@article {pmid41570009,
year = {2026},
author = {Tay, YL and Thomson, SB and Hnatova, S and Ng, S and Teo, SR and McCallum, R and Sim, B and Tarantini, L and Tai, FL and Bollati, V and Loh, M and Hayden, MR and Leavitt, BR and Pouladi, MA},
title = {Silencing of human HTT by targeted CRISPR/dCas9-mediated epigenetic editing.},
journal = {Journal of Huntington's disease},
volume = {15},
number = {3},
pages = {399-407},
doi = {10.1177/18796397251415368},
pmid = {41570009},
issn = {1879-6400},
mesh = {Humans ; *Huntingtin Protein/genetics ; *DNA Methylation/genetics ; *Gene Silencing ; *Huntington Disease/genetics/therapy ; DNA Methyltransferase 3A ; *Epigenesis, Genetic ; *CRISPR-Cas Systems ; Epigenome Editing ; DNA (Cytosine-5-)-Methyltransferases/genetics ; },
abstract = {BackgroundGene silencing is widely recognized as a promising therapeutic approach for dominant monogenic disorders. Current silencing strategies, many of which are transient, utilize RNA interference. Gene silencing may also be achieved through directed epigenetic editing using a CRISPR/dCas9 effector fused to DNA methyltransferase 3A (dCas9-DNMT3A). We used this system to direct DNA methylation to HTT, the causal gene underlying the autosomal dominant neurodegenerative disorder Huntington's disease, to assess the translational potential of this strategy for treating a genetic neurological disease.ObjectiveTo characterize the regulatory effect of targeted dCas9-DNMT3A-mediated DNA methylation at HTT.MethodsWe exploited DNA methylation profiles of high and low HTT-expressing tissues and targeted hypomethylated regions of HTT associated with high levels of HTT expression.ResultsDe novo DNA methylation of loci within defined upstream, promoter, intragenic and downstream regions of HTT resulted in robust, acute silencing of HTT. The best long-term silencing of HTT, which persisted up to 30 days, was observed when targeted DNA methylation was directed to the 5'UTR and promoter regions of HTT.ConclusionsHTT gene silencing may be achieved via targeted de novo DNA methylation within hypomethylated regulatory regions at the HTT locus. DNA methylation editing may be an attractive therapeutic approach for Huntington disease due to its potential for long-term silencing and reversibility.},
}

Humans
*Huntingtin Protein/genetics
*DNA Methylation/genetics
*Gene Silencing
*Huntington Disease/genetics/therapy
DNA Methyltransferase 3A
*Epigenesis, Genetic
*CRISPR-Cas Systems
Epigenome Editing
DNA (Cytosine-5-)-Methyltransferases/genetics

@article {pmid42045650,
year = {2026},
author = {Saha, A and Ocampo, RF and Wright, JT and Taylor, DW and Palermo, G},
title = {Molecular mechanisms and biotechnology applications of CRISPR-Cas12a.},
journal = {Nature reviews. Molecular cell biology},
volume = {27},
number = {8},
pages = {601-616},
pmid = {42045650},
issn = {1471-0080},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry ; *Biotechnology/methods ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Animals ; *Bacterial Proteins/genetics/metabolism/chemistry ; *Endodeoxyribonucleases/metabolism/genetics/chemistry ; },
abstract = {CRISPR-Cas12a is a versatile RNA-guided nuclease that has rapidly gained prominence for its dual functionality in genome editing and nucleic acid detection. In this Review, we discuss the structural, biochemical and mechanistic features of Cas12a that underpin its autonomous processing of the guide RNA and indiscriminate cleavage of single-stranded DNA, which enable Cas12a applications ranging from gene therapy to rapid diagnostics. We discuss key allosteric regulators and functional modules that orchestrate Cas12a activity, focusing on the core regulatory structural elements that control maturation of the guide RNA, target specificity, and both cis-cleavage and trans-cleavage activities, including the determinants of off-target cleavage. We provide a comparative analysis of Cas12a and the widely used Cas9, which further illuminates the distinctive attributes of Cas12a, and discuss recent advances in the characterization of its orthologues and in the development of engineered variants that expand its capabilities. Collectively, we present a comprehensive understanding of Cas12a and its increasing impact on biotechnology, therapeutics and molecular diagnostics.},
}

*CRISPR-Cas Systems/genetics
Humans
*CRISPR-Associated Proteins/metabolism/genetics/chemistry
*Biotechnology/methods
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
Animals
*Bacterial Proteins/genetics/metabolism/chemistry
*Endodeoxyribonucleases/metabolism/genetics/chemistry

@article {pmid42190664,
year = {2026},
author = {Zhang, H and Zhang, Z and Wang, P and Xu, T and Chen, X and Zhao, Y and Lin, S and Cai, W and Ren, P and Luo, C and Zhang, P and Wang, Y and Hou, S and Zhao, Y and Zeng, H and Liu, Z and Wang, C and Gao, Z and Feng, Y and Pan, D and Zeng, Z},
title = {Uncovering spatially resolved functional genomics with CRISPR screen sequencing.},
journal = {Cell},
volume = {189},
number = {15},
pages = {4594-4618.e48},
doi = {10.1016/j.cell.2026.04.049},
pmid = {42190664},
issn = {1097-4172},
mesh = {*Genomics/methods ; Animals ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Macrophages/metabolism/immunology ; Intercellular Adhesion Molecule-1/metabolism/genetics ; CD8-Positive T-Lymphocytes/metabolism/immunology ; Hyaluronan Receptors/metabolism ; Mice ; CRISPR-Cas Systems ; Cell Line, Tumor ; High-Throughput Nucleotide Sequencing/methods ; },
abstract = {Spatial omics has advanced our understanding of tissue-level biology, yet tools to systematically link gene functional perturbations to spatial phenotypes and signaling pathways remain limited. To address this, we developed spatial CRISPR screen sequencing (SPAC-seq), a high-throughput spatial CRISPR screen platform, and TARDIS (target prioritization toolkit for perturbation data in spatial omics), a statistical spatial perturbation analysis toolkit. Using SPAC-seq and TARDIS, we linked gene perturbations to spatial phenotypes and pathways, uncovering how Icam1 loss in tumor cells promotes metastasis via immune suppression and macrophage polarization. In CD8[+] T cells, we revealed Cd44's role in regulating spatial phenotypes by interacting with Spp1 on macrophages. We also demonstrated the model of the transcription factor-chemokine receptor axis coupling cell states with chemotaxis. SPAC-seq and TARDIS provide an effective framework to study spatially resolved functional genomics and pathways across diverse biological and disease contexts.},
}

*Genomics/methods
Animals
Humans
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Macrophages/metabolism/immunology
Intercellular Adhesion Molecule-1/metabolism/genetics
CD8-Positive T-Lymphocytes/metabolism/immunology
Hyaluronan Receptors/metabolism
Mice
CRISPR-Cas Systems
Cell Line, Tumor
High-Throughput Nucleotide Sequencing/methods

@article {pmid42192532,
year = {2026},
author = {Hwang, HY and Yi, H and Gwon, Y and Jeon, E and Kim, D},
title = {High-fidelity genome and prime editing enabled by the AI-designed openCRISPR-1.},
journal = {Genome medicine},
volume = {18},
number = {1},
pages = {},
pmid = {42192532},
issn = {1756-994X},
support = {RS-2025-00521074//National Research Foundation of Korea/ ; RS-2025-02214578, HR22C1363, RS-2024-02507183//Korea Health Industry Development Institute (KHIDI)/ ; },
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; HEK293 Cells ; *Genome, Human ; Induced Pluripotent Stem Cells/metabolism ; CRISPR-Associated Protein 9/genetics ; },
abstract = {BACKGROUND: RNA-guided nucleases such as CRISPR-Cas9 systems have revolutionized genome engineering by enabling programmable DNA modifications. Although structure-guided and evolution-derived high-fidelity Cas9 variants improve target specificity, they often compromise on-target activity or constrain guide RNA (gRNA) design.

METHODS: We performed head-to-head comparisons of OpenCRISPR-1 and Cas9 in human cells using amplicon sequencing, multiplex Digenome-seq, and off-target validation by targeted sequencing. Editing activity was assessed across 28 endogenous loci in HEK293T cells and further evaluated in human induced pluripotent stem cells (iPSCs) and MRC-5 fibroblasts. To test clinically relevant delivery, Cas9 and OpenCRISPR-1 ribonucleoproteins were delivered using engineered virus-like particles (eVLPs). We also generated OpenCRISPR-based prime editors, OpenCRISPR-PE2 and OpenCRISPR-PE7, and compared them with PE2max and PE7 using pegRNAs and engineered epegRNAs.

RESULTS: Here, we show that OpenCRISPR-1, an AI-designed, Cas9-like nuclease, retains Cas9-level editing efficiency across multiple genomic loci while significantly reducing off-target mutations. Using multiplex Digenome-seq and targeted deep sequencing, OpenCRISPR-1 exhibits up to a 553-fold reduction in off-target mutations compared to Cas9 and achieves off-target indices that match or surpass those of high-fidelity Cas9 variants. OpenCRISPR-1 also sustains robust editing across diverse gRNA formats (GX19, gX19, and gX20), highlighting its enhanced versatility. Furthermore, converting OpenCRISPR-1 into a prime editor yields comparable editing efficiencies while lowering the relative specificity ratio by up to 97%.

