen receptor (ESR1) promoters showed opposing influences on tamoxifen response and patient survival. These findings demonstrate the necessity of promoter-level analysis in functional genomics and suggest new strategies for therapeutic intervention through promoter-specific targeting.},
}

*Promoter Regions, Genetic
Humans
*Single-Cell Analysis/methods
Tamoxifen/pharmacology/therapeutic use
*Estrogen Receptor alpha/genetics
*Breast Neoplasms/genetics/drug therapy/pathology
CRISPR-Cas Systems
Female
Protein Isoforms/genetics
Cell Line, Tumor
Gene Expression Regulation, Neoplastic/drug effects

@article {pmid41728952,
year = {2026},
author = {Feng, H and Li, Z and Zhang, H and Zheng, Y and Xu, B and Zhang, Y and Zou, L and Wu, L},
title = {Characterization of gRNA-dependent and gRNA-independent off-target binding sites of PspCas13b and RfxCas13d in mammalian cells.},
journal = {Nucleic acids research},
volume = {54},
number = {4},
pages = {},
pmid = {41728952},
issn = {1362-4962},
support = {2021YFA1100201//National Key R&D Program of China/ ; 2022YFA1303301//National Key R&D Program of China/ ; XDB0570000//Chinese Academy of Sciences/ ; 82400181//National Natural Science Foundation of China/ ; 82270160//National Natural Science Foundation of China/ ; 2024M751998//China Postdoctoral Science Foundation/ ; 2023YFC2706401//Ministry of Science and Technology/ ; },
mesh = {Humans ; HEK293 Cells ; Binding Sites ; CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *RNA-Binding Proteins/metabolism/genetics ; Regulatory Factor X Transcription Factors ; Gene Editing ; Transcriptome ; },
abstract = {CRISPR-Cas13 systems, harnessed for RNA-guided transcriptome editing, hold significant promise for clinical and in vivo therapeutic applications. However, understanding their in vivo target specificity and recognition rules remains a challenge. In this study, we employed the uSpyCLIP method, which enhances sensitivity and specificity for identifying RNA-binding protein (RBP) binding sites, to map the transcriptome-wide binding sites of catalytically inactive PspCas13b (dPspCas13b) and RfxCas13d (dRfxCas13d) in HEK293T cells, using a variety of single guide RNAs (gRNAs). Surprisingly, we identified both gRNA-dependent and gRNA-independent off-target binding sites for both dCas13 complexes. These gRNA-independent off-target sites exhibited distinct RNA structural and sequence signatures: dPspCas13b's gRNA-independent binding was associated with specific RNA structural features, while dRfxCas13d's was linked to unique sequence motifs. Analysis of gRNA-dependent off-target sites revealed the crucial role of the DR-distal and middle regions of the gRNA in determining binding specificity. Further analysis demonstrated that some off-target binding events led to changes in gene expression at the messenger RNA and/or protein level. Collectively, our findings provide important insights into the characteristics of gRNA-dependent and gRNA-independent off-target binding for PspCas13b and RfxCas13d, offering valuable guidance for optimizing Cas13 and gRNA design in future applications.},
}

Humans
HEK293 Cells
Binding Sites
CRISPR-Cas Systems
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*RNA-Binding Proteins/metabolism/genetics
Regulatory Factor X Transcription Factors
Gene Editing
Transcriptome

@article {pmid41729380,
year = {2026},
author = {Liu, YY and Lv, YR and Jia, JT and Zhang, R and Yang, B and Xue, SY and Bayaer, H and Bagen, A and Chen, RB and Tunala, S and Wang, R and Ding, YL and Zhao, L and Liu, YH},
title = {Rapid and Simple Detection of Enterocytozoon Bieneusi Using Lateral Flow Assay Based on Recombinase Polymerase Amplification or Nested PCR Combined with CRISPR-Cas12a.},
journal = {Acta parasitologica},
volume = {71},
number = {2},
pages = {},
pmid = {41729380},
issn = {1896-1851},
support = {2025MS03139 and 2024LHMS03049//National Natural Science Foundation of Inner Mongolia/ ; 32260887//National Natural Science Foundation of China/ ; BR251303//Research and Innovation Team Building Special B-class Team/ ; YLXKZX-NND-012//Inner Mongolia Autonomous Region First Class Discipline Research Special Project/ ; },
mesh = {*Enterocytozoon/isolation & purification/genetics ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; Animals ; Feces/microbiology ; *Microsporidiosis/diagnosis/veterinary/microbiology ; *Polymerase Chain Reaction/methods ; Humans ; Recombinases/genetics ; DNA, Fungal/genetics ; *Nucleic Acid Amplification Techniques/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {BACKGROUND: Enterocytozoon bieneusi is an obligate intracellular microsporidian pathogen. It primarily causes diarrhea and weight loss in infected humans and animals, resulting in substantial economic losses to the livestock industry. Therefore, establishing a highly sensitive and specific detection method for E. bieneusi is critical for its prevention and control.

METHODS: crRNA and recombinase polymerase amplification (RPA) primers were designed based on partial sequences of the 18 S ribosomal RNA gene and the internal transcribed spacer (ITS) of E. bieneusi. DNA extracted from fecal samples was amplified using RPA or nested polymerase chain reaction (PCR). PCR amplicons were treated with a Tris-saturated phenol-chloroform-isoamyl alcohol mixture to obtain purified target DNA, which was subsequently introduced into the CRISPR-Cas12a reaction system. Post-reaction detection was performed via qPCR instrumentation, fluorescence visualization, and lateral flow strip (LFS) assays. The operational parameters for E. bieneusi detection were subsequently optimized using RPA/CRISPR-Cas12a or nested PCR/CRISPR-Cas12a platforms. The aforementioned methodology was concurrently validated using 50 clinical specimens with known E. bieneusi infection status.

RESULTS: The limits of detection were 7.13 copies/µL for RPA/CRISPR-Cas12a and 2.35 × 10[- 2] copies/µL for nested PCR/CRISPR-Cas12a. When the concentration of unamplified DNA in the CRISPR-Cas12a reaction system reached ≥ 1 × 10[- 4] µg/µL, the single-stranded DNA reporter was efficiently cleaved, resulting in a detectable fluorescence signal. The nested PCR/CRISPR-Cas12a technology was used to analyze 50 fecal samples with confirmed E. bieneusi-positive or -negative status. The results obtained from instrument-based detection, fluorescence observation, and lateral flow test strip detection were completely consistent.

CONCLUSIONS: We established the first integration of nested PCR with CRISPR-Cas12a for the detection of E. bieneusi. and were also the first to quantitatively explore the detection limit of Cas12a using non-amplified E. bieneusi DNA. This approach offers a rapid, specific, and highly sensitive diagnostic method. Furthermore, the wide selection of appropriate visualization methods facilitates adaptation to various laboratory conditions and sample template concentrations, enabling accurate result interpretation.},
}

*Enterocytozoon/isolation & purification/genetics
*CRISPR-Cas Systems
Sensitivity and Specificity
Animals
Feces/microbiology
*Microsporidiosis/diagnosis/veterinary/microbiology
*Polymerase Chain Reaction/methods
Humans
Recombinases/genetics
DNA, Fungal/genetics
*Nucleic Acid Amplification Techniques/methods
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41730290,
year = {2026},
author = {Chen, L and Murillo-de-Ozores, AR and Park, E and Ou, SM and Knepper, MA},
title = {GFP reporter system reveals cell-to-cell variability in aquaporin-2 expression.},
journal = {American journal of physiology. Cell physiology},
volume = {330},
number = {4},
pages = {C782-C793},
doi = {10.1152/ajpcell.00936.2025},
pmid = {41730290},
issn = {1522-1563},
support = {ZIA-HL001285//HHS | NIH | NHLBI | Division of Intramural Research (DIR)/ ; ZIA-HL006129//HHS | NIH | NHLBI | Division of Intramural Research (DIR)/ ; },
mesh = {*Aquaporin 2/genetics/metabolism ; *Green Fluorescent Proteins/genetics/metabolism ; Animals ; Genes, Reporter ; Cell Line ; Mice ; *Kidney Tubules, Collecting/metabolism/drug effects/cytology ; Deamino Arginine Vasopressin/pharmacology ; CRISPR-Cas Systems ; Gene Expression Regulation ; },
abstract = {Vasopressin regulates transcription of the aquaporin-2 gene (Aqp2) in collecting duct principal cells. To investigate regulatory mechanisms in Aqp2 gene transcription, we engineered an Aqp2 reporter cell line using CRISPR/Cas9 to insert a green fluorescent protein (GFP) cassette at the endogenous Aqp2 gene locus in mpkCCD cells. In the absence of dDAVP (1-desamino-8-D-arginine-vasopressin), a vasopressin analog, these cells exhibited low or undetectable GFP and Aqp2 expression in all cells. dDAVP stimulation (1 nM dDAVP for 48 h) markedly increased both GFP and Aqp2 expression together with reversal upon dDAVP removal. These observations demonstrate that GFP faithfully tracks Aqp2 expression. Interestingly, fewer than 50% of cells express GFP and Aqp2 after dDAVP or forskolin, indicating significant variability even though they were clonally derived. We flow-sorted the GFP[-] cells (Aqp2[-]) and GFP[+] cells (Aqp2[+]), regrew them, and restimulated them separately with dDAVP. Cells originating from GFP[-] cells gave rise to both GFP[-] cells and GFP[+] cells, and GFP[+] cells similarly regenerated both GFP[-] and GFP[+] populations in the same proportion. Flow cytometry analysis of the DNA content showed variability in cell cycle phases, with most GFP[+] cells in G0/G1, and most GFP[-] cells in G2/S. RNA-seq analysis of the GFP[-] and GFP[+] cells revealed increased abundance of cell cycle-related transcripts in the GFP[-] cells. We conclude that: 1) heterogeneity in Aqp2 expression is related to cell cycle state and 2) the newly generated reporter cell line will likely serve as a useful tool to study Aqp2 transcriptional regulation.NEW & NOTEWORTHY To investigate regulatory mechanisms in Aqp2 gene transcription, we engineered an Aqp2 reporter cell line using CRISPR/Cas9 to insert a green fluorescent protein (GFP) cassette at the endogenous Aqp2 gene locus in mpkCCD cells. We demonstrate that the GFP reporter accurately and dynamically tracks the expression and regulation of endogenous Aqp2. We reveal that Aqp2 heterogeneity in mpkCCD cells is at least partly driven by differences in cell cycle phase.},
}

*Aquaporin 2/genetics/metabolism
*Green Fluorescent Proteins/genetics/metabolism
Animals
Genes, Reporter
Cell Line
Mice
*Kidney Tubules, Collecting/metabolism/drug effects/cytology
Deamino Arginine Vasopressin/pharmacology
CRISPR-Cas Systems
Gene Expression Regulation

@article {pmid41730595,
year = {2026},
author = {Chang, X and Han, C and Ji, H and Zeng, Z and Yang, J and Liu, Q and Jia, C and Zhao, L and Zhou, C and Chen, S and Knoll, W and Li, J and Wang, Z and Zhang, L},
title = {SPARC: A programmable molecular diagnostic platform based on a signal-triggered, self-supplied crRNA and tiered PER-transcription-CRISPR cascade for early detection of hepatocellular carcinoma.},
journal = {Analytica chimica acta},
volume = {1394},
number = {},
pages = {345209},
doi = {10.1016/j.aca.2026.345209},
pmid = {41730595},
issn = {1873-4324},
mesh = {Humans ; *Liver Neoplasms/diagnosis/genetics ; *Carcinoma, Hepatocellular/diagnosis/genetics ; *MicroRNAs/genetics/analysis ; *Early Detection of Cancer/methods ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Limit of Detection ; *Molecular Diagnostic Techniques ; },
abstract = {BACKGROUND: Accurate quantification of microRNAs (miRNAs) is essential for early cancer detection, yet remains challenging due to their short length, low abundance, and high sequence similarity. Existing assays often struggle to achieve sufficient sensitivity, specificity, and robustness for reliable clinical deployment.

RESULTS: We introduce SPARC, a programmable molecular diagnostic platform that integrates a signal-triggered primer exchange reaction, self-supplied crRNA generation, and a tiered PER-transcription-CRISPR/Cas12a amplification cascade. Using miRNA-21 as a model, SPARC achieves an ultralow detection limit of 1.22 fM and a broad quantitative range from 1 fM to 100 nM. The system exhibits high specificity, strong analytical stability, and modular adaptability to diverse targets, including miRNA-122. Notably, the dual-directional profiling of oncogenic and tumor-suppressive miRNAs enhances diagnostic resolution. When applied to HCC cell lines and clinical tissues, SPARC accurately distinguished malignant from normal samples and showed excellent agreement with qRT-PCR measurements and histopathological assessments.