CONCLUSIONS: These findings establish generative AI-guided protein design as a powerful strategy to overcome the specificity-efficiency trade-off, expanding the genome editing toolkit for both research and therapeutic use, and ushering in a new era of rational protein design.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems
RNA, Guide, CRISPR-Cas Systems/genetics
HEK293 Cells
*Genome, Human
Induced Pluripotent Stem Cells/metabolism
CRISPR-Associated Protein 9/genetics

@article {pmid42218140,
year = {2026},
author = {Di Bernardo, M and Kern, RS and Cepeda Diaz, AK and Mallar, A and Choi, SJ and Nutter-Upham, A and Lourido, S and Blainey, PC and Cheeseman, I},
title = {Brieflow: an integrated computational pipeline for high-throughput analysis of optical pooled screening data.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42218140},
issn = {2041-1723},
support = {GM126930//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; 000955563//National Science Foundation (NSF)/ ; Data Science Internship Program//Massachusetts Life Sciences Center (MLSC)/ ; UROP Program//Massachusetts Institute of Technology (MIT)/ ; R01HG009283//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; R01AI144369//Division of Intramural Research, National Institute of Allergy and Infectious Diseases (Division of Intramural Research of the NIAID)/ ; },
mesh = {*Computational Biology/methods ; *High-Throughput Screening Assays/methods ; CRISPR-Cas Systems ; Reproducibility of Results ; Humans ; *Genomics/methods ; Phenotype ; Software ; Large Language Models ; Pooled Testing ; },
abstract = {Optical pooled screening (OPS) has emerged as a powerful technique for functional genomics, enabling researchers to link genetic perturbations with complex cellular morphological phenotypes at scale. However, OPS data analysis presents challenges due to massive datasets, complex multi-modal integration requirements, and the absence of standardized frameworks. Here, we present Brieflow, a computational pipeline for end-to-end analysis of fixed-cell optical pooled screening data. We demonstrate Brieflow's capabilities through reanalysis of a CRISPR-Cas9 screen encompassing 5072 fitness-conferring genes, processing more than 70 million cells with multiple phenotypic markers. To accelerate biological interpretation, we additionally present MozzareLLM, a framework leveraging large language models to identify biological processes within phenotypic clusters and prioritize gene candidates for experimental validation. Our combined analysis recovers coherent biological modules missed by existing analytical approaches, including five core mitochondrial sub-programs absent from the original study. The modular design and open-source implementation of Brieflow facilitates the integration of new analytical components while ensuring computational reproducibility and improved performance for the use of high-content phenotypic screening in biological discovery.},
}

*Computational Biology/methods
*High-Throughput Screening Assays/methods
CRISPR-Cas Systems
Reproducibility of Results
Humans
*Genomics/methods
Phenotype
Software
Large Language Models
Pooled Testing

@article {pmid42230636,
year = {2026},
author = {Snetkova, V and Galan, C and Lopez, R and Rios, AR and Kudo, T and Dorighi, K and Warming, S and Haley, BJ},
title = {A tunable Cas12a platform for single-cell perturbation screening and CRISPRi.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42230636},
issn = {2041-1723},
mesh = {*Single-Cell Analysis/methods ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *CRISPR-Associated Proteins/genetics/metabolism ; Humans ; *Gene Editing/methods ; *Bacterial Proteins/genetics/metabolism ; Single-Cell Gene Expression Analysis ; Degrons ; HEK293 Cells ; Gene Knockout Techniques/methods ; Transcriptome ; Animals ; Endodeoxyribonucleases ; },
abstract = {Single-cell perturbation (Perturb-seq) screens have primarily relied on Cas9 for inducing loss-of-function phenotypes, whereas Cas12a, despite its unique effectiveness for multiplex guide expression, remains underexplored. This may be due to Cas12a's guide RNA array (pre-crRNA) self-processing activity and the subsequent challenges associated with pre-crRNA sequence recovery during single-cell RNA sequencing library preparation. To overcome the self-processing constraint, we optimized pre-crRNA expression vectors and established a degron-based, enhanced Cas12a system for gene knock-out. As demonstrated across cell types, target genes, and with a minimized guide RNA library, this platform allows for accurate detection of pre-crRNAs and gene editing-induced effects on the transcriptome in single cells. Additionally, we show that HyperLbCas12a outperforms other existing variants for multiplexed gene suppression. While the rapid reversibility of this repressor highlights specific kinetic constraints for degron-based single-cell recording, the system provides a potent, modular tool for contexts requiring tunable, transient silencing. Together, this suite of technologies greatly expands the possibilities for future Perturb-seq efforts and broader application of Cas12a for genetic disruption at scale.},
}

*Single-Cell Analysis/methods
*CRISPR-Cas Systems/genetics
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*CRISPR-Associated Proteins/genetics/metabolism
Humans
*Gene Editing/methods
*Bacterial Proteins/genetics/metabolism
Single-Cell Gene Expression Analysis
Degrons
HEK293 Cells
Gene Knockout Techniques/methods
Transcriptome
Animals
Endodeoxyribonucleases

@article {pmid42236706,
year = {2026},
author = {Zhu, L and Nguyen, LT and Bell, AG and Krebel, T and Gillmann, KM and Cao, Q and Oatman, H and Hariri, J and Möglich, A and Myhrvold, C and Toettcher, JE},
title = {Multimodal control of Cas13d activity through domain insertion at an allosteric hotspot.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42236706},
issn = {2041-1723},
support = {R01GM144362//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Allosteric Regulation ; Humans ; *CRISPR-Associated Proteins/genetics/metabolism/chemistry ; Protein Domains ; Blue Light ; HEK293 Cells ; Sirolimus/pharmacology/analogs & derivatives ; },
abstract = {CRISPR-Cas13d RNA nucleases are powerful tools for programmable RNA targeting. A light-controlled RNA nuclease could be transformative by enabling researchers to selectively knock down transcripts at desired positions in a cell or tissue or at timepoints of interest. Here, we develop a set of RfxCas13d tools that can be multimodally controlled by either light or small molecule addition. By screening an RfxCas13d library containing insertions of the AsLOV2 photoswitchable domain, we identify an OptoCas13d-off variant that induced target RNA cleavage in the dark and switched to an inactive state under blue light. We show that the same allosteric hotspot can be exploited to generate an OptoCas13d-on with an inverted light response and a ChemoCas13d that is activated by rapamycin analogs, enabling knockdown of endogenous mRNA and protein targets. Overall, our study shows that engineered allostery can produce stimulus-controlled Cas13d variants to modulate RNA with high spatial and temporal precision.},
}

*CRISPR-Cas Systems/genetics
Allosteric Regulation
Humans
*CRISPR-Associated Proteins/genetics/metabolism/chemistry
Protein Domains
Blue Light
HEK293 Cells
Sirolimus/pharmacology/analogs & derivatives

@article {pmid42236735,
year = {2026},
author = {Turocy, J and Jerabek, S and Hur, W and Kim, J and Xu, S and Zhao, Q and Xu, J and Robles, A and Liu, X and Treff, N and Marin, D and Hadjantonakis, AK and Egli, D},
title = {Asymmetric attrition and secondary chromosome destabilization after double-strand breaks in human embryonic development.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42236735},
issn = {2041-1723},
mesh = {Humans ; *DNA Breaks, Double-Stranded ; *Embryonic Development/genetics ; *Chromosomal Instability/genetics ; Centromere/genetics/metabolism ; Chromatids/metabolism ; Telomere/genetics/metabolism ; DNA Repair ; Spindle Apparatus/metabolism ; CRISPR-Cas Systems ; },
abstract = {DNA repair in human embryos is poorly understood, and double-strand breaks (DSBs) can cause chromosome loss. We show that chromosomal alterations relative to an induced DSB are asymmetric: acentric arms show complementary gains and losses, while centric arms are biased toward losses. Centromeric to the cut site secondary breakage and attrition is extensive. In contrast, break sites at acentric arms are conserved with no secondary breakage. These differences reflect differential forces at the mitotic spindle. Telomeric arms detach from the pro-metaphase spindle while centric truncated chromosomes lag during anaphase, suggesting that the DSB impedes sister chromatid separation. Secondary breakage near the centromere concordant with extensive attrition at the DSB site indicates a DSB can destabilize a chromosome without end-joining of sister chromatids. These results highlight the risks of chromosomal-scale changes in CRISPR-Cas9 genome editing and show that a single DSB can destabilize a human embryo chromosome independent of fusion-breakage cycles.},
}

Humans
*DNA Breaks, Double-Stranded
*Embryonic Development/genetics
*Chromosomal Instability/genetics
Centromere/genetics/metabolism
Chromatids/metabolism
Telomere/genetics/metabolism
DNA Repair
Spindle Apparatus/metabolism
CRISPR-Cas Systems

@article {pmid42251067,
year = {2026},
author = {Gopal, N and Abay, T and Payne, C and Gomez, M and Rogers, MM and Fofanah, IU and Kallon, TPMS and Kamara, MS and Suk, HJ and Sandi, JD and Brock-Fisher, T and Stachler, E and Allan-Blitz, LT and Roach, DJ and Paye, MF and Wilkason, C and Grant, DS and Ozonoff, A and Sabeti, PC},
title = {Rapid development and field evaluation of a portable CRISPR-based assay for Mpox during the 2025 Sierra Leone outbreak.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42251067},
issn = {2041-1723},
mesh = {Sierra Leone/epidemiology ; Humans ; *Disease Outbreaks ; Sensitivity and Specificity ; Rapid Diagnostic Tests ; *CRISPR-Cas Systems/genetics ; *Hemorrhagic Fever, Ebola/epidemiology/diagnosis/virology ; *Ebolavirus/genetics/isolation & purification ; },
abstract = {The large 2025 Mpox clade IIb outbreak in Sierra Leone underscores the urgent need for portable, low-cost diagnostics in decentralized settings. While CRISPR-based assays offer high sensitivity and flexibility, their deployment during active outbreaks remains limited. Here we show the rapid development and field evaluation of Mpox SHINE, a CRISPR-Cas13 assay that integrates lyophilized reagents, ambient-temperature lysis, and automated fluorescence detection on the portable DxHub device. The assay achieves analytical sensitivity down to 10 copies/µL. Clinical validation in Sierra Leone, using 56 clinical specimens, confirms complete concordance with qPCR, demonstrating 100% sensitivity and 100% specificity. Crucially, Mpox SHINE also detects the virus directly from unextracted lesion swabs while maintaining 100% sensitivity and specificity. The mean time-to-result is fast, averaging 11.4 minutes for extracted samples and 27.9 minutes for unextracted samples. These findings demonstrate that CRISPR-based diagnostics translate quickly from genomic sequence to clinically validated, deployable tools within a single outbreak window.},
}

Sierra Leone/epidemiology
Humans
*Disease Outbreaks
Sensitivity and Specificity
Rapid Diagnostic Tests
*CRISPR-Cas Systems/genetics
*Hemorrhagic Fever, Ebola/epidemiology/diagnosis/virology
*Ebolavirus/genetics/isolation & purification

@article {pmid42269486,
year = {2026},
author = {Popsuj, S and Kalsang, T and Kim, K and Drummond, E and Manekar, P and Munagapati, P and Oleti, M and Sato, H and Vickery, I and Gigante, ED and Stolfi, A},
title = {Validated CRISPR/Cas9 guide RNAs targeting neurodevelopmental genes in the tunicate Ciona robusta.},
journal = {Differentiation; research in biological diversity},
volume = {150},
number = {},
pages = {100973},
doi = {10.1016/j.diff.2026.100973},
pmid = {42269486},
issn = {1432-0436},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Neurodevelopment/genetics ; *Ciona/genetics/growth & development ; Mutagenesis ; Transcription Factors/genetics ; Gene Expression Regulation, Developmental ; *Ciona intestinalis/genetics ; },
abstract = {The tunicate Ciona robusta provides a powerful and simplified model for dissecting the genetic control of developmental and cell biology. With a larval CNS composed of just over 200 neurons and sensory cells, it has also emerged as a model organism for neurobiology and the development of the nervous system. Although CRISPR/Cas9-mediated mutagenesis is now routinely used in Ciona as an important technique used to interrogate gene function in diverse biological processes, validated single-guide RNAs (sgRNAs) have yet to be validated for several key neural genes. Here, we report the design and experimental validation of 25 novel sgRNAs targeting eight conserved genes encoding conserved proteins involved in neurodevelopment and neural function, including six transcription factors (Cdx, Foxb, Sox1/2/3, Dmbx, Engrailed, and Mnx) and two neural effector genes (Tyrosinase and Slc18a3/VAChT). Candidate sgRNAs were selected and tested for mutagenesis efficiency using Illumina-based target site amplicon sequencing. All sgRNAs induced insertions or deletions at their target loci, with most genes yielding at least one sgRNA with mutagenesis efficacy exceeding 30%, with the exception of Dmbx, for which maximal efficacy reached 25%. We further compared measured mutagenesis rates to scores generated by different predictive algorithms, observing a modest but potentially improved correlation with predictions based on a newer algorithm. Based on these results, we recommend considering both scoring algorithms in combination, for improved predictive value for Ciona.},
}

Animals
*CRISPR-Cas Systems/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics
Neurodevelopment/genetics
*Ciona/genetics/growth & development
Mutagenesis
Transcription Factors/genetics
Gene Expression Regulation, Developmental
*Ciona intestinalis/genetics

@article {pmid42276496,
year = {2026},
author = {Wang, F and Zhang, M and Chen, S and Fu, S and Huai, M and Liu, M and Meng, G and Dong, C},
title = {CRISPR/Cas9-mediated disruption of Cmpks1 reveals its role as a key regulator of carotenoid biosynthesis and metabolic adaptation in Cordyceps militaris.},
journal = {International journal of biological macromolecules},
volume = {372},
number = {},
pages = {152987},
doi = {10.1016/j.ijbiomac.2026.152987},
pmid = {42276496},
issn = {1879-0003},
mesh = {*Cordyceps/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Carotenoids/metabolism ; Gene Expression Regulation, Fungal ; *Fungal Proteins/genetics/metabolism ; *Polyketide Synthases/genetics/metabolism ; Oxidative Stress ; *Adaptation, Physiological/genetics ; Light ; Mutation ; },
abstract = {Cordyceps militaris, a renowned edible mushroom, produces orange-yellow fruiting bodies (FBs), primarily due to carotenoid accumulation. However, genetic mechanisms and functional roles underlying carotenoid biosynthesis remain poorly understood. Here, we identified Cmpks1, a light-induced gene encoding a reducing type I polyketide synthase, as a key regulator of pigment biosynthesis. Transcription of Cmpks1 was CmWC-1-dependent and upregulated during FB development. CRISPR/Cas9-mediated loss-of-function mutants of Cmpks1 exhibited stable albino phenotypes but retained FB differentiation. In addition to abolishing carotenoid biosynthesis, the disruption of Cmpks1 increased sensitivity to high light and oxidative stress, indicating its role in redox homeostasis. Metabolomic profiling of the ΔCmpks1 mutant, including significantly reduced ergothioneine and elevated cordycepin, revealed extensive metabolic reprogramming, coupled with activation of compensatory survival mechanisms. These findings elucidate the genetic mechanisms governing pigment formation that influence the quality of Cordyceps products, offering new insights into the role of metabolites in fungal morphogenesis and stress adaptation.},
}

*Cordyceps/genetics/metabolism
*CRISPR-Cas Systems/genetics
Carotenoids/metabolism
Gene Expression Regulation, Fungal
*Fungal Proteins/genetics/metabolism
*Polyketide Synthases/genetics/metabolism
Oxidative Stress
*Adaptation, Physiological/genetics
Light
Mutation

@article {pmid42276945,
year = {2026},
author = {Meng, D and Zhang, Y and Zou, S and Wang, J and Tian, C and Gao, T and Liu, J and Yuan, X and Chen, S and Qiao, Y and Ma, N and Chang, H and Gao, X},
title = {Cas9-PALB2 fusion protein enhances CRISPR/Cas9 mediated gene knock-in efficiency.},
journal = {Journal of bioscience and bioengineering},
volume = {142},
number = {3},
pages = {187-195},
doi = {10.1016/j.jbiosc.2026.05.004},
pmid = {42276945},
issn = {1347-4421},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Knock-In Techniques/methods ; HEK293 Cells ; *Fanconi Anemia Complementation Group N Protein/genetics/metabolism ; *CRISPR-Associated Protein 9/genetics/metabolism ; *Gene Editing/methods ; *Recombinant Fusion Proteins/genetics/metabolism ; Recombinational DNA Repair ; },
abstract = {Over the past decade, CRISPR-based technologies have revolutionized our capacity to manipulate genomes, thereby reshaping the landscape of functional genomics research. Among the CRISPR toolkit, CRISPR/Cas9-mediated homology-directed repair (HDR) enables precise genome editing with predefined mutations, rendering it an indispensable tool for gene functional analysis, disease model construction, and the development of gene therapy strategies. Nevertheless, despite the robust efficiency of CRISPR/Cas9 in mediating gene knockouts, HDR-dependent gene knock-in (KI) remains a major bottleneck due to its inherently low efficiency. Herein, we report that the co-expression of PALB2 with the CRISPR/Cas9 nuclease could trigger an enhanced HDR effect. Specifically, the fusion of Cas9 with PALB2 elevated KI efficiency by approximately 1.7-fold in human HEK293T cells. Furthermore, this Cas9-PALB2 fusion strategy exhibited cross-cell-type efficacy, demonstrating its broad applicability beyond a single cell line. Notably, the combined application of the Cas9-PALB2 fusion system and Nocodazole further boosted KI efficiency to a remarkable 25.5%. Collectively, these findings establish the Cas9-PALB2 fusion as a highly potent and versatile strategy to augment HDR-mediated KI efficiency, highlighting its substantial potential for widespread utilization in applications that demand high-fidelity genome editing.},
}

Humans
*CRISPR-Cas Systems/genetics
*Gene Knock-In Techniques/methods
HEK293 Cells
*Fanconi Anemia Complementation Group N Protein/genetics/metabolism
*CRISPR-Associated Protein 9/genetics/metabolism
*Gene Editing/methods
*Recombinant Fusion Proteins/genetics/metabolism
Recombinational DNA Repair

@article {pmid42379534,
year = {2026},
author = {Lummerstorfer, M and Xue, Z and Zheng, D and König, L and Brunner, L and Lächelt, U},
title = {Lipid nanoparticles for Cas9 ribonucleoprotein delivery: design and evaluation of ionisable oligoamine-lipidoids.},
journal = {European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences},
volume = {224},
number = {},
pages = {107596},
doi = {10.1016/j.ejps.2026.107596},
pmid = {42379534},
issn = {1879-0720},
mesh = {*Nanoparticles/chemistry/administration & dosage ; *Lipids/chemistry/administration & dosage ; *Ribonucleoproteins/administration & dosage/chemistry ; *CRISPR-Associated Protein 9/administration & dosage/chemistry ; Humans ; *Amines/chemistry/administration & dosage ; CRISPR-Cas Systems ; Gene Editing ; Liposomes ; },
abstract = {Lipid nanoparticles (LNPs) are the most advanced RNA delivery technology and are used with CRISPR-RNA in multiple clinical in vivo genome editing trials. By contrast, systemic delivery of Cas9 ribonucleoproteins (RNPs) - despite their high intrinsic efficiency - has lagged, largely due to a lack of mature delivery systems, and RNA‑optimised LNPs cannot readily be translated to RNPs. Differences arise from the pH-sensitive protein and cargo-specific optimal lipid compositions. With regard to the fundamental ionisable lipid component, comparatively less optimisation has been carried out for Cas9-RNPs than for RNA. In this work, C12-200, developed as a potent ionisable lipidoid for RNA-LNPs and also well-suited for Cas9-RNPs, served as the lead structure. Using an analogue synthesis strategy, 17 alternative C12-lipidoids were generated from different oligoamine precursors with structural differences, including the number of nitrogens (2 to ≈40), architecture (linear, branched or containing an N-heterocycle), and separating alkyl spacers (ethyl, propyl) between ionisable groups. Employing the different lipidoids in analogous LNP formulations enabled a systematic assessment at relevant stages of Cas9-RNP delivery and the identification of structure-activity relationships. Two C12-lipidoids with piperazine ring, ethyl spacers and three (C12-AEP) or four nitrogens (C12-BAEP) were identified as the most effective, exhibiting potencies comparable to or exceeding C12-200 in the in vitro knockout model. This study reports a systematic evaluation of ionisable oligoamine-lipidoids in Cas9-RNP-LNP formulations, highlights critical delivery bottlenecks, and provides recommendations for the design of potent candidates.},
}