SIGNIFICANCE: This streamlined and self-amplifying cascade system provides a scalable, robust, and clinically compatible platform for ultrasensitive miRNA detection. SPARC holds strong potential for early hepatocellular carcinoma screening, molecular subtyping, and broader precision oncology applications.},
}

Humans
*Liver Neoplasms/diagnosis/genetics
*Carcinoma, Hepatocellular/diagnosis/genetics
*MicroRNAs/genetics/analysis
*Early Detection of Cancer/methods
*CRISPR-Cas Systems
Cell Line, Tumor
Limit of Detection
*Molecular Diagnostic Techniques

@article {pmid41731282,
year = {2026},
author = {du Rand, A and Buttle, B and Sheppard, H},
title = {Developing CRISPR-Based Therapies for Epidermolysis Bullosa: A Comprehensive Review of Current Strategies.},
journal = {Drugs},
volume = {86},
number = {4},
pages = {465-483},
pmid = {41731282},
issn = {1179-1950},
mesh = {Humans ; *Gene Editing/methods ; *Epidermolysis Bullosa/therapy/genetics ; *Genetic Therapy/methods ; *CRISPR-Cas Systems/genetics ; Animals ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Currently, there is no permanent treatment for the group of severe monogenic fragile skin conditions epidermolysis bullosa (EB). The recent US Food and Drug Administration (FDA)-approved in vivo gene replacement therapy beremagene geperpavec (Vyjuvek[®]) provides a promising solution, but it requires ongoing application and is not applicable to all forms of EB. Targeted gene editing approaches directly addressing pathogenic mutations hold great promise for the development of durable personalized therapies. Here, we comprehensively describe the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) gene editing landscape for EB, critically review the advantages and limitations of emerging therapeutic strategies, and present some future perspectives. We find that the widespread application of Cas9 nuclease is currently hindered by off-target genotoxicity, which can be mitigated using Cas9 nickases. Further, new tools including prime editing have recently emerged and hold significant potential for EB gene therapy. Ongoing developments in gene editing technologies focused on improving safety and editing precision offer significant promise for the future clinical translation of potentially lifelong treatments for people with EB.},
}

Humans
*Gene Editing/methods
*Epidermolysis Bullosa/therapy/genetics
*Genetic Therapy/methods
*CRISPR-Cas Systems/genetics
Animals
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid41732093,
year = {2026},
author = {Neupane, S and Pfrender, ME and Wang, L and Xu, S},
title = {Detection of CRISPR-Cas-induced mutations in Daphnia.},
journal = {G3 (Bethesda, Md.)},
volume = {16},
number = {5},
pages = {},
pmid = {41732093},
issn = {2160-1836},
support = {R35 GM133730/GM/NIGMS NIH HHS/United States ; 2324639//NSF EDGE program/ ; 2220696//NSF EDGE program/ ; 2348390//NSF Career program/ ; R35GM133730/GF/NIH HHS/United States ; },
mesh = {Animals ; *Daphnia/genetics ; *CRISPR-Cas Systems ; *Mutation ; INDEL Mutation ; Reproducibility of Results ; Gene Editing ; },
abstract = {CRISPR-Cas9 has established itself as a robust tool for conducting loss-of-function gene research in emerging model species, including the freshwater zooplankton Daphnia. However, sensitive detection of mutations, especially in genetic mosaic and pooled samples, remains a challenge. In this study, we evaluate 2 of the most widely used mutation screening techniques, the T7 Endonuclease I assay and fragment analysis for their sensitivity, accuracy, and practical use in detecting CRISPR-induced indels in 4 targeted genes, DNMT3A, DNMT3B, PERIOD2, and DMRT1 in Daphnia magna. Here, we show that T7 Endonuclease I, although it offers a quick and cost-effective screening method, often produces false positives, especially when examining pooled samples. Conversely, fragment analysis facilitates detecting allele size differences at a fine resolution, reproducibility in detecting indels, and distinguishing zygosity, and is more reliable as a method to detect mutation. Our comparative analyses convey the importance of carefully selecting the appropriate screening methods depending on research questions.},
}

Animals
*Daphnia/genetics
*CRISPR-Cas Systems
*Mutation
INDEL Mutation
Reproducibility of Results
Gene Editing

@article {pmid41732321,
year = {2026},
author = {He, Y and Tu, X and Xue, Y and Chen, Y and Ye, B and Li, X and Li, D and Zhong, Z and Zhong, Q},
title = {CRISPR screening redefines therapeutic target identification and drug discovery with precision and scalability.},
journal = {Journal of pharmaceutical analysis},
volume = {16},
number = {2},
pages = {101357},
pmid = {41732321},
issn = {2214-0883},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 screening technology is redefining the landscape of drug discovery and therapeutic target identification by providing a precise and scalable platform for functional genomics. The development of extensive single-guide RNA (sgRNA) libraries enables high-throughput screening (HTS) that systematically investigates gene-drug interactions across the genome. This powerful approach has found broad applications in identifying drug targets for various diseases, including cancer, infectious diseases, metabolic disorders, and neurodegenerative conditions, playing a crucial role in elucidating drug mechanisms and facilitating drug screening. Despite challenges like off-target effects, data complexity, and ethical or regulatory concerns, ongoing advancements in CRISPR technology and bioinformatics are steadily overcoming these limitations. Additionally, by integrating with organoid models, artificial intelligence (AI), and big data technologies, CRISPR screening expands the scale, intelligence, and automation of drug discovery. This integration boosts data analysis efficiency and offers robust support for uncovering new therapeutic targets and mechanisms. This review outlines the fundamental principles and applications of CRISPR screening technology, delves into specific case studies and technical challenges, and highlights its expanding role in drug discovery and target identification. It also examines the potential for clinical translation and addresses the associated ethical and regulatory considerations.},
}

@article {pmid41732389,
year = {2026},
author = {Fanarraga, ML and García Hevia, L},
title = {Silica nanoparticles as advanced platforms for nucleic acid delivery.},
journal = {Materials today. Bio},
volume = {37},
number = {},
pages = {102921},
pmid = {41732389},
issn = {2590-0064},
abstract = {Nucleic acid therapeutics, including siRNA, mRNA, plasmid DNA, and CRISPR/Cas systems, have demonstrated remarkable potential but continue to face translational barriers related to systemic instability, immune activation, and inefficient intracellular delivery. Conventional lipid and polymeric carriers, although clinically validated, often lack the structural resilience and versatility required for large or complex cargos. Silica-based nanoparticles, particularly mesoporous silica nanoparticles, provide a distinctive combination of mechanical rigidity, tunable porosity, and abundant surface chemistry that enables robust encapsulation, protection, and controlled release of diverse nucleic acids. This review adopts a problem-driven perspective, analyzing how specific nanoarchitectural designs, surface functionalizations, and ligand-mediated targeting strategies address key limitations in nucleic acid delivery. Emphasis is placed on overcoming systemic barriers such as premature degradation, immune recognition, and restricted biodistribution, as well as intracellular challenges including endosomal escape and nuclear access. Hybrid and biomimetic silica platforms are highlighted for their capacity to integrate combinatorial and theranostic functionalities, expanding the therapeutic scope toward complex payloads and multifunctional formulations. By linking synthesis approaches with translational requirements, an integrated roadmap is proposed that positions silica nanocarriers as advanced platforms for next-generation gene therapy. The evidence underscores the potential of silica architectures to combine structural durability with versatile chemical adaptability, thereby enabling safe, efficient, and clinically relevant delivery of nucleic acids.},
}

@article {pmid41733889,
year = {2026},
author = {Son, SH and Woo, S and Choi, A and Ahn, S and Yoo, HC},
title = {Advances in Engineered Virus-Like Particles for Genome Editing and Therapy.},
journal = {BioDrugs : clinical immunotherapeutics, biopharmaceuticals and gene therapy},
volume = {40},
number = {2},
pages = {317-333},
pmid = {41733889},
issn = {1179-190X},
support = {RS202400403169//Korea Basic Science Institute/ ; 2021R1C1C2006283//National Research Foundation/ ; RS-2024-00412879//National Research Foundation/ ; },
mesh = {*Gene Editing/methods ; Humans ; *Genetic Therapy/methods ; Animals ; Genetic Engineering/methods ; Genetic Vectors ; CRISPR-Cas Systems ; Gene Transfer Techniques ; },
abstract = {Engineered virus-like particles (eVLPs) have emerged as a promising class of delivery systems for genome editing agents. By combining the efficient cellular entry of viral vectors with the safety advantages of nonviral platforms, eVLPs enable transient delivery of ribonucleoproteins such as Cas9, base editors, and prime editors. Successive design strategies, from rational engineering to directed optimization, have progressively improved particle assembly, cargo stability, and editing efficiency. In parallel, pseudotyping approaches have expanded the versatility of eVLPs for cell-type-specific targeting. Recent preclinical studies highlight their potential across diverse applications, ranging from monogenic disease models to complex disorders, and support their advancement toward clinical translation. This review summarizes the structural and production principles of eVLPs, surveys key advances in their development, and discusses therapeutic opportunities and future challenges for their application in genome editing.},
}

*Gene Editing/methods
Humans
*Genetic Therapy/methods
Animals
Genetic Engineering/methods
Genetic Vectors
CRISPR-Cas Systems
Gene Transfer Techniques

@article {pmid41734599,
year = {2026},
author = {Kim, GH and Kim, MM},
title = {Effect of p53 gene mutation with loss of function on the expression of genes and proteins involved in cell proliferation.},
journal = {Mutation research},
volume = {832},
number = {},
pages = {111931},
doi = {10.1016/j.mrfmmm.2026.111931},
pmid = {41734599},
issn = {1873-135X},
mesh = {Humans ; *Cell Proliferation/genetics ; *Tumor Suppressor Protein p53/genetics/metabolism ; CRISPR-Cas Systems ; Cell Line, Tumor ; *Loss of Function Mutation ; Gene Expression Regulation, Neoplastic ; *Mutation ; Cell Cycle/genetics ; },
abstract = {The tumor suppressor gene TP53 plays a vital role in preserving genomic integrity by regulating cell cycle progression, DNA repair mechanisms, and apoptosis. This study aims to examine how CRISPR/Cas9-induced loss-of-function mutations in the p53 gene influence cellular processes on cell cycle regulation and tumorigenic signaling in HT1080 human fibrosarcoma cells. Successful TP53 gene disruption was confirmed by Sanger sequencing, and its structural modelling using AlphaFold2 and ChimeraX confirmed alterations in the predicted TP53 protein structure compared to that of wild type. Gene expression analyses, conducted via RT-PCR and qPCR, demonstrated a marked decrease in TP53 mRNA expression within the modified cells. Despite the mutation, the edited cells elevated activity of the senescence marker β-galactosidase (SA-β-gal). They decreased the production of collagen, suggesting that the structural disruption caused by CRISPR/Cas9 leads to the loss of functional p53 activity. Western blotting and immunofluorescence assays further revealed a remarkable downregulation of key cell cycle and tumorigenesis-related proteins, including TP53, phosphorylated TP53 (p-TP53), acetylated TP53 (ac-TP53), MMP-2, cyclin D, cyclin E, AKT, BAX, MDM2, and phosphorylated Rb (p-Rb) in the edited cells relative to the wild-type counterpart. Our results suggest that the TP53 mutation may disrupt essential pathways related to cell proliferation and stress responses. This provides new insights into TP53 functionality and underscores its potential as a therapeutic target in cancer biology.},
}

Humans
*Cell Proliferation/genetics
*Tumor Suppressor Protein p53/genetics/metabolism
CRISPR-Cas Systems
Cell Line, Tumor
*Loss of Function Mutation
Gene Expression Regulation, Neoplastic
*Mutation
Cell Cycle/genetics

@article {pmid41734974,
year = {2026},
author = {Ding, Y and Chen, X and Wu, K and Hou, H and Wang, Y and Yan, B and Khursheed, MHUR and Shang, C and Zhang, X and Pan, Y and Wu, L and Li, J},
title = {A Truncated WRKY Protein Enhances Drought Resistance in Wild Tomatoes Through the SlWRKY16-CIP2b-SlSYP121 Module.},
journal = {Plant biotechnology journal},
volume = {24},
number = {6},
pages = {3840-3860},
pmid = {41734974},
issn = {1467-7652},
support = {Nos.CSTB2023TIAD-KPX0026//Special Key Project of Technological Innovation and Application Development of Chongqing/ ; CSTB2023TIAD-KPX0025//Special Key Project of Technological Innovation and Application Development of Chongqing/ ; 31872123//National Natural Science Foundation of China/ ; 32172597//National Natural Science Foundation of China/ ; SWU-KQ22041//Fundamental Research Funds for the Central Universities/ ; CARS-23-B08//China Agriculture Research System/ ; },
mesh = {*Solanum lycopersicum/genetics/physiology/metabolism ; *Droughts ; *Plant Proteins/genetics/metabolism ; *Transcription Factors/genetics/metabolism ; Gene Expression Regulation, Plant ; Polymorphism, Single Nucleotide/genetics ; Stress, Physiological/genetics ; CRISPR-Cas Systems ; Drought Resistance ; },
abstract = {Drought stress is a major abiotic factor that severely affects plant growth and food production. Identifying drought-resistant genes and their regulatory mechanisms is essential for mitigating the negative impacts of drought on plants. In this study, we identified a natural single nucleotide polymorphism (SNP) mutation in SlWRKY16 that is closely linked to drought tolerance in tomato. This SNP leads to the expression of a truncated SlWRKY16 protein. The CRISPR/Cas9 knockout of SlWRKY16, which produces this truncated SlWRKY16 protein, exhibits enhanced drought tolerance, whereas the overexpression lines demonstrate the opposite effect. Yeast two-hybrid screening demonstrated that SlWRKY16 physically interacted with CONSTANS Interacting Protein 2b (CIP2b). The CIP2b knockout mutants displayed increased sensitivity to drought stress. Importantly, this drought-sensitive phenotype was rescued in double mutants (cip2b/slwrky16). RNA-seq analysis revealed that a syntaxin gene (SlSYP121) co-expressed with both SlWRKY16 and CIP2b. Electrophoretic mobility shift assays confirmed that SlWRKY16 directly binds to the promoter of SlSYP121 and represses its expression, while the truncated SlWRKY16 protein failed to bind. Moreover, SlSYP121 acts as a positive regulator of drought tolerance. Our findings further demonstrate that the interaction between CIP2b and SlWRKY16 reduces the binding affinity of SlWRKY16 to the SlSYP121 promoter. This study identified a key SNP associated with differences in drought tolerance between wild and cultivated tomato, elucidated the regulatory function of the SlWRKY16-CIP2b-SlSYP121 module in the tomato drought response, and enhanced our understanding of the molecular mechanisms underlying plant drought resistance.},
}

*Solanum lycopersicum/genetics/physiology/metabolism
*Droughts
*Plant Proteins/genetics/metabolism
*Transcription Factors/genetics/metabolism
Gene Expression Regulation, Plant
Polymorphism, Single Nucleotide/genetics
Stress, Physiological/genetics
CRISPR-Cas Systems
Drought Resistance

@article {pmid41735098,
year = {2026},
author = {Naruse, K and Loosli, F and Ansai, S and Birney, E and Wittbrodt, J},
title = {Medaka: a novel model for analyzing genome-environment interactions.},
journal = {Trends in genetics : TIG},
volume = {42},
number = {4},
pages = {350-361},
doi = {10.1016/j.tig.2025.12.005},
pmid = {41735098},
issn = {0168-9525},
mesh = {Animals ; *Oryzias/genetics ; *Gene-Environment Interaction ; Genome-Wide Association Study ; *Genome ; Quantitative Trait Loci ; CRISPR-Cas Systems ; },
abstract = {Medaka is an established vertebrate model system for biological and biomedical research. It possesses unique features that make it particularly suitable for studying genome-environment interactions. Endemic to habitats spanning from 4 to 40°C and varying salinities, it combines broad ecological adaptability with experimental tractability. Its exceptional tolerance to inbreeding enabled the creation of the Medaka Inbred Kiyosu-Karlsruhe panel-80 near-isogenic, fully sequenced lines derived from a single wild population. More than 100 wild-derived, fully sequenced strains, collected throughout East Asia for more than 40 years, show relatively low intra-strain variation (inbreeding coefficient of >0.75) but high inter-strain variability (SNP rates >4%). Advanced quantification methods facilitate genome-wide association studies and quantitative trait locus mapping. The system's amenability to clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 editing and emerging epigenomic profiling enables causal validation and regulatory-mechanism discovery. Collectively, medaka offers an unparalleled vertebrate framework for integrating genetics, environment, and epigenetics-bridging evolutionary, biomedical, and population-level perspectives.},
}

Animals
*Oryzias/genetics
*Gene-Environment Interaction
Genome-Wide Association Study
*Genome
Quantitative Trait Loci
CRISPR-Cas Systems

@article {pmid41735520,
year = {2026},
author = {Panji, JM and Germano, RFV and America, M and Lou, L and Lecordier, L and Tebabi, P and Martin, M and Vanhollebeke, B},
title = {Scalable and multimodal brain angiogenesis and blood-brain barrier genetics by somatic mutagenesis.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {},
pmid = {41735520},
issn = {2399-3642},
support = {Ctrl-BBB 865176//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; },
mesh = {Animals ; *Blood-Brain Barrier/metabolism ; Zebrafish/genetics ; *Mutagenesis ; *Neovascularization, Physiologic/genetics ; Mice ; CRISPR-Cas Systems ; *Brain/blood supply ; Mice, Transgenic ; *Angiogenesis/genetics ; },
abstract = {The endothelial blood-brain barrier (BBB) is a complex neuroprotective structure whose maturation begins during brain angiogenesis and is completed postnatally. Despite its clinical importance, our understanding of BBB ontogeny and regulation remains limited. This shortcoming results from the difficulty of modeling the BBB in vitro and the current limitation of in vivo BBB genetics to low-throughput Mendelian genetic approaches. In addition, a single animal model facilitating the study of the neurovascular events occurring during both embryogenesis and adulthood is lacking. Here, leveraging the complementary anatomical and experimental attributes of mice and zebrafish, we present an integrated somatic CRISPR-Cas9 gene disruption strategy that allows us to assess candidate genes for their role in brain angiogenesis and BBB permeability within two and six weeks, respectively. Phenotypic assessment of brain angiogenesis is conducted in the transparent embryonic zebrafish hindbrain, while BBB maintenance is evaluated at both the behavioral and the functional level by combining endothelial-specific Cas9 transgenic mice and BBB-targeting adeno-associated viruses. The quick turnaround time and multiplexing capacity of our multi-species approach enable multimodal BBB functional genomics.},
}

Animals
*Blood-Brain Barrier/metabolism
Zebrafish/genetics
*Mutagenesis
*Neovascularization, Physiologic/genetics
Mice
CRISPR-Cas Systems
*Brain/blood supply
Mice, Transgenic
*Angiogenesis/genetics

@article {pmid41735731,
year = {2026},
author = {Zhao, R and Chen, J and Li, Y and Chen, DY and Kang, X and Dong, S and Yuan, X and Li, X and Gao, L and Yang, G and Chu, X and Wang, JG},
title = {CRISPR/Cas9-mediated α-prolamin gene (Seita.8G190200) mutagenesis increases the content of functional amino acids in foxtail millet (Setaria italica).},
journal = {Plant cell reports},
volume = {45},
number = {3},
pages = {},
pmid = {41735731},
issn = {1432-203X},
support = {2023YFD1202702-6//National Key Research and Development Program of China/ ; 2025QNLJ203//"Youth Science and Technology Leadership Talent Training Program" of Shanxi Agricultural University/ ; },
mesh = {*Setaria Plant/genetics/metabolism ; *Amino Acids/metabolism ; *CRISPR-Cas Systems/genetics ; *Prolamins/genetics/metabolism ; Gene Editing ; *Mutagenesis/genetics ; *Plant Proteins/genetics/metabolism ; Plants, Genetically Modified ; Nutritive Value ; },
abstract = {The gene editing of α-prolamin gene (Seita.8G190200) exhibited significant increases in multiple functional aminoacids. The vast majority sugar components showed significant increases in the gene editing lines. Knockout of the prolamin gene not only improves the nutritional quality but also affects its eating quality. Proteins in foxtail millet exhibits a balanced amino acid composition that meets human dietary requirements.Prolamins are notably deficient in essential amino acids such as lysine, the structure, morphology, and functional characteristics of foxtail millet prolamin have been sufficiently elucidated, but the biological roles of prolamin genesstill need further exploration. This study demonstrates that the α-prolamin gene (Seita.8G190200) plays crucial roles in regulating prolamin content, functional amino acid and sugar components levels, potentially serving as an important target for improving both nutritional and eating quality of foxtail millet. These findings provide scientific foundations for developing novel nutritionally fortified functional foxtail millet products, breeding new foxtail millet varieties enriched with functional amino acids for human health benefits.},
}

*Setaria Plant/genetics/metabolism
*Amino Acids/metabolism
*CRISPR-Cas Systems/genetics
*Prolamins/genetics/metabolism
Gene Editing
*Mutagenesis/genetics
*Plant Proteins/genetics/metabolism
Plants, Genetically Modified
Nutritive Value

@article {pmid41736546,
year = {2026},
author = {Zhang, C and Ye, K and Shang, Y and Song, Y and Li, P and Jiang, X and Yang, C and Liang, A and Zhang, J and Meng, F and Zhang, M},
title = {Spatially concentrated adenine base editors efficiently correct PLP1 mutations in oligodendrocytes.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41736546},
issn = {1362-4962},
support = {32370853//National Natural Science Foundation of China/ ; },
mesh = {*Oligodendroglia/metabolism ; *Gene Editing/methods ; Animals ; *Myelin Proteolipid Protein/genetics/metabolism ; *Adenine/metabolism ; Mutation ; Mice ; *Pelizaeus-Merzbacher Disease/genetics/therapy/pathology ; Humans ; CRISPR-Cas Systems ; Adenosine Deaminase/metabolism/genetics ; },
abstract = {Oligodendrocytes (OLs), the myelinating cells of the central nervous system, are particularly prone to pathogenic G-to-A mutations, such as PLP1A243V, which causes Pelizaeus-Merzbacher disease (PMD), a lethal hypomyelinating disorder lacking effective therapy. Although adenine base editors (ABEs) can in principle correct such mutations, their application in OLs is limited by inefficient on-target editing. Here, we develop a spatially concentrated ABE (cABE) strategy that enhances editing by promoting nuclear translocation of tRNA adenosine deaminase (TadA*) from the cytoplasm. Using a SunTag-based multivalent recruitment system, TadA* is locally enriched at genomic targets (cABE-1.0), achieving robust editing in vitro. To enable in vivo delivery while preserving high efficiency and fidelity, SpCas9 is replaced with compact eNme2-C Cas9, generating an AAV-compatible variant (cABE-2.0). Notably, cABE-2.0 forms dynamic nuclear puncta with properties of liquid-liquid phase separation, enhancing on-target editing while substantially reducing transcriptome-wide RNA off-target effects. Functionally, cABE-2.0 efficiently corrects the PLP1A243V mutation in OLs, restores Plp subcellular localization, and rescues myelination-related phenotypes. These findings demonstrate that spatial reorganization, rather than increasing intrinsic catalytic activity of TadA*, provides a distinct principle for improving base editing in difficult-to-edit cell types, such as OLs, offering a mechanistic and technical framework for gene therapy of PMD and related myelin disorders.},
}

*Oligodendroglia/metabolism
*Gene Editing/methods
Animals
*Myelin Proteolipid Protein/genetics/metabolism
*Adenine/metabolism
Mutation
Mice
*Pelizaeus-Merzbacher Disease/genetics/therapy/pathology
Humans
CRISPR-Cas Systems
Adenosine Deaminase/metabolism/genetics

@article {pmid41736562,
year = {2026},
author = {Purvis, IJ and Ochoa Olmos, OE and Park, KU and Kaufman, ML and Henry, CM and Schaaf, C and Clise, OJ and Tesdahl, CD and Haas, A and Brzezinski, JA},
title = {A robust cis-regulatory network ensures Otx2 expression during retinal development.},
journal = {Development (Cambridge, England)},
volume = {153},
number = {6},
pages = {},
doi = {10.1242/dev.204881},
pmid = {41736562},
issn = {1477-9129},
support = {R01-EY024272/NH/NIH HHS/United States ; //Research to Prevent Blindness/ ; R01 EY024272/EY/NEI NIH HHS/United States ; F31-EY034794/NH/NIH HHS/United States ; F31 EY034794/EY/NEI NIH HHS/United States ; F31EY034794/NH/NIH HHS/United States ; //Gates Institute/ ; //Anschutz Medical Campus, University of Colorado/ ; //University of Colorado/ ; },
mesh = {*Otx Transcription Factors/genetics/metabolism ; Animals ; *Retina/metabolism/embryology ; *Gene Expression Regulation, Developmental ; Enhancer Elements, Genetic/genetics ; Mice ; *Gene Regulatory Networks/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {The transcription factor Otx2 is essential for photoreceptor and bipolar cell formation during retinal development. Otx2 expression is complex and underlies multiple cell fate decisions during development. To understand how Otx2 expression is regulated, we explored the activity and function of three of its enhancers (DHS2, DHS4 and DHS15). Enhancer reporter assays and lineage tracing show that DHS4 initiates Otx2 expression while DHS2 and DHS15 maintain expression in photoreceptors. Matched CRISPR/Cas9 and CRISPR interference systems were used to mutate or epigenetically silence enhancers, respectively. CRISPR reduced OTX2 expression acutely, but failed to significantly alter cell fate choice over the long term. In contrast, CRISPR interference of these enhancers caused permanent OTX2 loss and corresponding cell fate changes. While these data suggest that each enhancer is needed for normal Otx2 expression, it also highlights that the enhancers can interact and substitute for each other during development. This cis-regulatory element flexibility likely promotes Otx2 expression robustness. Such robustness may enable complex genes, like Otx2, to resist environmental stressors and regulatory disruptions to promote reproducible developmental outcomes.},
}

*Otx Transcription Factors/genetics/metabolism
Animals
*Retina/metabolism/embryology
*Gene Expression Regulation, Developmental
Enhancer Elements, Genetic/genetics
Mice
*Gene Regulatory Networks/genetics
CRISPR-Cas Systems/genetics

@article {pmid41738557,
year = {2026},
author = {Girard, V and Sorge, S and Kurth, J and Alexandre, C and Gould, AP},
title = {ShineGAL4 drivers for tissue and cell-type specific optogenetics in Drosophila.},
journal = {Development (Cambridge, England)},
volume = {153},
number = {4},
pages = {},
pmid = {41738557},
issn = {1477-9129},
support = {FC001088/CRUK_/Cancer Research UK/United Kingdom ; FC001088/MRC_/Medical Research Council/United Kingdom ; FC001088/WT_/Wellcome Trust/United Kingdom ; 223760/WT_/Wellcome Trust/United Kingdom ; 104566/WT_/Wellcome Trust/United Kingdom ; 543-2022//EMBO/ ; //Francis Crick Institute/ ; },
mesh = {Animals ; *Optogenetics/methods ; *Drosophila Proteins/genetics/metabolism ; Transcription Factors/genetics/metabolism ; *Drosophila melanogaster/genetics ; Organ Specificity/genetics ; CRISPR-Cas Systems/genetics ; Neurons/metabolism ; *Drosophila/genetics ; },
abstract = {An optogenetic split-GAL4 system, ShineGAL4, allows genes to be manipulated with unprecedented spatiotemporal precision. Here, we convert a panel of 14 GAL4 drivers widely used in Drosophila research into their ShineGAL4 counterparts. Homology assisted CRISPR knock-in (HACK) is used to replace GAL4 with the GAL4 DNA binding domain fused to a Magnet photoswitch. We show that the resulting ShineGAL4 drivers enable gene expression to be rapidly induced by light specifically in fat body, muscles, enterocytes, oenocytes, Malpighian tubules, neurons, neuroblast lineages, glial subtypes or in all glia. We also develop an optogenetic cassette for photoactivation of GAL4 in 'silent' FLP-out clones. This panel of optogenetic tools will enable precise spatiotemporal control of gene expression in a wide range of different Drosophila tissues and cell-types.},
}

Animals
*Optogenetics/methods
*Drosophila Proteins/genetics/metabolism
Transcription Factors/genetics/metabolism
*Drosophila melanogaster/genetics
Organ Specificity/genetics
CRISPR-Cas Systems/genetics
Neurons/metabolism
*Drosophila/genetics

@article {pmid41738762,
year = {2026},
author = {Irfan, M and Duran-Pinedo, A and Solbiati, J and Rocha, FG and Gibson, FC and Frias-Lopez, J},
title = {A CRISPR array orchestrates virulence and host response in Porphyromonas gingivalis.},
journal = {Microbiology spectrum},
volume = {14},
number = {4},
pages = {e0283425},
pmid = {41738762},
issn = {2165-0497},
support = {R01 DE029775/DE/NIDCR NIH HHS/United States ; DE029775/DE/NIDCR NIH HHS/United States ; },
mesh = {*Porphyromonas gingivalis/genetics/pathogenicity ; Animals ; Virulence/genetics ; Biofilms/growth & development ; Macrophages/microbiology/immunology ; Humans ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Moths/microbiology ; Bacteroidaceae Infections/microbiology/immunology ; Cytokines/metabolism ; Host-Pathogen Interactions ; THP-1 Cells ; Larva/microbiology ; },
abstract = {CRISPR-Cas systems are primarily recognized for their defensive role against foreign DNA. Recent studies, however, indicate involvement in regulatory functions. The persistence of a large spacer array without apparent phage targets in Porphyromonas gingivalis ATCC 33277 remains unexplained. This study demonstrates that deletion of the non-coding CRISPR array 30.1 in P. gingivalis ATCC 33277 results in increased biofilm formation, elevated virulence in a Galleria mellonella infection model, and significant alterations in the macrophage transcriptomic response. The ΔCRISPR-30.1 mutant forms twice as much biofilm as the wild type, induces 50% mortality in Galleria larvae within 130 h compared to 200 h for the wild type (P < 0.0001), and elicits a cytokine profile characterized by increased IL-6, CXCL1, CXCL2, and CXCL9 secretion. Dual RNA sequencing of THP-1 macrophages infected with wild-type and ΔCRISPR 30.1 strains reveals that the loss of the array activates bacterial metabolic and secretion pathways while suppressing host innate and adaptive immune signaling. Single-primer amplification (SPA) identifies numerous self-genome loci bound by individual 30.1 spacers, supporting a direct, spacer-mediated regulatory mechanism. These findings establish CRISPR array 30.1 as a previously unrecognized regulator of P. gingivalis physiology and host-pathogen interactions.IMPORTANCECRISPR-Cas systems are established as adaptive immune elements, yet spacer arrays without known targets are frequently observed in bacteria and often lack a defined function. In P. gingivalis, a keystone periodontal pathogen, a non-coding CRISPR array has been shown to regulate biofilm formation, virulence in an invertebrate model, and the macrophage transcriptional response. This expands the recognized functions of CRISPR arrays to include the direct regulation of bacterial physiology and the modulation of host immune responses, identifying CRISPR spacers as potential targets for antimicrobial interventions. Furthermore, elucidating the role of CRISPR arrays in P. gingivalis may have broader clinical implications, given the established associations between periodontal health and systemic inflammatory diseases. Targeting spacer arrays to modulate bacterial virulence could influence the management of these conditions and enhance the translational relevance of such therapeutic strategies.},
}