*Nanoparticles/chemistry/administration & dosage
*Lipids/chemistry/administration & dosage
*Ribonucleoproteins/administration & dosage/chemistry
*CRISPR-Associated Protein 9/administration & dosage/chemistry
Humans
*Amines/chemistry/administration & dosage
CRISPR-Cas Systems
Gene Editing
Liposomes

@article {pmid42406843,
year = {2026},
author = {Morianou, I and Phillimore, L and Khatri, BS and Marston, L and Gribble, M and Burt, A and Bernardini, F and Hammond, AM and Nolan, T and Crisanti, A},
title = {Engineering resilient gene drives for sustainable malaria control by predicting, testing and overcoming target site resistance in Anopheles gambiae.},
journal = {PLoS biology},
volume = {24},
number = {7},
pages = {e3003879},
pmid = {42406843},
issn = {1545-7885},
mesh = {Animals ; *Anopheles/genetics ; *Gene Drive Technology/methods ; Female ; *Mosquito Control/methods ; *Malaria/prevention & control/transmission ; Mosquito Vectors/genetics ; *Insecticide Resistance/genetics ; Alleles ; CRISPR-Cas Systems ; },
abstract = {CRISPR-based gene drives are selfish genetic elements with the potential to spread through entire insect populations for sustainable vector control. Gene drives designed to disrupt the reproductive capacity of females can suppress laboratory populations of the malaria mosquito, Anopheles gambiae. However, any suppressive intervention will inevitably exert an evolutionary pressure for resistance, and the likelihood of resistance emerging at natural population scales remains poorly defined. Here, we present a pipeline to quantify the evolutionary space for resistance, enabling accelerated discovery, engineering, and testing of both natural and drive-induced variants that could reverse gene drive spread. We applied our approach to stress-test a best-in-class suppression gene drive that has evaded resistance in all laboratory-contained releases to date, known as Ag(QFS)1. We showed that previously undetected resistant alleles can arise at low frequency, including a novel type of partially resistant alleles that can perturb drive-invasion dynamics. Integrating experimentally derived resistance rates with population genetic modeling shows that single-target suppression drives are unlikely to be robust at natural mosquito population sizes, even at highly constrained loci. Here, we engineer and validate multiplexed gene drives in Anopheles gambiae, that target multiple conserved sites, actively removing resistant alleles. Our models predict that such gene drives could supress large natural mosquito populations in the field.},
}

Animals
*Anopheles/genetics
*Gene Drive Technology/methods
Female
*Mosquito Control/methods
*Malaria/prevention & control/transmission
Mosquito Vectors/genetics
*Insecticide Resistance/genetics
Alleles
CRISPR-Cas Systems

@article {pmid42454502,
year = {2026},
author = {Madhusudan, S and Eskici, N and Gomez-Sanchez, C and Pulli, K and Vaaralahti, K and Yellapragada, V and Conway, JRW and Wang, Y and Raivio, T},
title = {CRISPR activation of DLX5 drives neural progenitors to the GnRH cell fate.},
journal = {Journal of molecular endocrinology},
volume = {77},
number = {1},
pages = {},
doi = {10.1530/JME-26-0040},
pmid = {42454502},
issn = {1479-6813},
mesh = {*Gonadotropin-Releasing Hormone/metabolism ; Humans ; *Homeodomain Proteins/genetics/metabolism ; Cell Differentiation/genetics ; *Neural Stem Cells/metabolism/cytology ; Neurons/metabolism/cytology ; Animals ; Cell Movement/genetics ; *CRISPR-Cas Systems/genetics ; Fibroblast Growth Factor 8/metabolism/genetics ; Mice ; Hypothalamic-Pituitary-Gonadal Axis ; *Cell Lineage/genetics ; Transcription Factors ; },
abstract = {Gonadotropin-releasing hormone (GnRH) neurons regulate the hypothalamic-pituitary-gonadal (HPG) axis and are required for puberty onset and reproductive competence. However, the transcriptional regulators governing GnRH neuron specification and migration remain poorly defined. The homeodomain transcription factor DLX5 is expressed in fetal human GnRH neurons, its expression precedes that of GNRH1 in human pluripotent stem cell (hPSC)-derived GnRH neurons, and in mice, it serves as a guidance cue for GnRH neuron migration. We hypothesized that DLX5 may act as an upstream regulator of human GnRH neuron fate specification and migratory capacity. Using CRISPR activation, we upregulated DLX5 during FGF8b-directed differentiation of hPSCs to GnRH neurons via dual SMAD inhibition and Notch inhibition, as previously described. DLX5 activation increased neural progenitor motility (P < 0.001), upregulated FGF8 (P < 0.05), and induced GABAergic markers, including GAD1 and GAD2. Notably, DLX5 activation induced GNRH1 in the absence of exogenous FGF8b (P < 0.05), suggesting that in GnRH neurons, DLX5 regulates FGF8. When combined with exogenous FGF8b, DLX5 activation produced distinct neuronal patterning accompanied by upregulation of extracellular matrix genes, such as SPARC, which has been implicated in neurite outgrowth. Collectively, these data indicate that activation of DLX5 promotes GnRH neurogenesis from hPSCs, by driving GABAergic fate, inducing FGF8, and remodeling the extracellular matrix.},
}

*Gonadotropin-Releasing Hormone/metabolism
Humans
*Homeodomain Proteins/genetics/metabolism
Cell Differentiation/genetics
*Neural Stem Cells/metabolism/cytology
Neurons/metabolism/cytology
Animals
Cell Movement/genetics
*CRISPR-Cas Systems/genetics
Fibroblast Growth Factor 8/metabolism/genetics
Mice
Hypothalamic-Pituitary-Gonadal Axis
*Cell Lineage/genetics
Transcription Factors

@article {pmid42458955,
year = {2026},
author = {Kigaru, A and Ateka, EM and Pappu, HR and Nganga, EM and Murori, R and Toili, ME and Runo, S},
title = {Enhanced Rice Yellow Mottle Virus Resistance via CRISPR/Cas9-Targeted Mutagenesis of the Rice eIF(iso)4G Gene.},
journal = {Molecular plant pathology},
volume = {27},
number = {7},
pages = {e70312},
pmid = {42458955},
issn = {1364-3703},
support = {//African Union Commission/ ; },
mesh = {*Oryza/genetics/virology ; *CRISPR-Cas Systems/genetics ; *Disease Resistance/genetics ; *Plant Diseases/virology/genetics ; *Plant Viruses/physiology/pathogenicity ; *Mutagenesis/genetics ; *Eukaryotic Initiation Factor-4G/genetics/metabolism ; *Plant Proteins/genetics/metabolism ; Plants, Genetically Modified ; *Genes, Plant ; Mutation/genetics ; },
abstract = {Rice is a staple crop primarily recognised for its high content of carbohydrates and proteins. Rice yellow mottle disease (RYMD) is a destructive disease affecting rice fields in sub-Saharan Africa and is caused by the rice yellow mottle virus (RYMV). Development of virus-resistant genotypes is a highly recommended and effective approach to controlling RYMV. A genetic approach that exploits recessive mutations in susceptibility (S) genes may enhance resistance to the virus. Reports indicate that most rice genotypes grown in Kenya are vulnerable to RYMV infection. Genome editing has shown promise in enhancing agronomic traits in crops. We obtained enhanced resistance to RYMV in the Indica rice cv. IR2793-80-01 using the CRISPR-Cas9 system. The eIF(iso)4G susceptibility gene was targeted because natural mutations in this gene confer recessive resistance to RYMV. A Cas9-OseIF(iso)4G-gRNA-expressing vector targeting the eIF(iso)4G gene was introduced into rice calli via Agrobacterium-mediated transformation. Ten T2 homozygous mutant plant lines were assessed for their reaction to RYMV, and infection was significantly reduced. There were no significant differences in the agronomic characteristics between the T2 mutant lines and the wild-type plants. CRISPR/Cas9-mediated knockout alleles of the eIF(iso)4G gene conferred enhanced resistance to RYMV, which may be classified as partial. Findings underscore the need to embrace precise editing strategies, such as prime editing, to generate superior resistance alleles. Overall, the study provides an alternative resistance enhancement strategy that can create knockout resistance alleles that can be incorporated into breeding programmes for RYMV resistance.},
}

*Oryza/genetics/virology
*CRISPR-Cas Systems/genetics
*Disease Resistance/genetics
*Plant Diseases/virology/genetics
*Plant Viruses/physiology/pathogenicity
*Mutagenesis/genetics
*Eukaryotic Initiation Factor-4G/genetics/metabolism
*Plant Proteins/genetics/metabolism
Plants, Genetically Modified
*Genes, Plant
Mutation/genetics

@article {pmid42459963,
year = {2026},
author = {Dasgupta, R and Das, K},
title = {CasPINS: an integrated web-based platform for CRISPR/TALEN gRNA design, primer generation, and indel decomposition analysis.},
journal = {Bioinformatics advances},
volume = {6},
number = {1},
pages = {vbag189},
pmid = {42459963},
issn = {2635-0041},
abstract = {Genome editing researchers currently navigate multiple disconnected tools for guide RNA (gRNA) design, primer generation, and editing analysis-a fragmented workflow that introduces errors and limits reproducibility. CasPINS (Cas-Primer-Indel Suite) addresses this gap as an open-source, unified platform integrating the complete genome editing computational workflow into a single interactive web application accessible without programming expertise. The platform supports 90+ species, 14 CRISPR-Cas variants, TALEN design, and six editing modes. Primer design integrates with Ensembl and NCBI databases relative to cut sites, while indel quantification utilizes Non-Negative Least Squares (NNLS) decomposition of Sanger chromatograms with maximum signal extraction and R 2 -corrected conservative modes. Benchmarking demonstrates strong concordance with established tools, including a 68.8% recovery of CHOPCHOP gRNAs and 67.2% of CRISPOR gRNAs across five human benchmark genes, alongside an algorithmic agreement within 2.6 percentage points on gold-standard TIDE data. Ultimately, a step-count analysis shows that CasPINS significantly streamlines usability, reducing discrete user actions from 25 to 8 steps compared to the traditional sequential-tool pipeline.},
}

@article {pmid42460575,
year = {2026},
author = {Li, M and Huang, D and Xu, C and Fang, M and He, P and He, Y and Wang, X and Xu, Z},
title = {Thermally Unlocked One-Pot RPA-CRISPR Cas12b Assay Integrated with the Centrifugal Microfluidic Chip for Multiplex Detection of Porcine Viruses.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.6c02126},
pmid = {42460575},
issn = {1520-6882},
abstract = {Highly contagious porcine viruses, represented by African swine fever virus (ASFV), porcine reproductive and respiratory syndrome virus (PRRSV), and pseudorabies virus (PRV), inflict severe economic losses on the swine industry and pose significant threats to global food security. Consequently, developing rapid, convenient, and efficient point-of-care testing (POCT) methods is essential for viral disease control. Although recombinase polymerase amplification (RPA) coupled with CRISPR/Cas systems demonstrates significant POCT potential, its practical application is currently restricted by operational complexity and limited throughput. Herein, a thermally unlocked one-pot RPA-CRISPR Cas12b assay integrated with the centrifugal microfluidic chip (TORCH) platform was developed in this paper. In this strategy, a thermal gating switch was utilized to physically isolate CRISPR reagents from the RPA during the initial phase, effectively addressing the inherent incompatibility in one-pot reactions. By employing the centrifugal microfluidic chip with a portable device, a highly integrated workflow enables fully automated processing ranging from sample lysis to multiplexed detection. Validated using pseudovirus-spiked porcine blood samples, TORCH successfully achieved multiplexed detection of ASFV, PRV, and PRRSV with the limits of detection as low as 0.5 copies/μL, while exhibiting exceptional resistance to interference and robust reagent stability. Overall, TORCH stands as a robust and user-friendly diagnostic solution, holding significant potential for early warning intervention and decentralized biosecurity control in resource-scarce environments.},
}

@article {pmid42462008,
year = {2026},
author = {Skopintsev, P and Esain-Garcia, I and DeTurk, EC and Yoon, PH and Zhou, Z and Weiss, T and Kamalu, M and Chamraj, A and Loi, KJ and Langeberg, CJ and Boger, RS and Nisonoff, H and Karp, HM and Chen, LX and Shi, H and Vohra, K and Banfield, JF and Cate, JHD and Jacobsen, SE and Doudna, JA},
title = {Structure and evolution-guided design of minimal RNA-guided nucleases.},
journal = {Science (New York, N.Y.)},
volume = {393},
number = {6808},
pages = {313-318},
doi = {10.1126/science.aed6123},
pmid = {42462008},
issn = {1095-9203},
mesh = {Humans ; *CRISPR-Cas Systems ; Cryoelectron Microscopy ; *Directed Molecular Evolution/methods ; DNA/chemistry ; *Gene Editing ; Models, Molecular ; Protein Conformation ; *Protein Engineering/methods ; RNA/chemistry ; *RNA, Guide, CRISPR-Cas Systems/chemistry ; *Bacterial Proteins/chemistry/genetics ; *Endodeoxyribonucleases/chemistry/genetics ; *CRISPR-Associated Proteins/chemistry/genetics ; },
abstract = {The design of RNA-guided nucleases with properties not limited by evolution can expand programmable genome-editing capabilities. However, generating diverse multidomain proteins with robust enzymatic properties remains challenging. Here, we use a protein design strategy that couples a structure-guided inverse-folding model with evolution-informed residue constraints to generate active, divergent variants of TnpB, a minimal CRISPR-Cas12-like nuclease, termed SynTnpBs. High-throughput screening of artificial intelligence-generated variants yielded editors that retained or exceeded wild-type activity in bacterial, plant, and human cells. Cryo-electron microscopy-based structure determination of the most divergent variant revealed stabilizing contacts in the RNA-DNA interfaces across conformations, demonstrating the design potential of this approach. Together, these results establish a strategy for creating non-natural RNA-guided nucleases and conformationally active nucleic acid binders, enlarging the designable protein space.},
}

Humans
*CRISPR-Cas Systems
Cryoelectron Microscopy
*Directed Molecular Evolution/methods
DNA/chemistry
*Gene Editing
Models, Molecular
Protein Conformation
*Protein Engineering/methods
RNA/chemistry
*RNA, Guide, CRISPR-Cas Systems/chemistry
*Bacterial Proteins/chemistry/genetics
*Endodeoxyribonucleases/chemistry/genetics
*CRISPR-Associated Proteins/chemistry/genetics

@article {pmid42462085,
year = {2026},
author = {Effah, CY and Li, X and Zhang, Q and Ding, L and Drokow, EK and Zhang, X and Wu, Y},
title = {Integrating CRISPR/Cas Biosensors with Advanced Platforms: A Holistic Path Toward Preamplification-free, Multiplexed, and Continuous Molecular Monitoring.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.6c00860},
pmid = {42462085},
issn = {2379-3694},
abstract = {The paradigm of molecular diagnostics has been transformed by the repurposing of CRISPR-Cas systems from being gene-editing tools to nucleic acid detection engines with remarkable specificity and programmability. Both the SHERLOCK and DETECTR platforms have shown high sensitivity and specificity; however, the requirement of a pre-amplification step to achieve clinically relevant detection limits adds another layer of complexity and cost and is also a potential source of contamination, precluding their use as true point-of-care (POC) tools. The next frontier for CRISPR diagnostics will be the design of biosensors that enable preamplification-free, multiplex, and continuous direct detection of targets. Achieving this goal will involve the very close integration of CRISPR biology with nano-biotechnology, microfluidics, orthogonal Cas enzyme systems, and artificial intelligence (AI). This review aims to provide a comprehensive overview of recent advancements and strategic thinking related to this integration. This review discusses how nanomaterials facilitate signal generation and transduction, how microfluidics automates, multiplexes, and miniaturizes "all-in-one" devices, and how orthogonal CRISPR systems can enable robust multiplexing. We will also probe into the emerging application of AI to accelerate guide RNA design and optimize the performance of CRISPR biosensors. Furthermore, the roles of orthogonality and nanomaterials in real-time, continuous molecular monitoring will be assessed. The review will finally discuss the transformative future applications of high-throughput biomarker discovery and theranostics potential through massively parallelized CRISPR sensing.},
}

@article {pmid42462700,
year = {2026},
author = {Gao, Y and Wang, B},
title = {Programmable cell killer: CRISPR-Cas12a2 eliminates cells via RNA identity.},
journal = {Molecular cell},
volume = {86},
number = {14},
pages = {2662-2664},
doi = {10.1016/j.molcel.2026.06.029},
pmid = {42462700},
issn = {1097-4164},
mesh = {*CRISPR-Cas Systems ; Humans ; *RNA/genetics/metabolism ; *CRISPR-Associated Proteins/genetics/metabolism ; Saccharomyces cerevisiae/genetics ; *Gene Editing/methods ; },
abstract = {In a recent issue of Nature, Scholz et al.[1] apply the RNA-triggered DNA shredding activity of CRISPR-Cas12a2 in eukaryotic cells to enable programmable elimination of yeast and human cells expressing target transcripts with single-nucleotide resolution specificity and non-detectable off-target activity.},
}

*CRISPR-Cas Systems
Humans
*RNA/genetics/metabolism
*CRISPR-Associated Proteins/genetics/metabolism
Saccharomyces cerevisiae/genetics
*Gene Editing/methods

@article {pmid42462716,
year = {2026},
author = {Hong, A and Liu, M and Truta, A and Talaie, A and Smith, GR and Bondy-Denomy, J},
title = {Gabija restricts phage circularization and DNA replication.},
journal = {Cell host & microbe},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.chom.2026.06.016},
pmid = {42462716},
issn = {1934-6069},
abstract = {Anti-bacteriophage systems such as restriction-modification and CRISPR-Cas have DNA substrate specificity mechanisms that enable the identification of invaders. How Gabija, a highly prevalent nuclease-helicase antiphage system, limits phage replication while executing self- vs. non-self-discrimination remains unknown. Here, we show that phage-encoded DNA end-binding proteins that antagonize host RecBCD sensitize phages to Gabija. When targeting a temperate lambda-like phage in Pseudomonas aeruginosa, Gabija prevents phage genome circularization and subsequent replication. DNA end-binding complexes, including a phage exonuclease and a single-stranded DNA (ssDNA)-annealing protein or GamMu dimers that prevent loading of the host repair complex RecBCD, are necessary and sufficient to license phage and plasmid sensitivity to Gabija. Mutant escape phages lacking these DNA end-binding proteins become protected from Gabija by RecBCD translocation activities. RecBCD activity on the bacterial genome, presumably whenever it is linearized, also prevents Gabija from targeting self-DNA. Therefore, we propose that Gabija antagonizes the circularization and replication of linear DNA devoid of RecBCD as a mechanism to identify and antagonize foreign invaders.},
}