*Porphyromonas gingivalis/genetics/pathogenicity
Animals
Virulence/genetics
Biofilms/growth & development
Macrophages/microbiology/immunology
Humans
*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
Moths/microbiology
Bacteroidaceae Infections/microbiology/immunology
Cytokines/metabolism
Host-Pathogen Interactions
THP-1 Cells
Larva/microbiology

@article {pmid41739553,
year = {2026},
author = {Valinsky, WC and Ray, RP and Schaefer, KS and Grimm, JB and Nicolini, C and Lavis, LD and Clapham, DE},
title = {Phenotypic CRISPR screens identify NLRX1 as an essential activator of the human mitochondrial permeability transition.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {9},
pages = {e2535298123},
pmid = {41739553},
issn = {1091-6490},
support = {NA//Janelia Research Campus (Janelia)/ ; },
mesh = {Humans ; Calcium/metabolism ; *Mitochondria/metabolism/genetics ; *Mitochondrial Proteins/metabolism/genetics ; Membrane Potential, Mitochondrial ; CRISPR-Cas Systems ; Mitochondrial Membrane Transport Proteins/metabolism/genetics ; Mitochondrial Membranes/metabolism ; Phenotype ; Clustered Regularly Interspaced Short Palindromic Repeats ; Permeability ; Calcium Channels/metabolism/genetics ; },
abstract = {The mitochondrial permeability transition (mPT) is an evolutionarily conserved destructive process that permeabilizes the inner mitochondrial membrane in response to calcium overload. The molecular mechanism underlying the mPT is not established. To unambiguously identify essential proteins, we designed two phenotypic assays for mitochondrial calcium overload and applied them to FACS-based CRISPR screening in human cells, ultimately evaluating 19,113 genes. The first screen studied mitochondrial membrane potential (MMP) collapse in response to calcium overload. Top-ranked genes were the essential proteins of the mitochondrial calcium uniporter complex, MCU and EMRE, reflecting that the calcium-induced MMP collapse results from mitochondrial calcium entry and not the mPT. The second screen measured the permeability of the inner mitochondrial membrane. Here, the fluorescent interaction of a membrane impermeant ~600 Da dye and a mitochondrial-targeted HaloTag protein was studied under mPT activating conditions; calcium overload and the thiol-reactive molecule phenylarsine oxide. With secondary validation, we identified four protein-encoding genes that delayed or prevented the mPT under knockout: NF2, REST, BPTF, and NRLX1. Knockout of the nonmitochondrial proteins BPTF, NF2, or REST increased mitochondrial calcium retention capacity (CRC). However, calcium release or sensitivity to cyclosporin A (CsA) persisted, indicative of mPT sensitizers. Only knockout of the mitochondrial matrix protein, NLRX1, increased CRC, abolished calcium release, and was CsA-insensitive. This top-ranked hit of the mitochondrial permeability screen meets the definition of an essential mPT activator. Integral membrane proteins, including all previously proposed mPT candidates, were not essential activators.},
}

Humans
Calcium/metabolism
*Mitochondria/metabolism/genetics
*Mitochondrial Proteins/metabolism/genetics
Membrane Potential, Mitochondrial
CRISPR-Cas Systems
Mitochondrial Membrane Transport Proteins/metabolism/genetics
Mitochondrial Membranes/metabolism
Phenotype
Clustered Regularly Interspaced Short Palindromic Repeats
Permeability
Calcium Channels/metabolism/genetics

@article {pmid41740027,
year = {2026},
author = {Duarte, DF and Lucena, LP and Gonçalves, MHO and Benko-Iseppon, AM and Aburjaile, F and Azevedo, V and Brenig, B and Gama, MAS and Souza, EB},
title = {Phylogenomic analysis of Paracidovorax citrulli strains reveals the presence of two lineages in Brazil.},
journal = {Genetics and molecular biology},
volume = {48},
number = {4},
pages = {e20250046},
pmid = {41740027},
issn = {1415-4757},
abstract = {Paracidovorax citrulli is the causative agent of bacterial fruit blotch in melons and watermelons. This study used comparative genomic approaches of 17 Brazilian P. citrulli strains obtained from melons and watermelons to classify them into groups I and II and try to understand their genomic differences. The genomes of P. citrulli presented general characteristics similar to those shown for the genomes of the type strain of P. citrulli and reference strains of groups I and II. A phylogenomic analysis revealed two distinct groups of P. citrulli, in which most Brazilian P. citrulli strains were grouped with the strain representing group I. CRISPR-Cas analysis revealed the presence of two proteins, Cas3 and Cas10, in all Brazilian P. citrulli genomes. In addition, we observed the presence of two plasmids (pAMC6 and pAC53) in three Brazilian P. citrulli strains, all closely related to group I. The prediction of effector proteins revealed the XopE/AvrPphe protein as a differential between the strains of groups I and II. The present study will contribute to a more detailed understanding of aspects of host-pathogen interactions and will help improve the detection of strains from these groups, thus elucidating the population dynamics of Brazilian strains of P. citrulli.},
}

@article {pmid41740122,
year = {2026},
author = {Yıldırım, K and Kavas, M},
title = {The role of CRISPR in modern plant breeding: overcoming breeding barriers and legislative challenges through transgene-free genome editing.},
journal = {Genome},
volume = {69},
number = {},
pages = {1-17},
doi = {10.1139/gen-2025-0063},
pmid = {41740122},
issn = {1480-3321},
mesh = {*Plant Breeding/methods/legislation & jurisprudence ; *Gene Editing/methods/legislation & jurisprudence ; *CRISPR-Cas Systems ; Plants, Genetically Modified/genetics ; *Crops, Agricultural/genetics ; *Genome, Plant ; Transgenes ; },
abstract = {Traditional plant breeding techniques-such as crossbreeding, mutation breeding, and marker-assisted selection-have significantly contributed to crop improvement over the past century. However, these methods are often limited by long breeding cycles, low precision, and the unintended transfer of undesirable traits. To address these challenges, transgenic breeding emerged as a powerful tool, enabling the introduction of specific foreign genes to confer desirable traits such as pest resistance or herbicide tolerance. While highly effective and precise, transgenic approaches face considerable regulatory and public acceptance barriers, particularly in regions with strict GMO legislation. The advent of CRISPR/Cas genome editing has revolutionized plant breeding by enabling precise, efficient, and targeted modification of native genes, significantly accelerating the development of improved crops. Among CRISPR-based methods, transgene-free genome editing has gained prominence for producing enhanced plant varieties without integrating foreign DNA, thus avoiding many regulatory constraints associated with GMOs. This review aims to provide a comprehensive overview of CRISPR-mediated genome editing technologies in crop innovation, emphasizing especially the use of transgene-free approaches in modern plant breeding and their transformative potential to overcome the limitations of conventional methods while providing a regulation-friendly pathway for crop improvement.},
}

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

@article {pmid41740934,
year = {2026},
author = {Sun, W and Zhu, S and Dong, Q and Tang, S and Liu, Q and Sha, Y and Chen, T and Wang, R and Chen, Y and Ying, H},
title = {Engineering Escherichia coli cell Factories for continuous 5'-cytidine monophosphate production via biofilm-anchored dual-enzyme cascade catalysis.},
journal = {Bioresource technology},
volume = {447},
number = {},
pages = {134267},
doi = {10.1016/j.biortech.2026.134267},
pmid = {41740934},
issn = {1873-2976},
mesh = {*Biofilms/growth & development ; *Escherichia coli/metabolism/genetics/enzymology ; *Cytidine Monophosphate/biosynthesis ; Biocatalysis ; CRISPR-Cas Systems/genetics ; Uridine Kinase/metabolism ; },
abstract = {5'-cytidine monophosphate (5'-CMP) serves as a crucial intermediate for diverse nucleotide derivatives and finds extensive applications in the food and pharmaceutical industries. However, existing enzymatic production processes suffer from low catalytic efficiency and poor economic feasibility. In this study, we developed a continuous 5'-CMP production system based on a cell-enzyme co-immobilized biocatalytic platform. First, the pgaABCD gene cluster was integrated into ClearColi BL21(DE3) using CRISPR-Cas9, enhancing its biofilm-forming capacity by 168.93% and enabling robust cell immobilization on the carrier. Second, a dual-anchoring strategy utilizing ice-nucleation protein (INP) and autotransporter (AIDA-I) enabled surface display of uridine kinase (UDK) and acetate kinase (AckA) on the cell surface. This approach successfully addressed the instability and recovery issues of free enzymes by using biofilm engineering to co-immobilize cells and enzymes. The modified strain achieved a 5'-CMP productivity of 1.77 mmol/L/h, 5.98-fold higher than free intracellular enzyme catalysis, and was reused for ten consecutive cycles under the tested conditions while maintaining a cytidine conversion rate above 73.79%, and a 5'-CMP yield above 59.26%. This work demonstrates the first successful realization of continuous 5'-CMP biosynthesis and establishes an efficient route for its industrial production.},
}

*Biofilms/growth & development
*Escherichia coli/metabolism/genetics/enzymology
*Cytidine Monophosphate/biosynthesis
Biocatalysis
CRISPR-Cas Systems/genetics
Uridine Kinase/metabolism

@article {pmid41741438,
year = {2026},
author = {Liu, T and Lin, Y and Liu, Q and Liao, W and Lin, Y and Zhang, Y and Zhang, J and Cao, W and Yang, L and Hong, Z and Lu, Z},
title = {Target-stabilized base editors enable robust high-fidelity RNA editing.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41741438},
issn = {2041-1723},
support = {82327805//National Natural Science Foundation of China (National Science Foundation of China)/ ; KQTD20210811090117032//Shenzhen Science and Technology Innovation Commission/ ; },
mesh = {*RNA Editing/genetics ; *Adenosine Deaminase/genetics/metabolism ; Humans ; Animals ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; HEK293 Cells ; RNA-Binding Proteins/genetics/metabolism ; Mutation ; Gene Editing/methods ; Mice ; Amyotrophic Lateral Sclerosis/genetics ; },
abstract = {RNA base editing using engineered deaminases represents a powerful tool to correct mutations at the RNA level. However, widespread off-target effects, primarily arising from dissociated free deaminases, remain a significant challenge. Here, we devise the RECODE (RNA editing with conditionally stable and enhanced ADAR1 deaminase variants) system, which employs designer degron-tagged ADAR1 deaminase (ADAR1d) with guide RNA (gRNA)-regulated stability. By promoting degradation of gRNA-unbound ADAR1d, RECODE markedly reduces transcriptome-wide edits while maintaining high on-target efficacy. Engineering gRNA for target RNA-induced conformational switching confines ADAR1d stabilization to intended editing sites, further enhancing editing precision. With structure-guided rational engineering of ADAR1d, RECODE efficiently corrects an Amyotrophic Lateral Sclerosis-relevant FUS mutation and installs a therapeutic mutation to Angptl3 in vivo, which mitigate FUS mislocalization to neuronal axons and lower plasma lipids, respectively. These findings establish RECODE as a highly stringent and efficient RNA editing technology and underscore a general principle for enhancing the specificity of RNA-guided protein effectors.},
}

*RNA Editing/genetics
*Adenosine Deaminase/genetics/metabolism
Humans
Animals
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
HEK293 Cells
RNA-Binding Proteins/genetics/metabolism
Mutation
Gene Editing/methods
Mice
Amyotrophic Lateral Sclerosis/genetics

@article {pmid41741470,
year = {2026},
author = {Aird, EJ and Serrano-Benitez, A and Siegner, SM and Cannavo, E and Belotserkovskaya, R and Gueorguieva, N and Fielden, J and Cullot, G and Ammann, S and Bader, AS and Gupta, V and Andrieux, G and Raab, R and Del Rey González, M and Cathomen, T and Cejka, P and Corn, JE and Jackson, SP},
title = {ERCC6L2 ensures repair fidelity for staggered-end DNA double-strand breaks.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41741470},
issn = {2041-1723},
support = {855741-DDREAMM-ERC-2019-SyG//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; 855741-DDREAMM-ERC-2019-SyG//EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)/ ; },
mesh = {*DNA Breaks, Double-Stranded ; Humans ; *DNA Helicases/metabolism/genetics ; *DNA Repair ; DNA-Binding Proteins/metabolism/genetics ; Endodeoxyribonucleases/metabolism/genetics ; MRE11 Homologue Protein/metabolism/genetics ; CRISPR-Cas Systems ; Gene Editing ; Cell Cycle Proteins/metabolism/genetics ; DNA Repair Enzymes/metabolism/genetics ; Poly-ADP-Ribose Binding Proteins/metabolism ; HEK293 Cells ; Nuclear Proteins/metabolism ; Bacterial Proteins ; Acid Anhydride Hydrolases ; CRISPR-Associated Proteins ; },
abstract = {DNA double-strand breaks (DSBs) both pose threats to genome integrity and are commonly used for genome editing applications. Structural features of DSB ends play key roles in determining DNA repair pathway usage and outcomes during genome editing, but the cellular factors involved in these processes are only partially known. Through genome-wide CRISPRi screening, we identify ERCC6L2 as critical for repairing Cas12a-induced staggered DSBs but irrelevant for Cas9-induced blunt DSBs. We show that ERCC6L2 acts as a protection factor for staggered DSBs with either 5' or 3' polarity, preventing large deletions and translocations stemming from DNA damage induced by Cas12a, TALENs, or dual Cas9 nicks. Furthermore, ERCC6L2 loss hyper-sensitizes cells to multiple staggered DSBs induced by promiscuous Cas12a activity or etoposide-induced TOP2 trapping. By combining genetics and biochemical reconstitution, we find that ERCC6L2 counteracts MRE11-RAD50-NBS1 (MRN)-mediated resection by binding and melting staggered DNA ends, thereby promoting accurate end joining. Our data reveal a protective role of ERCC6L2 in staggered-end DSB repair, which suggests the molecular underpinnings of pathology in patients with ERCC6L2 mutations and cautions against using overhang-inducing genome editing tools for their treatment.},
}

*DNA Breaks, Double-Stranded
Humans
*DNA Helicases/metabolism/genetics
*DNA Repair
DNA-Binding Proteins/metabolism/genetics
Endodeoxyribonucleases/metabolism/genetics
MRE11 Homologue Protein/metabolism/genetics
CRISPR-Cas Systems
Gene Editing
Cell Cycle Proteins/metabolism/genetics
DNA Repair Enzymes/metabolism/genetics
Poly-ADP-Ribose Binding Proteins/metabolism
HEK293 Cells
Nuclear Proteins/metabolism
Bacterial Proteins
Acid Anhydride Hydrolases
CRISPR-Associated Proteins