@article {pmid42463576,
year = {2026},
author = {Li, Q and Wang, H and He, Y and Hong, G and Bao, G},
title = {Gene editing of hematopoietic stem cells: applications and advances.},
journal = {International journal of hematology},
volume = {},
number = {},
pages = {},
pmid = {42463576},
issn = {1865-3774},
abstract = {Allogeneic hematopoietic stem cell transplantation remains the standard treatment for various hematologic genetic disorders resulting from single or multiple genes. However, this strategy is hindered by two main problems: failure to find a matching donor and the risk of graft-versus-host disease (GVHD) after transplantation. Recent advances in gene editing, particularly nucleases exemplified by clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) and related derivative tools, have overcome the limitations posed by the poor specificity of traditional gene modification techniques. A robust groundwork has been established for developing efficient, precise, and diverse gene editing strategies, facilitating the clinical application of ex vivo modified autologous hematopoietic stem cells (HSCs). In contrast, autologous HSC transplantation does not have the previously mentioned problems associated with allogeneic transplantation. Consequently, gene editing involving ex vivo genetic modification of HSCs and subsequent reinfusion in a single patient has emerged, with related research progressing from investigation into fundamental mechanisms and proof-of-concept studies to clinical trials.},
}

@article {pmid42463755,
year = {2026},
author = {Nevard, K and Gonzalez, E and Harvey-Samuel, T and Sanders, C and Homem, RA},
title = {CRISPR-Cas9 mediated knockout of the white gene in the bluetongue virus vector, Culicoides sonorensis (biting midge).},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {42463755},
issn = {2045-2322},
mesh = {Animals ; *Ceratopogonidae/genetics/virology ; *CRISPR-Cas Systems ; *Bluetongue virus ; Female ; *Insect Vectors/genetics/virology ; *Gene Knockout Techniques ; *Insect Proteins/genetics ; Gene Editing ; },
abstract = {Culicoides biting midges are small blood feeding insects responsible for the transmission of important arthropod-borne viruses (arboviruses) such as bluetongue virus (BTV), Schmallenberg virus (SBV) and epizootic hemorrhagic disease virus (EHDV), which cause major losses to livestock production worldwide. Culicoides sonorensis is the primary vector of BTV in North America and one of the few Culicoides species to be colonised and reared in artificial conditions. Gene editing technology has been used to explore virus-vector interactions in other vector groups, particularly within mosquitoes. Despite the availability of a reference genome since 2018, to date there have been no reports of gene editing in C. sonorensis. Here, we report the first instance of gene editing in C. sonorensis, achieved by intrathoracic injection of adult females with Cas9 and sgRNAs targeting the white gene. We generated heritable mutations in the white gene which produced both white eye and red eye phenotypes and went on to establish a homozygous knockout line carrying a single mutation. We observed gene editing efficiencies of up to 12.3%, making this an efficient protocol for genetic manipulation of Culicoides biting midges, opening the door to functional genomics studies and the development of control strategies in these important and understudied disease vectors.},
}

Animals
*Ceratopogonidae/genetics/virology
*CRISPR-Cas Systems
*Bluetongue virus
Female
*Insect Vectors/genetics/virology
*Gene Knockout Techniques
*Insect Proteins/genetics
Gene Editing

@article {pmid42463825,
year = {2026},
author = {Taha, BA and Addie, AJ and Haider, AJ and Ibnaouf, K and Arsad, N},
title = {Reversing cancer cell behavior using AI-guided CRISPR and quantum nanobiology: a systems-based approach to epigenetic reprogramming.},
journal = {Gene therapy},
volume = {},
number = {},
pages = {},
pmid = {42463825},
issn = {1476-5462},
abstract = {Treatment effectiveness is hindered by the phenotypic plasticity of cancer and the genetic complexity of tumors. However, CRISPR-Cas-based medicines face challenges with specificity, off-target effects, and tumor heterogeneity adaptability. This work investigates the possible combination of quantum biological processes, artificial intelligence, and nanomaterials to improve CRISPR gene editing and modulate or reverse selected malignant phenotypes. Quantum machine learning (QML) can be used to simulate quantum processes like electron tunneling in DNA repair and spin-dependent enzyme activity. To enable exact tumor phenotypic reversal, these models will be combined with optimization approaches powered by AI to direct CRISPR editing in oncogenic signaling networks. Graphene, gold nanoparticles, and lipid-based vectors are some of the nanomaterials that will be used as carriers to effectively and deliver CRISPR systems in a biocompatible manner to the cancer microenvironment. We hypothesize that selected homeostatic gene-expression states may be partially restored in experimental cancer models through the integration of quantum-informed AI, CRISPR gene alteration, and nanomaterial delivery. This integrated strategy could support future cancer therapies that move beyond tumor suppression toward controlled modulation of malignant cell states, although substantial preclinical and clinical validation remains necessary.},
}

@article {pmid42464282,
year = {2026},
author = {Zheng, W and Wang, M and Tu, Q and Bian, X and Zhang, Y and Wang, X},
title = {Establishment of SRLC: a multiplex genome editing technology for Saccharomyces cerevisiae and its application in metabolic engineering of malonyl-CoA pathway.},
journal = {Microbial cell factories},
volume = {},
number = {},
pages = {},
doi = {10.1186/s12934-026-03068-w},
pmid = {42464282},
issn = {1475-2859},
support = {2023YFC3402003//National Key R&D Program of China/ ; },
abstract = {The development of advanced genome engineering tools is crucial for optimizing metabolic pathways in Saccharomyces cerevisiae and achieving efficient biomanufacturing. This study proposes an enhancing multiplex genome editing strategy in S. cerevisiae by employing Escherichia coli-derived single-stranded annealing proteins (SSAPs) combined with S. cerevisiae-derived homologous recombinases (Rad51 and Rad52). The strategy utilizes an SSAP-Rad-Linearized CRISPR (SRLC) platform, which supports efficient simultaneous editing of multiple genomic loci without constructing complex multi-gRNA expression vectors. Co-overexpressing Rad51/Rad52 and E. coli SSAP proteins significantly enhances homologous recombination (HR), allowing precise multi-locus genome editing mediated by short homologous arms. Furthermore, SRLC employs a linearized CRISPR-Cas system to stimulate homologous recombination and enable counter-selection in S. cerevisiae, thereby improving precise multiplex genome editing efficiency. We applied SRLC to engineer the malonyl-CoA metabolic pathway in S. cerevisiae. Through a single round of editing and screening, we constructed a chassis strain with 9 targets simultaneously modification and achieved a 9.6-fold increase in intracellular malonyl-CoA. Using this chassis, 3-hydroxypropionic acid production increased 4.5-fold relative to wild-type S. cerevisiae. This platform offers a robust and scalable tool for S. cerevisiae manipulation and a practical pathway-engineering strategy for building for malonyl-CoA-derived factories.},
}

@article {pmid42115602,
year = {2026},
author = {Larrosa-Godall, M and Shackleford, L and Edgington, MP and Leftwich, PT and Luk, JCY and Southworth, J and Rosell, S and Creasey, JT and Aked, JM and Nevard, K and Dodds, A and Mckee, M and Adedeji, E and Gonzalez, E and Ang, JXD and Anderson, MAE and Alphey, L},
title = {Integrating multiplexing into confineable gene drives effectively overrides resistance in Anopheles stephensi.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42115602},
issn = {2041-1723},
support = {INV-008549/GATES/Gates Foundation/United States ; BBS/E/I/00007033, BBS/E/I/00007038, BBS/E/I/00007039//RCUK | Biotechnology and Biological Sciences Research Council (BBSRC)/ ; INV-008549/GATES/Gates Foundation/United States ; },
mesh = {Animals ; *Anopheles/genetics ; CRISPR-Cas Systems/genetics ; Alleles ; *Gene Drive Technology/methods ; *Mosquito Vectors/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Insecticide Resistance/genetics ; Malaria/transmission/prevention & control ; *Mosquito Control/methods ; },
abstract = {Anopheles stephensi is a major malaria vector mainly present in southern Asia and the Arabian Peninsula. Since 2012 it has invaded several countries of eastern Africa, stimulating urgent efforts to develop more efficient strategies for vector control such as CRISPR/Cas9-based homing gene drives. Target site resistance due to end-joining repair is a significant challenge to the deployment of these systems. The use of multiple sgRNAs has the potential to solve this issue. Here we perform experimental crosses to assess the homing and cutting efficiency of both classical (e.g. four adjacent sgRNAs all in one construct) and additive (e.g. separate constructs each expressing a single sgRNA) multiplexing strategies targeting the cardinal locus, in the presence and absence of a resistance allele. We find resistance alleles at one sgRNA target site can be mitigated by the presence of the additional sgRNAs with either strategy, and do not significantly reduce the homing efficiency for either strategy, validating their effectiveness. Further modelling using parameters derived from the strains generated indicates that while both strategies can overcome resistance allele formation, the fitness of the drive-carrying alleles is a critical factor in determining the overall performance and persistence of a split drive.},
}

Animals
*Anopheles/genetics
CRISPR-Cas Systems/genetics
Alleles
*Gene Drive Technology/methods
*Mosquito Vectors/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
*Insecticide Resistance/genetics
Malaria/transmission/prevention & control
*Mosquito Control/methods

@article {pmid42154535,
year = {2026},
author = {Ziegler, M and Günter, C and Alecu, JE and Xue, X and Kim, HM and Saffari, A and Davies, AK and Sahin, M and Ebrahimi-Fakhari, D},
title = {CRISPR/Cas9 loss-of-function screen in a neuronal model of AP-4 deficiency identifies ATG9A trafficking modulators.},
journal = {JCI insight},
volume = {11},
number = {14},
pages = {},
doi = {10.1172/jci.insight.202204},
pmid = {42154535},
issn = {2379-3708},
mesh = {Humans ; *Membrane Proteins/metabolism/genetics ; *Vesicular Transport Proteins/metabolism/genetics ; CRISPR-Cas Systems ; *Autophagy-Related Proteins/metabolism/genetics ; *Neurons/metabolism ; Protein Transport/genetics ; *Adaptor Protein Complex 4/genetics/deficiency/metabolism ; trans-Golgi Network/metabolism ; *Spastic Paraplegia, Hereditary/genetics/metabolism ; Loss of Function Mutation ; },
abstract = {Biallelic loss-of-function variants in adaptor protein complex 4 (AP-4) disrupt trafficking of transmembrane proteins at the trans-Golgi network, including autophagy-related protein 9A (ATG9A), leading to childhood-onset hereditary spastic paraplegia (AP-4-HSP). AP-4-HSP is characterized by features of both a neurodevelopmental and a degenerative neurological disease. To investigate the molecular mechanisms underlying AP-4-HSP and identify potential therapeutic targets, we conducted an arrayed CRISPR/Cas9 loss-of-function screen of 8,478 genes, targeting the "druggable genome," in a human neuronal model of AP-4 deficiency. Through this phenotypic screen and subsequent experiments, key modulators of ATG9A trafficking were identified, and complementary pathway analyses provided insights into the regulatory landscape of ATG9A transport. Knockdown of ANPEP and NPM1 enhanced ATG9A availability outside the trans-Golgi network, suggesting that they regulate ATG9A localization. These findings deepen our understanding of ATG9A trafficking in the context of AP-4 deficiency and offer a framework for the development of targeted interventions for AP-4-HSP.},
}

Humans
*Membrane Proteins/metabolism/genetics
*Vesicular Transport Proteins/metabolism/genetics
CRISPR-Cas Systems
*Autophagy-Related Proteins/metabolism/genetics
*Neurons/metabolism
Protein Transport/genetics
*Adaptor Protein Complex 4/genetics/deficiency/metabolism
trans-Golgi Network/metabolism
*Spastic Paraplegia, Hereditary/genetics/metabolism
Loss of Function Mutation

@article {pmid42156946,
year = {2026},
author = {di Lillo, A and Tavella, S and Iannelli, F and Crisafulli, G and Gioia, U and Trastus, LA and Cabrini, M and d'Adda di Fagagna, F},
title = {Site-specific DNA double-strand break induces local transcription in cis and protein expression.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {},
pmid = {42156946},
issn = {2399-3642},
support = {21762//Associazione Italiana per la Ricerca sul Cancro (Italian Association for Cancer Research)/ ; 22458//Associazione Italiana per la Ricerca sul Cancro (Italian Association for Cancer Research)/ ; 30471//Associazione Italiana per la Ricerca sul Cancro (Italian Association for Cancer Research)/ ; },
mesh = {*DNA Breaks, Double-Stranded ; *Transcription, Genetic ; Humans ; Promoter Regions, Genetic ; CRISPR-Cas Systems ; },
abstract = {The DNA damage response is a complex network of pathways that cells activate to safeguard genome integrity following DNA damage, including DNA double-strand breaks. We and others previously reported that RNA polymerase II, together with components of the preinitiation complex, is recruited to exposed DNA ends. This results in the assembly of a fully competent transcriptional apparatus and the synthesis of damage-induced long non-coding RNAs, which are necessary for full DNA damage response activation. Thus, DNA double-strand breaks could act as transcriptional promoters. Whether such DNA breaks, generated upstream of an open reading frame lacking a transcriptional promoter and followed by a polyadenylation signal, can induce the transcription of a coding RNA that is subsequently translated into a protein product remains unknown. Here, taking advantage of the CRISPR/Cas9 technology, we generate a sequence-specific double-strand break upstream of a promoter-less, and therefore silent, reporter gene in two distinct cellular systems. In both cell models, a DNA double-strand break is sufficient to trigger the expression of polyadenylated transcripts and a protein product. Collectively, our results demonstrate that DNA double-strand breaks can act as functional promoters capable of driving protein synthesis, revealing an additional mechanism through which DNA damage can regulate gene expression.},
}

*DNA Breaks, Double-Stranded
*Transcription, Genetic
Humans
Promoter Regions, Genetic
CRISPR-Cas Systems

@article {pmid42191711,
year = {2026},
author = {Feng, X and Ding, J and Liu, Y and Lopez Del Amo, V and Gantz, VM and Chen, XX and Champer, J and Liu, F},
title = {Self-limiting population suppression gene drive design in the West Nile vector mosquito, Culex quinquefasciatus.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42191711},
issn = {2041-1723},
support = {82372289//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82202559//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32270672//National Natural Science Foundation of China (National Science Foundation of China)/ ; MS25C140016//Natural Science Foundation of Zhejiang Province (Zhejiang Provincial Natural Science Foundation)/ ; },
mesh = {Animals ; *Culex/genetics/virology ; Female ; *Mosquito Vectors/genetics/virology ; *Gene Drive Technology/methods ; West Nile virus ; Male ; CRISPR-Cas Systems ; *West Nile Fever/transmission/virology ; Mosquito Control/methods ; Alleles ; },
abstract = {Culex quinquefasciatus is a major vector of West Nile virus and other pathogens, yet genetic population suppression tools for this species remain limited. Here, we develop a self-limiting, CRISPR-based suppression gene drive system targeting doublesex, close to the male-determining locus, promoting male transmission. A recoded dsxM sequence converts females into sterile intersexes, preventing population-level spread. The drive achieves super-Mendelian inheritance (~ 71%) and generates resistance alleles that are fully or partially dominant female sterile. Single-release cage trials show extended but self-limiting population suppression. Population modeling of this RIDD (Release of Insects carrying a Dominant-sterile Drive) system further indicates that repeated releases can substantially reduce fertile female numbers at low release ratios and intrinsic growth rates, outperforming non-drive strategies under comparable conditions. Together, these results establish a self-limiting suppression gene drive platform for Culex, providing a confined and sustainable framework for vector population control.},
}

Animals
*Culex/genetics/virology
Female
*Mosquito Vectors/genetics/virology
*Gene Drive Technology/methods
West Nile virus
Male
CRISPR-Cas Systems
*West Nile Fever/transmission/virology
Mosquito Control/methods
Alleles

@article {pmid42192102,
year = {2026},
author = {Gong, Y and Shi, S and Li, L and Qian, Y and Lu, T and Zhang, Z and Jiang, L and Liu, G and Cui, M and Li, S and Li, Z and Lin, H},
title = {PIWIL3-piRNA pathway controls rabbit oogenesis and embryogenesis via broad regulation of the transcriptome and proteome.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42192102},
issn = {2041-1723},
mesh = {Animals ; Female ; *Oogenesis/genetics ; *Piwi-Interacting RNA/metabolism/genetics ; *Argonaute Proteins/metabolism/genetics ; *Embryonic Development/genetics ; Rabbits ; Oocytes/metabolism ; *Transcriptome/genetics ; *Proteome/metabolism/genetics ; *RNA, Small Interfering/metabolism/genetics ; Humans ; Gene Expression Regulation, Developmental ; CRISPR-Cas Systems ; Fertility/genetics ; },
abstract = {Female infertility often arises from oogenic defects, yet the underlying molecular mechanisms remain elusive. The Piwi-piRNA pathway is crucial for gametogenesis, but its role in mammalian female fertility remains unclear, partly due to reliance on mouse models lacking PIWIL3. PIWIL3 exits in most other placental mammals and is highly expressed in human oocytes, but its function remains largely unexplored. Here, we show that rabbit PIWIL3 closely resembles its human counterpart and is the predominant PIWI protein in oocytes. Using CRISPR-Cas9 knockout, we demonstrate that PIWIL3 is essential for female fertility in rabbits, its loss leads to severe defects in oogenesis. Embryos lacking maternal PIWIL3 arrest by the 8-cell stage. Mechanistically, PIWIL3 binds ~18-nucleotide piRNAs, supports piRNA biogenesis, and regulates transcriptomic, proteomic, and transposable element dynamics during oocyte maturation and early embryogenesis. These findings establish PIWIL3 as an essential regulator of female fertility in non-rodent mammals, potentially including humans.},
}

Animals
Female
*Oogenesis/genetics
*Piwi-Interacting RNA/metabolism/genetics
*Argonaute Proteins/metabolism/genetics
*Embryonic Development/genetics
Rabbits
Oocytes/metabolism
*Transcriptome/genetics
*Proteome/metabolism/genetics
*RNA, Small Interfering/metabolism/genetics
Humans
Gene Expression Regulation, Developmental
CRISPR-Cas Systems
Fertility/genetics