@article {pmid41741636,
year = {2026},
author = {Mathiowetz, AJ and Meymand, ES and Parlakgül, G and van Hilten, N and Torres, EF and Artico, LL and Deol, KK and Lange, M and Pang, SP and Doubravsky, CE and Roberts, MA and Jorgens, DM and Zalpuri, R and Kang, M and Boone, C and Parks, BW and Zhang, Y and Morgens, DW and Newman, ET and Zhou, Y and Talukdar, S and Grabe, M and Ku, G and Levine, TP and Arruda, AP and Olzmann, JA},
title = {CLCC1 promotes hepatic neutral lipid flux and nuclear pore complex assembly.},
journal = {Nature},
volume = {652},
number = {8109},
pages = {462-470},
pmid = {41741636},
issn = {1476-4687},
support = {R01 HL147097/HL/NHLBI NIH HHS/United States ; R01 CA305423/CA/NCI NIH HHS/United States ; P30 DK098722/DK/NIDDK NIH HHS/United States ; R01 DK128099/DK/NIDDK NIH HHS/United States ; R01 GM137109/GM/NIGMS NIH HHS/United States ; R01 GM112948/GM/NIGMS NIH HHS/United States ; R01 DK140367/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; Mice ; *Nuclear Pore/metabolism ; *Liver/metabolism/cytology ; Hepatocytes/metabolism ; Lipid Droplets/metabolism ; Humans ; Molecular Dynamics Simulation ; Male ; Fatty Liver/metabolism/pathology/genetics ; Membrane Fusion ; *Lipid Metabolism ; CRISPR-Cas Systems ; Mice, Knockout ; Lipoproteins/metabolism ; Nuclear Envelope/metabolism ; Female ; },
abstract = {Imbalances in lipid storage and secretion lead to hepatic steatosis, the accumulation of lipid droplets in hepatocytes[1,2]. Our understanding of the mechanisms that govern the channelling of neutral lipids in hepatocytes towards cytosolic lipid droplets or secreted lipoproteins remains incomplete[3,4]. Here we performed a series of CRISPR-Cas9 screens under different metabolic states that led to the identification of CLCC1 as a critical regulator of neutral lipid storage and secretion in hepatocytes. Loss of CLCC1 resulted in the buildup of large lipid droplets in hepatoma cells and Clcc1 knockout in mice caused liver steatosis. Lipid droplets were present in the lumen of the endoplasmic reticulum of the Clcc1-knockout hepatocytes and exhibited properties of lipoproteins, indicating a profound shift in neutral lipid flux. The loss of CLCC1 also led to the accumulation of nuclear membrane herniations accompanied by a reduction in nuclear pores. Remote homology searches identified a domain in CLCC1 that is homologous to yeast Brl1 and Brr6, factors that promote nuclear envelope fusion during nuclear pore complex assembly. Molecular dynamics simulations and mutagenesis studies support a model in which CLCC1 mediates membrane bending and fusion. We propose that CLCC1 mediates membrane fusion to promote hepatic neutral lipid flux and nuclear pore complex assembly.},
}

Animals
Mice
*Nuclear Pore/metabolism
*Liver/metabolism/cytology
Hepatocytes/metabolism
Lipid Droplets/metabolism
Humans
Molecular Dynamics Simulation
Male
Fatty Liver/metabolism/pathology/genetics
Membrane Fusion
*Lipid Metabolism
CRISPR-Cas Systems
Mice, Knockout
Lipoproteins/metabolism
Nuclear Envelope/metabolism
Female

@article {pmid41741641,
year = {2026},
author = {Yang, L and Renauer, PA and Tang, K and Saskin, J and Zhou, L and Zou, C and Lee, SH and Fox, M and Johnson-Noya, S and Weiss, B and Deng, S and Fang, P and Chen, B and Sferruzza, G and Fooladi, S and Zhao, K and Park, D and Zhang, F and Tu, J and Chen, J and Moliterno, J and Gunel, M and Peng, L and Chen, S},
title = {OR7A10 GPCR engineering boosts CAR-NK therapy against solid tumours.},
journal = {Nature},
volume = {652},
number = {8110},
pages = {740-751},
pmid = {41741641},
issn = {1476-4687},
support = {DP2 CA238295/CA/NCI NIH HHS/United States ; R01 CA231112/CA/NCI NIH HHS/United States ; R33 CA281702/CA/NCI NIH HHS/United States ; R33 CA281702/CA/NCI NIH HHS/United States ; T32 GM136651/GM/NIGMS NIH HHS/United States ; T32 HD007149/HD/NICHD NIH HHS/United States ; T32 GM007499/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; Animals ; *Killer Cells, Natural/immunology/metabolism/cytology/transplantation ; Mice ; Female ; *Receptors, Chimeric Antigen/genetics/immunology/metabolism ; *Neoplasms/therapy/immunology/pathology ; Tumor Microenvironment/immunology ; *Immunotherapy, Adoptive/methods ; Male ; Cell Line, Tumor ; *Receptors, G-Protein-Coupled/genetics/metabolism/immunology ; Xenograft Model Antitumor Assays ; Cell Proliferation ; CRISPR-Cas Systems/genetics ; },
abstract = {Chimeric antigen receptor (CAR)-natural killer (NK) cell therapies hold promise for solid tumours but remain limited because of poor tumour infiltration, persistence and resistance in the tumour microenvironment[1-4]. Here, to identify gain-of-function targets that enhance CAR-NK cell efficacy, we performed an unbiased in vivo CRISPR activation screen followed by a barcoded targeted in vivo open reading frame screen in primary human CAR-NK cells. We identified and comprehensively validated OR7A10, a G protein-coupled receptor (GPCR), as the top candidate. Engineering CAR-NK cells with OR7A10 cDNA (a CRISPR-independent method with a simple manufacturing strategy) enhanced their proliferation, activation, degranulation, cytokine production, death ligand expression, chemokine receptor expression, cytotoxicity, persistence, metabolic fitness and tumour microenvironment resistance. Moreover, exhaustion in primary human NK cells derived from multiple peripheral blood and cord blood donors was reduced. OR7A10 gain-of-function CAR-NK cells displayed strong in vivo efficacy across multiple solid tumour models. For example, 100% complete response with long-term tumour control and survival benefit in an orthotopic breast cancer mouse model were achieved. These findings establish OR7A10-engineered CAR-NK cells as a highly potent and scalable off-the-shelf therapeutic for solid tumours.},
}

Humans
Animals
*Killer Cells, Natural/immunology/metabolism/cytology/transplantation
Mice
Female
*Receptors, Chimeric Antigen/genetics/immunology/metabolism
*Neoplasms/therapy/immunology/pathology
Tumor Microenvironment/immunology
*Immunotherapy, Adoptive/methods
Male
Cell Line, Tumor
*Receptors, G-Protein-Coupled/genetics/metabolism/immunology
Xenograft Model Antitumor Assays
Cell Proliferation
CRISPR-Cas Systems/genetics

@article {pmid41741653,
year = {2026},
author = {Saxton, DS and DeWeirdt, PC and Doering, CR and Roney, IJ and Laub, MT},
title = {A membrane-bound nuclease directly cleaves phage DNA during genome injection.},
journal = {Nature},
volume = {653},
number = {8115},
pages = {861-869},
pmid = {41741653},
issn = {1476-4687},
mesh = {*Bacteriophage lambda/genetics/physiology ; Cell Membrane/enzymology/metabolism ; DNA Cleavage ; *DNA, Viral/metabolism/genetics ; *Escherichia coli/virology/enzymology/cytology/metabolism ; Escherichia coli Proteins/metabolism ; Genome, Viral ; },
abstract = {From mammals to bacteria, the direct recognition and cleavage of viral nucleic acids is a potent defence strategy against viral infection, but it requires mechanisms for distinguishing self from non-self[1,2]. In bacteria, CRISPR-Cas and restriction-modification systems achieve this discrimination by recognizing specific DNA sequences or DNA modifications, respectively. Alternative mechanisms probably remain to be discovered. Here, we characterize SNIPE, an anti-bacteriophage defence system that constitutively localizes to the bacterial cell membrane in Escherichia coli to block phage λ infection. Using radiolabelled phage DNA and time-lapse microscopy to track phage genomes, we demonstrate that SNIPE directly cleaves phage DNA during genome injection. Based on proximity labelling, we find that SNIPE associates with host proteins essential for λ genome entry and with the λ tape measure protein, which facilitates λ genome injection across the inner membrane. SNIPE also defends against diverse siphoviruses, probably through direct interactions with their tape measure proteins. Our findings establish SNIPE as a widespread bacterial defence system that exploits the spatial organization of phage genome injection to specifically target viral DNA, representing a previously unknown strategy for distinguishing self from non-self in prokaryotic immune systems.},
}

*Bacteriophage lambda/genetics/physiology
Cell Membrane/enzymology/metabolism
DNA Cleavage
*DNA, Viral/metabolism/genetics
*Escherichia coli/virology/enzymology/cytology/metabolism
Escherichia coli Proteins/metabolism
Genome, Viral

@article {pmid41741655,
year = {2026},
author = {Lei, J and Yang, K and Cao, W and Qi, S and Du, X and Li, H and Wang, Y and Gan, J and Feng, Y and Li, Y and Zhang, W and Bai, B and Lin, X and Su, X and Zhang, Q and Liang, T and Yu, G},
title = {Pancreatic-targeted lipid nanoparticles based on organ capsule filtration.},
journal = {Nature},
volume = {652},
number = {8108},
pages = {220-229},
pmid = {41741655},
issn = {1476-4687},
support = {//Beijing Municipal Science & Technology Commission/ ; //National Key R&D Program of China/ ; },
mesh = {Animals ; *Nanoparticles/chemistry ; Mice ; *Pancreas/metabolism ; Humans ; *Lipids/chemistry ; Pancreatic Neoplasms/therapy/genetics ; RNA, Messenger/genetics/administration & dosage/metabolism ; Female ; Gene Editing ; Male ; Endocytosis ; Cancer Vaccines/immunology/administration & dosage ; Cytokines/genetics ; CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Capsules/chemistry ; Liposomes ; },
abstract = {Achieving pancreatic-targeted delivery marks a breakthrough in treating pancreatic diseases, yet precise delivery remains challenging[1]. Here we identify an explicit and universal principle for pancreatic-selective delivery and propose a pancreatic-targeted lipid nanoparticle (AH-LNP) for mRNA delivery. AH-LNP exhibits size enlargement after assembly with proteins, facilitating capsule-filter-mediated pancreas-selective accumulation and receptor-mediated endocytosis, thereby boosting the pancreatic-targeted ability. Benefiting from this, AH-LNP enables precise and efficient genome editing in the pancreas through the delivery of Cas9 mRNA and single guide RNA (sgRNA), exhibiting promising potential in the treatment of autoimmune pancreatic diseases. Furthermore, pancreatic-targeted delivery of mRNA encoding therapeutic cytokines through AH-LNP demonstrates superior antitumour efficacy when combined with a cancer vaccine or chimeric antigen receptor T cell therapy in multiple pancreatic cancer models. The safety and pancreatic mRNA delivery of AH-LNP were verified in multiple animal models, including non-human primates, demonstrating great promise for clinical translation. Our findings highlight the transformative potential of this pancreatic-targeted mechanism and the derived LNP platform, opening avenues for developing precision therapeutics against diverse pancreatic diseases.},
}

Animals
*Nanoparticles/chemistry
Mice
*Pancreas/metabolism
Humans
*Lipids/chemistry
Pancreatic Neoplasms/therapy/genetics
RNA, Messenger/genetics/administration & dosage/metabolism
Female
Gene Editing
Male
Endocytosis
Cancer Vaccines/immunology/administration & dosage
Cytokines/genetics
CRISPR-Cas Systems/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Capsules/chemistry
Liposomes

@article {pmid41741764,
year = {2026},
author = {Shivgotra, R and Soni, B and Bakrey, H and Kaur, P and Jain, SK},
title = {Bridging Gaps in Fungal Keratitis Management: Novel Diagnostics, Drug Delivery Systems, and Gene Therapies.},
journal = {AAPS PharmSciTech},
volume = {27},
number = {3},
pages = {},
pmid = {41741764},
issn = {1530-9932},
abstract = {Globally, fungal keratitis has become a major health concern, particularly in tropical and humid regions. The disease burden is exacerbated because of the underlying complexity of fungal pathogens, delayed or insufficient diagnosis, and limitations in existing therapeutic strategies, often leading to progressive corneal damage and impaired vision. Additional challenges include insufficient therapy optimization and variable efficacy of topical and systemic antifungal treatments, which may be influenced by factors such as rapid ocular drug clearance, ineffective ocular penetration, and the increasing prevalence of antifungal resistance. Conventional diagnostic techniques, such as microscopy and culture, continue to serve as reference standards but have limitations due to comparatively modest sensitivity and the prolonged turnaround times. In comparison, recent advances in diagnostic techniques, including CRISPR-based assays, PCR, MALDI-ToF MS, and in vivo confocal microscopy, as well as the novel drug-delivery nanocarriers, have been reported to show noticeable improvements in diagnostic accuracy and therapeutic outcomes. Furthermore, emerging DNA-based gene therapies and RNA-based therapeutics, along with advanced ocular drug carriers, have shown promising outcomes in preclinical research and early-phase clinical trials, suggesting potential advantages in enhanced tissue targeting and reduced therapeutic resistance. However, before widespread clinical acceptability, extensive clinical validation, long-term safety evaluations, and cost assessments are required, as existing findings are mainly limited to short-term and experimental research. This review focuses on the pathophysiology of fungal keratitis while underscoring the unmet diagnostic and therapeutic needs. It further explores the potential for developing translatable technologies aimed at predictive diagnosis and the effective management of this sight-threatening condition.},
}

@article {pmid41742301,
year = {2026},
author = {Lee, YI and Hart, CC and Henley-Beasley, CS and Herr, JS and Zerpa, E and Barton, ER and Hammers, DW and Sweeney, HL},
title = {Functional and structural pathologies in skeletal muscle of a rat model of Duchenne muscular dystrophy.},
journal = {Skeletal muscle},
volume = {16},
number = {1},
pages = {},
pmid = {41742301},
issn = {2044-5040},
support = {P50-AR052646/AR/NIAMS NIH HHS/United States ; P50-HD119693/NH/NIH HHS/United States ; },
mesh = {Animals ; *Muscular Dystrophy, Duchenne/pathology/physiopathology/genetics/metabolism ; *Muscle, Skeletal/pathology/physiopathology/metabolism ; Rats, Sprague-Dawley ; Disease Models, Animal ; Dystrophin/genetics/metabolism/deficiency ; Rats ; Male ; Muscle Contraction ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Duchenne muscular dystrophy (DMD) is a lethal pediatric degenerative muscle disease for which there is no cure. Robust preclinical models that recapitulate major clinical features of DMD are required to investigate efficacy of potential DMD therapeutics. Rat models of DMD have emerged as promising small animal models to accomplish this; however, there have been no comprehensive studies investigating the functional skeletal muscle decrements associated with the modeling of DMD in rats.

METHODS: CRISPR/Cas9 gene editing was used to generate a dystrophin-deficient Sprague-Dawley muscular dystrophy rat (MDR). Biochemical and immunofluorescent analyses were performed to confirm loss of dystrophin in striated muscles of this rat model. In situ and ex vivo muscle function was assessed in wild-type (WT) and MDR muscles at 3, 6, and 12 months of age, followed by histopathological analyses.

RESULTS: MDR muscle tissues exhibited loss of full-length dystrophin and reduced content of other dystrophin glycoprotein complex members. MDR extensor digitorum longus (EDL) muscles and diaphragms displayed pronounced and progressive muscle weakness beginning at 3 months of age, compared to WT littermates. EDLs also exhibit susceptibility to eccentric contraction-induced damage. Functional deficits in soleus muscles were less severe and were associated with a right shift in force-frequency relationship. MDR muscles display progressive histopathology including degenerative lesions, fibrosis, regenerative foci, and modest adipose deposition.

CONCLUSIONS: MDR is a preclinical model of DMD that exhibits many translational features of the human disease, including a large dynamic range of muscle decrements, that has high utility for the evaluation of potential therapeutics for DMD.},
}

Animals
*Muscular Dystrophy, Duchenne/pathology/physiopathology/genetics/metabolism
*Muscle, Skeletal/pathology/physiopathology/metabolism
Rats, Sprague-Dawley
Disease Models, Animal
Dystrophin/genetics/metabolism/deficiency
Rats
Male
Muscle Contraction
CRISPR-Cas Systems

@article {pmid41742365,
year = {2026},
author = {Yao, W and Li, Y and Du, Y and Gao, R and Tan, J and Fan, J and Liu, Y},
title = {Aptamer-Mediated Protein Corona In Situ Multiplex Microfluidic Detection for Cancer Diagnosis.},
journal = {Analytical chemistry},
volume = {98},
number = {9},
pages = {7123-7135},
doi = {10.1021/acs.analchem.6c00170},
pmid = {41742365},
issn = {1520-6882},
mesh = {Humans ; *Aptamers, Nucleotide/chemistry ; *Neoplasms/diagnosis/blood ; *Protein Corona/chemistry ; *Microfluidic Analytical Techniques ; *Biomarkers, Tumor/blood ; *Lab-On-A-Chip Devices ; CRISPR-Cas Systems ; Silicon Dioxide/chemistry ; },
abstract = {Liquid biopsy faces critical limitations in detecting low-abundance protein biomarkers due to serum complexity, single-target constraints, and insufficient sensitivity. Here, we present an integrated microfluidic protein aptamer corona-CRISPR (PACC) platform that synergizes nanoparticle protein corona (PC) enrichment, multiplexed aptamer recognition, and CRISPR-Cas12a-driven signal generation for facile and rapid point-of-care testing. Fe3O4@SiO2 nanoparticles capable of streamlining PC isolation via magnetic separation were employed to concentrate cancer-associated proteins from serum. A 6-plex aptamer panel, which selectively bound target proteins via forming protein-aptamer coronas (PACs), was used to initiate the in situ activation of CRISPR-Cas12a via complementary crRNAs for fluorescence signal generation. This entire workflow was miniaturized into a 3D-printed microfluidic chip and portable reader, achieving a fast sample-to-answer process. Clinical validation with 112 serum specimens demonstrated excellent diagnostic performance, achieving an area under the curve (AUC) of 0.95 and 88.24% accuracy. By integrating these modalities, the PACC platform overcomes serum matrix interference and biomarker scarcity, providing a scalable and low-cost avenue for detecting low-abundance protein biomarkers and advancing liquid biopsy toward precision oncology.},
}

Humans
*Aptamers, Nucleotide/chemistry
*Neoplasms/diagnosis/blood
*Protein Corona/chemistry
*Microfluidic Analytical Techniques
*Biomarkers, Tumor/blood
*Lab-On-A-Chip Devices
CRISPR-Cas Systems
Silicon Dioxide/chemistry

@article {pmid41742419,
year = {2026},
author = {Zhu, M and Yuan, J and Meng, Q and Yu, J and Xu, X and Xu, M and Ren, X and Hu, Y and Wei, G and Jia, Z and Yuan, G and Zang, L and Liu, S and Yang, Y and Zheng, Y and Wang, J and Cong, T and Xie, W and Lan, X and Cong, L and Ma, T and Ding, S and Guo, W and Zhang, X and Li, Y},
title = {Minimizing far-extending chromatin perturbation in genome editing preserves stem cell identity.},
journal = {Cell stem cell},
volume = {33},
number = {3},
pages = {470-486.e14},
doi = {10.1016/j.stem.2026.01.015},
pmid = {41742419},
issn = {1875-9777},
mesh = {*Gene Editing/methods ; *Chromatin/metabolism/genetics ; Animals ; Mice ; CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; *Neural Stem Cells/metabolism/cytology ; Mouse Embryonic Stem Cells/metabolism/cytology ; CCCTC-Binding Factor/metabolism ; },
abstract = {Although CRISPR-Cas9 holds therapeutic promise, broader application demands an understanding of complications in vast non-coding regions. We found that CRISPR-Cas9 can cause premature differentiation of neural stem cells in vivo and mouse embryonic stem cells in vitro, even when cleavage occurred at distant sites tens of kilobases away from the nearest regulatory elements. To investigate this, we employed an integrated assay for transposase-accessible chromatin (ATAC)/RNA sequencing (AR-seq) approach and identified editing-induced chromatin accessibility changes, with their scale varying by cell type. Cells with stemness are most affected, experiencing perturbations that extend over a hundred kilobases. Furthermore, even local DNA perturbations can disrupt CTCF- and condensate-associated chromatin architecture, causing distal transcriptional rewiring and, ultimately, loss of stemness identity. To minimize chromatin perturbations and preserve cell identity, we refined gene-editing strategies, including distance-aware sgRNA design, pharmacological attenuation of DNA resection, and alternative editing systems. This work paves the way for the safer and broader application of genome-editing technologies.},
}

*Gene Editing/methods
*Chromatin/metabolism/genetics
Animals
Mice
CRISPR-Cas Systems/genetics
Cell Differentiation/genetics
*Neural Stem Cells/metabolism/cytology
Mouse Embryonic Stem Cells/metabolism/cytology
CCCTC-Binding Factor/metabolism

@article {pmid41742729,
year = {2026},
author = {Krupa, LS and Villamayor, PR and Bandara, S and Zhang, Y and Palmiotti, A and von Lintig, J and Bendesky, A},
title = {Retinoic acid production via the ray-finned fish gene bco1l is essential for juvenile development.},
journal = {Genetics},
volume = {233},
number = {1},
pages = {},
pmid = {41742729},
issn = {1943-2631},
support = {R35 GM143051/GM/NIGMS NIH HHS/United States ; R35GM143051/NH/NIH HHS/United States ; EY020551/GF/NIH HHS/United States ; EY028121/GF/NIH HHS/United States ; //Columbia University SURF program/ ; },
mesh = {Animals ; *beta-Carotene 15,15'-Monooxygenase/genetics/metabolism ; *Tretinoin/metabolism ; *Fish Proteins/genetics/metabolism ; beta Carotene/metabolism ; Vitamin A/metabolism ; *Fishes/genetics/growth & development/metabolism ; CRISPR-Cas Systems ; },
abstract = {In vertebrates, vitamin A (VA) is crucial for development, tissue homeostasis, vision, and immunity. Retinal, a form of VA, can be produced via enzymatic cleavage of β-carotene by beta-carotene oxygenase 1 (bco1) and bco1-like (bco1l), but the developmental and tissue-specific functions of these genes are poorly understood. While bco1 is found across vertebrate taxa, bco1l is a paralog of bco1 that we discover to have evolved in the ray-finned fishes, the most abundant, speciose, and commercially important group of fishes. We investigated the function of bco1l in ray-finned Siamese fighting fish, commonly known as betta, an emerging model for genetics and development. Using CRISPR/Cas9 knockouts, we find that lack of bco1l results in reduced VA and elevated β-carotene in larvae, starting when animals have exhausted their yolk supply of retinal, followed by stunted growth and death during juvenile development. Exogenous retinoic acid largely rescues the mutation, demonstrating its deficiency causes these defects. bco1l is 7× more abundant than bco1 in the intestine. This, coupled with the inability of bco1 to sustain VA production in the bco1l mutant, indicates that bco1l is the primary enzyme for dietary carotenoid conversion into retinal. Our results show that VA production by bco1l is required for post-embryonic development, and that bco1l became essential after evolving via duplication of bco1.},
}

Animals
*beta-Carotene 15,15'-Monooxygenase/genetics/metabolism
*Tretinoin/metabolism
*Fish Proteins/genetics/metabolism
beta Carotene/metabolism
Vitamin A/metabolism
*Fishes/genetics/growth & development/metabolism
CRISPR-Cas Systems

@article {pmid41744735,
year = {2026},
author = {Hu, X and Su, J and Song, S},
title = {CRISPR/Cas System-Based Biosensors.},
journal = {Biosensors},
volume = {16},
number = {2},
pages = {},
pmid = {41744735},
issn = {2079-6374},
abstract = {Over the past decade, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins, originally identified as adaptive immune systems in bacteria and archaea that defend against invading nucleic acids, have revolutionized biological research [...].},
}

@article {pmid41747377,
year = {2026},
author = {Pan, MX and Lv, MM and Nie, YG and Su, M and Zha, CJ and Mei, RY and Ying, ZM},
title = {Ultrasensitive miRNA detection via magnetic bead-confined catalytic hairpin assembly enabling transcription-driven crRNA assembly and CRISPR/Cas12a activation.},
journal = {Biosensors & bioelectronics},
volume = {302},
number = {},
pages = {118559},
doi = {10.1016/j.bios.2026.118559},
pmid = {41747377},
issn = {1873-4235},
mesh = {*MicroRNAs/genetics/isolation & purification/analysis ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *CRISPR-Associated Proteins/genetics/chemistry ; DNA/chemistry/genetics ; *Endodeoxyribonucleases/chemistry/genetics ; Limit of Detection ; *Bacterial Proteins/chemistry/genetics ; Nucleic Acid Hybridization ; Transcription, Genetic ; },
abstract = {The integration of CRISPR/Cas12a with catalytic hairpin assembly (CHA), a strategy that predominantly relies on CHA to generate dsDNA activators for direct Cas12a activation, has emerged as a powerful tool in molecular diagnostics. However, two major challenges remain: the strict protospacer adjacent motif (PAM) dependence of the dsDNA and background leakage from hairpin hybridization. Herein, we report a bead-confined platform that transcription mediates crRNA reassembly and template activation of Cas12a for ultrasensitive miRNA detection. The target-triggered CHA assembly dynamically constructed a T7 transcription template from three initially locked hairpins (H1, H2, and H3), which not only transcribed scaffold RNA but also hybridized with its own product to form a DNA/RNA complex that activates Cas12a. The integration of the split T7 promoter with CHA effectively suppressed background suppression and enhanced detection sensitivity. Additionally, the magnetic beads increase local concentration and reaction kinetics, collectively contributing to a substantially enhanced detection sensitivity. Moreover, a crRNA assembly strategy designed for transcription-powered Cas12a not only circumvents the conventional PAM-dependent dsDNA activation pathway of Cas12a but also enables self-supplied crRNA without requiring additional activators. We demonstrated that the biosensor exhibits exceptional sensitivity for miRNA-21 detection, achieving a limit of 65.3 aM. Furthermore, the practicality of this method was preliminarily confirmed through accurately quantifying target levels in cell lines and human serum. Our method presents a viable solution with transformative potential, designed to address complex challenges in contemporary diagnostic applications.},
}

*MicroRNAs/genetics/isolation & purification/analysis
*Biosensing Techniques/methods
*CRISPR-Cas Systems/genetics
Humans
*CRISPR-Associated Proteins/genetics/chemistry
DNA/chemistry/genetics
*Endodeoxyribonucleases/chemistry/genetics
Limit of Detection
*Bacterial Proteins/chemistry/genetics
Nucleic Acid Hybridization
Transcription, Genetic

@article {pmid41747624,
year = {2026},
author = {Ming Wu, SC and Boheler, KR},
title = {Generation of an induced pluripotent stem cell and isogenic control line from a vascular Ehlers-Danlos Syndrome (vEDS) patient harboring a pathogenic c.755G>T in the COL3A1 gene.},
journal = {Stem cell research},
volume = {92},
number = {},
pages = {103945},
doi = {10.1016/j.scr.2026.103945},
pmid = {41747624},
issn = {1876-7753},
mesh = {Humans ; *Ehlers-Danlos Syndrome/genetics/pathology/metabolism ; *Induced Pluripotent Stem Cells/metabolism/cytology/pathology ; *Collagen Type III/genetics/metabolism ; Cell Line ; Cell Differentiation ; Fibroblasts/metabolism ; CRISPR-Cas Systems ; Ehlers-Danlos Syndrome, Type IV ; },
abstract = {We report the generation and characterization of a collagen III-mutant human iPSC line (JHUi007-A) and an isogenic gene-edited control (JHUi007-A-1). Reprogramming of dermal fibroblasts, obtained from a patient with vascular Ehlers-Danlos syndrome (vEDS) carrying the COL3A1 c.755G>T variant, was performed using integration-free Sendai virus. Isogenic controls were produced by CRISPR/Cas9 gene editing. Both lines displayed typical morphologies, expressed stemness factors, formed derivatives of all three germ layers, and maintained a normal karyotype. These lines readily differentiated into vascular smooth muscle cells with cytoskeletal differences between vEDS and control cells, confirming the utility of this resource to study disease processes.},
}

Humans
*Ehlers-Danlos Syndrome/genetics/pathology/metabolism
*Induced Pluripotent Stem Cells/metabolism/cytology/pathology
*Collagen Type III/genetics/metabolism
Cell Line
Cell Differentiation
Fibroblasts/metabolism
CRISPR-Cas Systems
Ehlers-Danlos Syndrome, Type IV

@article {pmid41747626,
year = {2026},
author = {Yoo, DH and Bayarsaikhan, D and Lee, J and Im, YS and Bayarsaikhan, G and Kang, HA and Lee, B and Kim, YO},
title = {Generation and characterization of SOX17-specific EGFP expressing human induced pluripotent stem cell line, KSCBi017-A-4, using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {92},
number = {},
pages = {103943},
doi = {10.1016/j.scr.2026.103943},
pmid = {41747626},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *SOXF Transcription Factors/metabolism/genetics ; *Green Fluorescent Proteins/metabolism/genetics ; Cell Line ; Cell Differentiation ; Endoderm/cytology/metabolism ; },
abstract = {We generated a human induced pluripotent stem cell (hiPSC) reporter line in which EGFP was inserted in-frame at the C-terminus of the endogenous SOX17 locus using CRISPR/Cas9-mediated homologous recombination. The targeted clone, KSCBi017-A-4, was isolated by puromycin selection and validated by PCR and Sanger sequencing. This SOX17-EGFP hiPSC line retains a normal karyotype and pluripotency and displays specific EGFP expression upon directed definitive endoderm differentiation. This reporter line provides a reliable tool for monitoring SOX17 expression during human endoderm specification.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*CRISPR-Cas Systems/genetics
*SOXF Transcription Factors/metabolism/genetics
*Green Fluorescent Proteins/metabolism/genetics
Cell Line
Cell Differentiation
Endoderm/cytology/metabolism

@article {pmid41747766,
year = {2026},
author = {Cai, Y and Zhuang, L and Wang, Z and He, L and Li, X and Liu, BF and Li, T and Zhang, G and Zhou, H and Huang, X and Li, Y},
title = {Gravity-Driven Formation of Water-in-Wax Spheres for Efficient One-Pot CRISPR Diagnostics.},
journal = {ACS nano},
volume = {20},
number = {9},
pages = {8055-8067},
doi = {10.1021/acsnano.6c01232},
pmid = {41747766},
issn = {1936-086X},
mesh = {*Gravitation ; *Water/chemistry ; Humans ; *Waxes/chemistry ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Microspheres ; CRISPR-Cas Systems ; Human papillomavirus 16/genetics/isolation & purification ; },
abstract = {Rapid, decentralized molecular diagnostics are urgently needed for effective infectious disease control. Here, we present "Wax-Sphere CRISPR" (WS-CRISPR), a paradigm-shifting platform for CRISPR-based diagnostics centered on a gravity-driven, interfacial phase-change self-encapsulation mechanism. This system fundamentally decouples bioreagent engineering from specific reaction vessels, transforming conventional, labor-intensive manual encapsulation into a standardized, physics-driven assembly process that generates discrete wax microspheres. Guided by fluid mechanics and interfacial thermodynamics, aqueous CRISPR droplets spontaneously traverse air/molten wax/ethanol to self-encapsulate and solidify, enabling standardized, high-throughput fabrication without manual wax handling. Upon temperature modulation, the wax phase change triggers sequential recombinase polymerase amplification (RPA) and CRISPR detection within a sealed, one-pot vessel. As a clinically oriented demonstration, WS-CRISPR enables multiplexed detection and risk stratification of all 14 high-risk HPV genotypes (HPV16/18 vs others) with a detection limit of 1 × 10[-18] M in under 30 min. Integrated with widely accessible devices─including a thermocycler, hand-held fluorescence reader, and microfluidic platform─the system demonstrated 97.4% sensitivity and 100% specificity across 70 clinical samples. By solving the engineering bottlenecks of scalability and universality, WS-CRISPR offers a robust tool for decentralized, large-scale pathogen surveillance.},
}

*Gravitation
*Water/chemistry
Humans
*Waxes/chemistry
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Microspheres
CRISPR-Cas Systems
Human papillomavirus 16/genetics/isolation & purification

@article {pmid41748831,
year = {2026},
author = {Moreno, DS and Carvalho, JP and Murray, E and Colombo, NSR and Lamas, A and Cobas, AC and Hill, C and Azeredo, J and Domingues, L},
title = {Evaluation of the delivery of an anti-Listeria endolysin via CRISPR-Cas9 engineered probiotic Saccharomyces boulardii.},
journal = {Applied microbiology and biotechnology},
volume = {110},
number = {1},
pages = {},
pmid = {41748831},
issn = {1432-0614},
mesh = {*Endopeptidases/genetics/pharmacology/metabolism ; *Probiotics/metabolism ; Humans ; *CRISPR-Cas Systems ; *Saccharomyces boulardii/genetics/metabolism ; *Listeria monocytogenes/drug effects ; Bacteriophages/genetics/enzymology ; Listeriosis/prevention & control/microbiology ; Saccharomyces cerevisiae/genetics ; },
abstract = {Listeriosis is a foodborne infection caused by Listeria monocytogenes that causes febrile gastroenteritis and central nervous system infections and that can often lead to fatality. Upon consumption of contaminated food, Listeria is able to survive a number of gastrointestinal stressors, including competition with the host microbiota. The emergence of antibiotic-resistant clones of L. monocytogenes, together with the side effects of antibiotic treatment, highlights the need for alternatives or additives for its treatment and prevention. Saccharomyces boulardii is a probiotic yeast that is often used alongside antibiotics to minimize side effects since it is not affected by them as a result of its eukaryotic nature. Furthermore, it can be engineered to produce a wide range of molecules. We previously engineered Saccharomyces cerevisiae through CRISPR-Cas9 integration to produce Ply511, a bacteriophage endolysin active against L. monocytogenes, showing the potential of engineered yeast to produce endolysins for biocontrol. In this study, we extended this approach to the probiotic yeast S. boulardii and directly compared the two yeasts as secretion hosts for Ply511. Using a simulated human gastrointestinal environment, we evaluated their ability to retain endolysin activity and reduce L. monocytogenes levels. We then tested the cell extracts from both yeasts in a bacterial consortium termed SImplified HUman intestinal MIcrobiota (SIHUMI), confirming a specificity for Listeria. Finally, we evaluated their activity in a simulated intestinal fermentation using fecal samples from human donors. Overall, this study demonstrates the potential of delivering endolysins to the gut via engineered probiotic S. boulardii. KEY POINTS: CRISPR-Cas9-engineered S. boulardii and S. cerevisiae were compared, both allowing the expression and activity of endolysin Ply511 against L. monocytogenes. Endolysin Ply511 retained its activity against L. monocytogenes in simulated gastrointestinal digestion and was specific against Listeria in a bacterial consortium termed SImplified HUman intestinal MIcrobiota (SIHUMI). Using fecal samples from human donors, the anti-Listeria effect was reduced potentially due to the lower metabolic activity of S. boulardii and the higher competition with the intestinal microbiome.},
}

*Endopeptidases/genetics/pharmacology/metabolism
*Probiotics/metabolism
Humans
*CRISPR-Cas Systems
*Saccharomyces boulardii/genetics/metabolism
*Listeria monocytogenes/drug effects
Bacteriophages/genetics/enzymology
Listeriosis/prevention & control/microbiology
Saccharomyces cerevisiae/genetics

@article {pmid41750315,
year = {2026},
author = {Schulze, A and Kainz, K and Bauer, MA and Carmona-Gutierrez, D},
title = {Editing Candida: Origins and Advances of CRISPR Tools.},
journal = {Biomolecules},
volume = {16},
number = {2},
pages = {},
pmid = {41750315},
issn = {2218-273X},
support = {10.55776/P37278//FWF Austrian Science Fund/ ; not applicable//University of Graz/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Candida/genetics/pathogenicity ; Humans ; Virulence/genetics ; Genome, Fungal ; Candidiasis/microbiology/genetics ; },
abstract = {Pathogens causing candidiasis encompass a diverse group of ascomycetous yeasts that have become essential models for studying fungal adaptability, pathogenicity, and host-pathogen interactions. Although many candidiasis-promoting species exist as commensals within host microbiota, several have acquired virulence traits that enable opportunistic infections, positioning them as a leading cause of invasive fungal disease in humans. Deciphering the molecular and genetic determinants that underpin the biology of organisms responsible for candidiasis has long been a central objective in medical and molecular mycology. However, research progress has been constrained by intrinsic biological challenges, including noncanonical codon usage and the absence of a complete sexual cycle in diploid species, which have complicated traditional genetic manipulation. CRISPR-Cas9 genome editing has overcome many of these limitations, providing a precise, efficient, and versatile framework for targeted genomic modification. This system has facilitated functional genomic studies ranging from single-gene deletions to high-throughput mutagenesis, yielding new insights into the mechanisms governing virulence, antifungal resistance, and stress adaptation. Since its initial application in Candida albicans, CRISPR-Cas9 technology has been refined and adapted for other clinically and industrially relevant species, including Nakaseomyces glabratus (formerly referred to as Candida glabrata), Candida parapsilosis, and Candida auris. The present work provides an overview of the evolution of genetic approaches employed in research directed against candidiasis-associated species, with a particular focus on the development and optimization of CRISPR-based systems. It highlights how recent advancements have improved the genetic tractability of these pathogens and outlines emerging opportunities for both fundamental and applied studies in fungal biology.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
*Candida/genetics/pathogenicity
Humans
Virulence/genetics
Genome, Fungal
Candidiasis/microbiology/genetics

@article {pmid41751014,
year = {2026},
author = {Wang, Q and Zheng, L and You, G and Dong, H and Chen, S and Wang, S and Chen, S},
title = {Navigating the Complexity: Advancing Diagnostic Strategies for Avian Reovirus in Chinese Poultry.},
journal = {Animals : an open access journal from MDPI},
volume = {16},
number = {4},
pages = {},
pmid = {41751014},
issn = {2076-2615},
support = {XTCXGC2021018, XTCXGC2021012//the '5511' Collaborative Innovation Project of Fujian Academy of Agricultural Sciences, China/ ; },
abstract = {Avian reovirus (ARV) infections pose a significant and evolving threat to China's poultry industry, the world's largest. Diverse farming systems-ranging from modern intensive operations to traditional waterfowl-poultry polyculture-foster a unique ecological niche for ARV, defined by complex serotypic and genotypic diversity, marked regional variations, potential interspecies transmission between chickens and waterfowl, and recurrent co-infections. Collectively, these factors undermine the efficacy of conventional diagnostic approaches. This review systematically outlines the current epidemic landscape of ARV in China, highlighting the molecular characteristics of prevailing strains (particularly those from waterfowl) and their roles in diagnostic evasion. We critically assess the performance and limitations of existing diagnostic techniques (virus isolation, ELISA, PCR/qPCR) within the Chinese epidemiological setting. Furthermore, we discuss innovative technologies-including multiplex qPCR, CRISPR-Cas systems, and next-generation sequencing (NGS)-that offer potential for developing next-generation diagnostics tailored to China's specific challenges. Finally, we propose future directions, with an emphasis on standardization, data sharing, and interdisciplinary collaboration to bridge the gap between cutting-edge innovation and on-farm application for precise ARV control.},
}

@article {pmid41751536,
year = {2026},
author = {Hawkins, V and Rudiger, SR and McLaughlan, CJ and Kelly, JM and Lehnert, K and Jacobsen, JC and Handley, RR and Henare, K and Verma, PJ and Snell, RG},
title = {Foundations of an Ovine Model of Fragile X Syndrome.},
journal = {Genes},
volume = {17},
number = {2},
pages = {},
pmid = {41751536},
issn = {2073-4425},
support = {3914//Curekids/ ; 20/259//Health Research Council of New Zealand/ ; },
mesh = {Animals ; *Fragile X Syndrome/genetics/pathology ; *Disease Models, Animal ; *Fragile X Messenger Ribonucleoprotein 1/genetics ; Sheep/genetics ; Male ; Female ; CRISPR-Cas Systems ; Gene Editing ; Gene Knockout Techniques ; Humans ; },
abstract = {BACKGROUND: Fragile X Syndrome (FXS) is an X-linked neurodevelopmental disorder characterised by intellectual disability, developmental delays, anxiety, and social and behavioural challenges. Currently, no effective treatments exist to address the root cause of FXS. Mouse models are the most widely used for studying molecular pathogenesis and conducting preclinical treatment testing. However, therapeutic interventions that show promise in rodent models have yet to succeed in clinical trials. After evaluating the current models, we have developed an ovine model to address this clinical translation gap. We expect this model to more accurately reflect the human condition in brain size, structure, and neurodevelopmental trajectory. We aim to establish this model as a valuable preclinical platform for testing therapies for FXS.

METHODS: To generate the sheep model, we used CRISPR-Cas9 dual-guide editing to knock out the Fragile X Messenger Ribonucleoprotein 1 (FMR1) gene in ovine embryos.

RESULTS: Two founder animals were created, one ram (male) and one ewe (female), both of which carried FMR1 gene knockouts. The ewe carries inactivating mutations on both alleles, with the edits in both animals resulting in no detectable Fragile X Messenger Ribonucleoprotein (FMRP) as expected. Both founders have undergone molecular characterisation and basic health checks, with the female founder showing increased joint flexibility, a characteristic of FXS. The ram has been used for breeding, with the successful transmission of the edited allele to his offspring. Importantly, specific lamb cohorts for postnatal treatment testing can be produced efficiently utilising accelerated breeding methods and preimplantation selection.},
}

Animals
*Fragile X Syndrome/genetics/pathology
*Disease Models, Animal
*Fragile X Messenger Ribonucleoprotein 1/genetics
Sheep/genetics
Male
Female
CRISPR-Cas Systems
Gene Editing
Gene Knockout Techniques
Humans

@article {pmid41751548,
year = {2026},
author = {Machel Gica, NG and Gica, WT and La, H and Mi, Y and Zhou, Y},
title = {Precision Breeding for a Global Staple Food: A Systematic Review with a Strategic Framework for CRISPR-Cas Applications in Rice (Oryza sativa L.).},
journal = {Genes},
volume = {17},
number = {2},
pages = {},
pmid = {41751548},
issn = {2073-4425},
support = {2025GBJ002388//Chinese Government Scholarship Council/ ; },
mesh = {*Oryza/genetics/growth & development ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Plant Breeding/methods ; Plants, Genetically Modified/genetics ; Genome, Plant ; Crops, Agricultural/genetics ; },
abstract = {Background: Rice is one of the world's main staple crops, and improving its productivity and resilience is important to achieving food security under varying climatic conditions. Objectives: This systematic review synthesizes the existing evidence on the application, technical limitations, and potential of the development of genome editing technologies (CRISPR-Cas) in rice (Oryza sativa L.), as well as presents a novel approach called the CRISPR Trait Prioritization and Readiness Framework (CTPRF). Methods: Peer-reviewed articles that reported applications of genome editing based on the CRISPR-Cas system in the genome of rice for trait improvement or functional genomics were identified through searches fromPubMed, Scopus, Web of Science, and Google Scholar with studies published between 2012 and 2025. Studies were screened on predefined inclusion criteria related to experimental validation, reporting of editing efficiency, and clear phenotypic results. Data on CRISPR systems, target genes, methods of delivery, traits modified, and phenotypic results were extracted and synthesized by comparative analysis. Results: A wide variety of different CRISPR systems have been used in rice, and our results indicate that NHEJ-mediated knockouts are effective in average genotypes with editing efficiencies in the range of 70-90%, but HDR and prime editing are still under 10%. The CTPRF is being introduced as a strategic decision support tool to evaluate traits from four dimensions: technical feasibility, phenotypic predictability, impact potential, and regulatory pathway. We use this framework for case studies in pioneering countries (USA, Japan, China) and show how it can be useful for guiding research investment and policy. Conclusions: CRISPR-Cas technologies have transformed rice breeding, but their introduction requires overcoming genotype-dependent barriers to transformation and negotiating patchwork regulatory environments. The CTPRF offers a roadmap for the acceleration of the development of climate-resilient and nutritious rice varieties for the action plan.},
}

*Oryza/genetics/growth & development
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Plant Breeding/methods
Plants, Genetically Modified/genetics
Genome, Plant
Crops, Agricultural/genetics

@article {pmid41751560,
year = {2026},
author = {Lee, S and Park, S and Bang, H and Kim, SU and Park, YH and Wee, G and Chae, U and Kim, E},
title = {VPS35 Deficiency Markedly Reduces the Proliferation of HEK293 Cells.},
journal = {Genes},
volume = {17},
number = {2},
pages = {},
pmid = {41751560},
issn = {2073-4425},
mesh = {Humans ; *Cell Proliferation/genetics ; *Vesicular Transport Proteins/genetics/deficiency/metabolism ; HEK293 Cells ; Apoptosis/genetics ; CRISPR-Cas Systems ; Mitochondrial Dynamics/genetics ; Mitochondria/metabolism/genetics ; Gene Knockout Techniques ; },
abstract = {Background/Objectives: The retromer protein complex is involved in various physiological processes, especially endosomal trafficking, and its dysregulation has been linked to Alzheimer's disease and Parkinson's disease, as well as VPS35 knockout (KO), causing early embryonic lethality. We aimed to investigate the cellular consequences of VPS35 deficiency. Methods: To investigate the effects of VPS35 loss, we used CRISPR/Cas9 to generate VPS35 KO human embryonic kidney 293 (HEK293) cells. We analyzed changes in retromer component expression, cell proliferation, apoptosis, and mitochondrial dynamics using Western blotting, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and confocal microscopy. Results: VPS35 KO led to a significant reduction in cell proliferation and decreased expression of VPS29 and VPS26, both essential for retromer complex assembly. Consequently, retromer formation was impaired. Compared to control cells, KO cells exhibited elevated levels of cleaved caspase-3, poly(ADP-ribose) polymerase, cytochrome C, and p21, while the expression of Ki-67, CDK4, and cyclin D was reduced. Additionally, VPS35 deletion also promoted mitochondrial fragmentation, associated with increased expression of mitochondrial fission-related proteins. Finally, the rescue experiment using the human VPS35 gene confirmed that the recovery of VPS35 not only led to the recovery of the essential elements constituting the retromer but also the recovery of molecules related to the cell cycle, restoring cell death to a normal level. Conclusions: These findings suggest that VPS35 plays a critical role in cell growth and survival by modulating apoptosis, mitochondrial dynamics, and cell cycle progression.},
}

Humans
*Cell Proliferation/genetics
*Vesicular Transport Proteins/genetics/deficiency/metabolism
HEK293 Cells
Apoptosis/genetics
CRISPR-Cas Systems
Mitochondrial Dynamics/genetics
Mitochondria/metabolism/genetics
Gene Knockout Techniques

@article {pmid41751614,
year = {2026},
author = {Sun, Q and Guo, Y and Wang, L and Jia, L and Wei, P and Ma, S},
title = {CRISPR-Mediated Silkworm: The Oncoming Agricultural Revolutions and a Rising Model Organism.},
journal = {Genes},
volume = {17},
number = {2},
pages = {},
pmid = {41751614},
issn = {2073-4425},
support = {32570591//National Natural Science Foundation of China/ ; },
mesh = {*Bombyx/genetics/growth & development ; Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Agriculture/methods ; Silk/genetics ; Genome, Insect ; Genomics/methods ; },
abstract = {The silkworm (Bombyx mori) is essential to sericulture and is also becoming a key model organism in genomics and agriculture. For decades, genetic studies of the silkworm were limited by inefficient and inflexible genome tools. CRISPR genome editing allows precise and scalable alterations to genes regulating development, physiology, and industrial traits. This review summarizes silkworm genome-editing breakthroughs, highlighting CRISPR's evolution from simple gene knockouts to large-scale genome-wide screening. We highlight how these advancements contribute to disease resistance, higher yields, and the development of new silk-based materials, as well as how they influence the development and growth rate of the sericulture. The creation of high-quality reference genomes, pangenomes, and genome-wide screening systems has made the silkworm a major model for integrating multiple biological datasets and approaches, such as genomic, transcriptomic, and proteomic. By considering the unique biological characteristics of the silkworm, this provides new insights for research on silk biology, piRNA synthetic biology, and hormonal signaling regulation. Finally, we examine new areas at the intersection of CRISPR, pangenomics, and artificial intelligence (AI) and suggest future paths for molecular breeding, pest control, and synthetic biology. Moreover, AI-assisted prediction of CRISPR outcomes is utilized to inform the design of targeted trait modifications, representing an approach to enhancing biomanufacturing efficiency and eco-friendly silk production. Together, these advances have made the silkworm a flexible genetic platform and an important part of sustainable agriculture and biomanufacturing.},
}

*Bombyx/genetics/growth & development
Animals
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Agriculture/methods
Silk/genetics
Genome, Insect
Genomics/methods

@article {pmid41751799,
year = {2026},
author = {Cai, X and Liang, X and Zou, P and Xiao, R and Wang, Y},
title = {CRISPRi Screening Identifies Essential E. coli Virulence Factors for Placental Barrier Breach in a Maternal-Fetal Infection Model.},
journal = {International journal of molecular sciences},
volume = {27},
number = {4},
pages = {},
pmid = {41751799},
issn = {1422-0067},
support = {82574169//National Natural Science Foundation of China/ ; 7232009, 7244289//Natural Science Foundation of Beijing Municipality/ ; 20240484724//Cross-cooperation project of Beijing Science and Technology New Star Program/ ; Subject leaders-03-02//High Level Public Health Technical Personnel Construction Project/ ; //Research Foundation of Capital Institute of Pediatrics/ ; //Beijing Chaoyang District Postdoctoral Research Foundation 2024/ ; },
mesh = {Female ; Pregnancy ; Animals ; *Virulence Factors/genetics/metabolism ; *Escherichia coli Infections/microbiology/genetics ; *Escherichia coli/pathogenicity/genetics ; Rats ; *Placenta/microbiology/metabolism ; Humans ; Disease Models, Animal ; Escherichia coli Proteins/genetics/metabolism ; Host-Pathogen Interactions ; Trophoblasts/microbiology/metabolism ; CRISPR-Cas Systems ; *Pregnancy Complications, Infectious/microbiology ; Neonatal Sepsis/microbiology ; },
abstract = {Early-onset neonatal sepsis caused by Escherichia coli (E. coli) threatens neonates' lives due to the pathogen's high virulence and multidrug resistance. The mechanisms that enable its placental barrier breach are poorly understood. Using a clinically isolated ST95 ExPEC strain from a neonatal sepsis case, along with a pregnant rat model and an in vitro placental barrier model, we performed CRISPR interference screening. This screen targeted 264 virulence factor genes and identified virulence factors for motility, iron acquisition, hemolysin secretion, and adherence/invasion as critical. We demonstrated that hlyB is essential for uterine infection, and we elucidated a mechanism for ibeA that facilitates syncytial trophoblast cell layer penetration by interacting with the host receptor(s) PSF/VIM to enhance bacterial internalization. Host cells countered ibeA+ E. coli infection via a novel host defense pathway involving upregulation of ASPHD1. This study systematically mapped the virulence factors required for E. coli placental translocation and delineated key host-pathogen interactions.},
}

Female
Pregnancy
Animals
*Virulence Factors/genetics/metabolism
*Escherichia coli Infections/microbiology/genetics
*Escherichia coli/pathogenicity/genetics
Rats
*Placenta/microbiology/metabolism
Humans
Disease Models, Animal
Escherichia coli Proteins/genetics/metabolism
Host-Pathogen Interactions
Trophoblasts/microbiology/metabolism
CRISPR-Cas Systems
*Pregnancy Complications, Infectious/microbiology
Neonatal Sepsis/microbiology

@article {pmid41751840,
year = {2026},
author = {Begum, SN and Hasan, SK},
title = {Prime Editing Driven Functional Genomics: Bridging Genotype to Phenotype in the Post-Genomic Era.},
journal = {International journal of molecular sciences},
volume = {27},
number = {4},
pages = {},
pmid = {41751840},
issn = {1422-0067},
mesh = {Humans ; *Gene Editing/methods ; CRISPR-Cas Systems ; *Genomics/methods ; Phenotype ; Genotype ; Animals ; },
abstract = {The post-genomic era, defined by large-scale sequencing initiatives, has generated an unprecedented catalogue of human genetic variation. Yet, the vast majority of genetic variants remain classified as variants of uncertain significance or are located within poorly characterized non-coding regions, thereby hindering the effective translation of genomic data into meaningful biological understanding and clinical application. Bridging this genotype-to-phenotype gap requires precise, high-throughput functional genomics. Early CRISPR-Cas9 knockout and CRISPR interference/activation (CRISPRi/a) screens mapped gene-level functions but could not assess single nucleotide variants (SNVs). Bridging this genotype-to-phenotype gap demands precise, high-throughput functional genomics. Multiplexed assays of variant effect (MAVEs), like saturation genome editing, systematically test all possible mutations using CRISPR-Cas9 and donor libraries. Base editors allow targeted single-base changes without double-strand breaks but are limited in scope, while prime editing can introduce any small substitution, insertion, or deletion without double-strand breaks (DSBs) or donor templates. This review traces the evolution of functional screens from gene-level knockouts to saturation genomic editing (SGE), and highlights how prime editing is driving a new paradigm for the systematic functional characterization of thousands of variants across disease-relevant genes. We also detail the architecture, mechanism, and progressive optimization of PE systems and their delivery methods. Collectively, prime editing stands as a transformative platform poised to accelerate precision functional genomics and advance the diagnosis and treatment of genetic diseases.},
}

Humans
*Gene Editing/methods
CRISPR-Cas Systems
*Genomics/methods
Phenotype
Genotype
Animals

@article {pmid41751979,
year = {2026},
author = {Eskildsen, J and Dong, M and Hanak, T and Madsen, CK and Holme, I and Plaszkó, T and Vestergård, M and Nicolaisen, M and Thordal-Christensen, H and Brinch-Pedersen, H},
title = {Novel CRISPR/Cas9-Derived mlo Alleles in Barley: Resistance to Powdery Mildew and Microbiome Implications.},
journal = {International journal of molecular sciences},
volume = {27},
number = {4},
pages = {},
pmid = {41751979},
issn = {1422-0067},
support = {NNF19OC0056580//Novo Nordisk Foundation/ ; BarleyMicroBreed, 101060057//EU Horizon research and innovation/ ; },
mesh = {*CRISPR-Cas Systems ; *Hordeum/genetics/microbiology ; *Disease Resistance/genetics ; *Plant Diseases/microbiology/genetics ; *Ascomycota/pathogenicity ; *Microbiota/genetics ; Alleles ; Plant Roots/microbiology/genetics ; *Plant Proteins/genetics ; Mutation ; },
abstract = {Barley grown in temperate regions is often challenged by powdery mildew disease. An effective solution is mildew resistance locus o (mlo)-based resistance, which is monogenic, durable, and broad-spectrum. While the pleiotropic effects of mlo mutations on above-ground tissues are well documented, their impact on the root-associated microbiome remains underexplored. We utilized CRISPR/Cas9 to generate novel mlo mutant lines and evaluated their resistance to causal fungus Blumeria hordei. We further examined if mlo knockout has any impact on the overall root microbiome diversity and composition under field-like conditions and applied DESeq2 to compare the abundance of microbial taxa between mutants and wild type. We created five novel resistant mlo lines, including the first mutants with amino acid alterations in the protein's extracellular region. Mutant lines showed significantly reduced B. hordei colony formation (0.5-5%). While microbial alpha and beta diversity were not significantly altered, a few microbial taxa displayed time-dependent shifts in abundance. Overall, our study demonstrates the effectiveness of CRISPR/Cas9 in generating mlo-based resistance. Moreover, the study revealed functionally important residues in the protein's extracellular region. Finally, we present the first evidence of limited mlo-associated effects on root microbiome diversity and relative abundance of microbial taxa.},
}

*CRISPR-Cas Systems
*Hordeum/genetics/microbiology
*Disease Resistance/genetics
*Plant Diseases/microbiology/genetics
*Ascomycota/pathogenicity
*Microbiota/genetics
Alleles
Plant Roots/microbiology/genetics
*Plant Proteins/genetics
Mutation

@article {pmid41752145,
year = {2026},
author = {Fayed, S and Amer, S and Badawy, M and Bou Malhab, L and Omran, N and Khoder, G and Ghemrawi, R and Haider, M and Hamoudi, R and Harati, R},
title = {The Role of CRISPR and Its Therapeutic Applications in Glioblastoma.},
journal = {International journal of molecular sciences},
volume = {27},
number = {4},
pages = {},
pmid = {41752145},
issn = {1422-0067},
support = {210111350//University of Sharjah/ ; 2201110368//University of Sharjah/ ; 23010902146//University of Sharjah/ ; VRI-20-10//ASPIRE Precision Medicine Research Institute Abu Dhabi/ ; },
mesh = {Humans ; *Glioblastoma/therapy/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Brain Neoplasms/therapy/genetics ; Animals ; *Genetic Therapy/methods ; },
abstract = {Glioblastoma multiforme (GBM) remains the most aggressive and treatment-refractory form of primary brain tumor in adults, characterized by rapid proliferation, intratumoral heterogeneity and resistance to current therapies. Despite therapeutic advancements in surgical resection, radiotherapy and chemotherapy, clinical outcomes remain poor, underscoring the need for innovative molecular strategies. This review examines the therapeutic potential of CRISPR/Cas9 genome-editing technologies in GBM, highlighting their ability to model, dissect and potentially correct the genetic alterations that drive GBM tumorigenesis. Key molecular targets, such as EGFR, PTEN, TP53, NF1 and PIK3CA, are discussed within the context of GBM's mutational and signaling landscape. We further outline emerging CRISPR applications in preclinical models, the current status of CRISPR-based clinical trials and the major barriers hindering translation, including off-target effects, immunogenicity and the challenge of delivering gene-editing systems across the blood-brain barrier. Particular emphasis is placed on delivery technologies, viral and non-viral vectors, including lipid nanoparticles, polymeric systems, inorganic nanocarriers and DNA nanostructures, which are rapidly evolving to improve precision, safety and CNS penetrance. Collectively, this review highlights CRISPR/Cas9 as a powerful tool whose integration with molecular neuro-oncology and precision medicine may ultimately shift GBM treatment toward more personalized and durable therapeutic interventions.},
}

Humans
*Glioblastoma/therapy/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
*Brain Neoplasms/therapy/genetics
Animals
*Genetic Therapy/methods