@article {pmid42202890,
year = {2026},
author = {Li, L and Zhao, N and Ding, K and Zuo, L and Gan, Y and Liu, Y and Kong, L and Zhang, X and Zhao, Y},
title = {Development of a CRISPR/RspCas13d-based on-site rapid detection system for GII Norovirus.},
journal = {Journal of virological methods},
volume = {345},
number = {},
pages = {115421},
doi = {10.1016/j.jviromet.2026.115421},
pmid = {42202890},
issn = {1879-0984},
mesh = {*Norovirus/genetics/isolation & purification ; Humans ; Sensitivity and Specificity ; *Caliciviridae Infections/diagnosis/virology ; Feces/virology ; Rapid Diagnostic Tests ; *Gastroenteritis/virology/diagnosis ; RNA, Viral/genetics ; DNA Primers/genetics ; *CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; },
abstract = {Noroviruses (NoVs) are major cause of acute viral gastroenteritis and a serious public health concern. Current detection methods are limited in rapidity, equipment requirements, or sensitivity. In this study, we developed a rapid, sensitive, and specific detection assay for GII NoV by combining RT-RAA, T7 transcription, and the RspCas13d system. The RspCas13d protein was expressed and purified. RT-RAA primers and crRNA were designed against the conserved region of GII Nov. The assay was optimized and evaluated for specificity, sensitivity, and clinical performance. Results showed that the RT-RAA-RspCas13d method exhibited high specificity without cross-reactivity to other common enteric viruses. The limit of detection was 5 copies/μL. In clinical fecal samples, the assay showed high consistency with RT-qPCR. This method is rapid, simple, sensitive, and specific, providing a reliable tool for the rapid on-site detection of GII NoV.},
}

*Norovirus/genetics/isolation & purification
Humans
Sensitivity and Specificity
*Caliciviridae Infections/diagnosis/virology
Feces/virology
Rapid Diagnostic Tests
*Gastroenteritis/virology/diagnosis
RNA, Viral/genetics
DNA Primers/genetics
*CRISPR-Cas Systems
*Molecular Diagnostic Techniques/methods

@article {pmid42270039,
year = {2026},
author = {Krishnamurthy, KA and Xiao, R and Rutten, MGS and Bos, T and Bleeker, A and Zhang, M and de Vries, HI and Koster, M and Huijkman, N and Smit, M and Kloosterhuis, N and Boer, T and Schomakers, B and van Weeghel, M and van de Sluis, B and Wolters, JC and Bakker, BM and Oosterveer, MH},
title = {Hepatocyte-specific Cas9-mediated editing of G6pc and Slc37a4 elicits comparable biochemical and regulatory responses between glycogen storage disease (GSD) type Ia and Ib mice.},
journal = {Molecular metabolism},
volume = {110},
number = {},
pages = {102393},
pmid = {42270039},
issn = {2212-8778},
mesh = {Animals ; Mice ; *Glycogen Storage Disease Type I/metabolism/genetics ; *Hepatocytes/metabolism ; Liver/metabolism/pathology ; *Glucose-6-Phosphatase/genetics/metabolism ; Gene Editing/methods ; CRISPR-Cas Systems ; Male ; Female ; Humans ; Disease Models, Animal ; Proteomics ; *Antiporters/genetics/metabolism ; },
abstract = {BACKGROUND/OBJECTIVE: Glycogen storage disease type I (GSD I) is an autosomal recessive inborn error of carbohydrate metabolism. Patients with GSD type Ia and Ib exhibit overlapping and distinct symptoms and complications. Notably, GSD Ia patients show more severe hypertriglyceridemia and higher risk of hepatic tumors than GSD Ib patients.

METHODS: Given the liver's pivotal role in these processes, this study utilized hepatocyte-specific CRISPR/Cas9-mediated somatic gene editing to explore the pathophysiological and biochemical adaptations in hepatic GSD Ia and Ib side-by-side. Additionally, hepatic histology, transcriptomics, and proteomics analysis was performed.

RESULTS: Compared to controls, hepatic GSD Ia and Ib mice showed hepatomegaly, fasting hypoglycemia, hyperlactatemia, and increased uric acid in plasma, which was somewhat more pronounced in GSD Ia than Ib. Both GSD I subtypes showed similar reductions in hepatic acetyl-CoA precursor pool enrichment and increases in de novo biosynthesis of hepatic stearate and oleate. Interestingly, only GSD Ia mice showed mildly elevated plasma triglyceride and hepatic phosphate sugars. Metabolic changes were reflected at the transcriptomic and proteomic levels, with largely similar responses between GSD Ia and Ib livers. Moreover, altered mRNAs and protein levels related to nucleotide-binding oligomerization domain (NOD) signaling pathways, infection and inflammation, liver disease, and chemical carcinogenesis were somewhat more pronounced in hepatic GSD Ia than in GSD Ib mice.

CONCLUSIONS: Overall, the metabolic disturbance was more severe in hepatocyte-specific GSD Ia than in GSD Ib mice, consistent with the clinical phenotype in patients. The metabolic disorders and specific metabolites, genes, and proteins identified in this study provided new insights into the pathophysiological and biochemical phenotypes of GSD I subtypes in the liver.},
}

Animals
Mice
*Glycogen Storage Disease Type I/metabolism/genetics
*Hepatocytes/metabolism
Liver/metabolism/pathology
*Glucose-6-Phosphatase/genetics/metabolism
Gene Editing/methods
CRISPR-Cas Systems
Male
Female
Humans
Disease Models, Animal
Proteomics
*Antiporters/genetics/metabolism

@article {pmid42455481,
year = {2026},
author = {Marques, BS and Mendes, M and Alves, JL and Pais, A and Vitorino, C},
title = {Decoding and Overcoming Temozolomide Resistance Through CRISPR/Cas Technologies.},
journal = {Molecular diagnosis & therapy},
volume = {},
number = {},
pages = {},
pmid = {42455481},
issn = {1179-2000},
support = {2022.06174.PTDC//Fundação para a Ciência e a Tecnologia/ ; UID/PRR/00313/2025//Fundação para a Ciência e a Tecnologia/ ; UID/00313/2025//Fundação para a Ciência e a Tecnologia/ ; },
abstract = {Intrinsic and acquired resistance to temozolomide (TMZ), the standard chemotherapeutic agent for glioblastoma (GBM), is highly common and results in poor clinical outcomes. This review highlights the emerging dual role of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein (Cas) technologies in addressing this challenge. First, it examines genome-wide CRISPR screens that revealed DNA damage repair networks, stress adaptations, stemness maintenance, and tumor heterogeneity as key drivers of resistance to TMZ. Second, it examines CRISPR/Cas-based strategies, including targeted gene disruption and epigenetic silencing of O[6]-methylguanine-DNA methyltransferase (MGMT), to restore TMZ sensitivity. Finally, it explores CRISPR/Cas-engineered brain tumor models. Alongside these approaches, CRISPR/Cas technologies highlight the value of decoding the multifactorial basis of TMZ resistance and guiding rational therapeutic strategies. Continued refinement of CRISPR/Cas tools may ultimately contribute to more effective treatments for GBM.},
}

@article {pmid42455488,
year = {2026},
author = {Wu, H and Jiang, F and Tian, S},
title = {Research progress on detection technologies for Mycoplasma pneumoniae.},
journal = {European journal of clinical microbiology & infectious diseases : official publication of the European Society of Clinical Microbiology},
volume = {},
number = {},
pages = {},
pmid = {42455488},
issn = {1435-4373},
abstract = {Mycoplasma pneumoniae (M. pneumoniae) is a primary pathogen responsible for community-acquired pneumonia (CAP), particularly prevalent among children and adolescents. The recent global resurgence of infection cases, coupled with the rapid dissemination of macrolide-resistant M. pneumoniae (MRMP), underscores the critical clinical need for rapid and precise diagnostic technologies. This article systematically reviews the evolutionary trajectory of detection technologies, covering the transition from traditional culture and serological testing to modern molecular diagnostic techniques. It critically analyzes emerging platforms, including isothermal amplification, CRISPR/Cas-based diagnostics, microfluidic chips, and biosensors. It explores their potential in facilitating point-of-care testing (POCT) and simultaneous antimicrobial resistance profiling. Despite continuous technological advancements, challenges remain regarding the differentiation between active infection and colonization, as well as the balancing of cost-effectiveness. The future of M. pneumoniae diagnostics lies in the deep integration of multidisciplinary biotechnologies with artificial intelligence, aiming to construct intelligent "sample-to-answer" solutions to optimize clinical antimicrobial stewardship.},
}

@article {pmid42455881,
year = {2026},
author = {Gao, X and Hao, J and Lu, S and Wang, S and Sun, Y and Ke, X and Gao, X and Su, Y and Sun, Y and Tian, Y and Yan, W and Wang, J and Zheng, Z and Hai, R and Zhang, Q and Wang, J and Hu, W and Wang, G},
title = {Genome-wide CRISPR screen reveals PEX11B as a host restriction factor against ORFV through membrane fluidity regulation.},
journal = {PLoS pathogens},
volume = {22},
number = {7},
pages = {e1013767},
pmid = {42455881},
issn = {1553-7374},
mesh = {Animals ; Sheep ; *Membrane Fluidity ; *Host-Pathogen Interactions ; *Parvoviridae Infections/genetics/virology/metabolism/veterinary ; *Membrane Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Virus Replication ; Male ; Clustered Regularly Interspaced Short Palindromic Repeats ; Antigens, Differentiation ; },
abstract = {Host-pathogen interactions are shaped by cellular restriction factors that direct antiviral defenses. We built the first ovine genome-wide CRISPR knockout library in sheep testis (OA3.Ts) cells, targeting all protein-coding genes. Using this platform, we identified PEX11B, a peroxisomal membrane regulatory protein, as a strong restriction factor against orf virus (ORFV) infection. Removing PEX11B increased viral susceptibility and triggered severe cytopathic effects with membrane fusion and syncytia formation. Mechanistic studies showed that PEX11B knockout harmed peroxisomal integrity and disrupted lipid metabolism. This led to greater plasma membrane fluidity, creating a proviral environment that allowed more viral entry and replication. These results reveal a new antiviral function for PEX11B in blocking viral infection and underscore the importance of peroxisomal regulation in host-virus interactions.},
}

Animals
Sheep
*Membrane Fluidity
*Host-Pathogen Interactions
*Parvoviridae Infections/genetics/virology/metabolism/veterinary
*Membrane Proteins/genetics/metabolism
CRISPR-Cas Systems
Virus Replication
Male
Clustered Regularly Interspaced Short Palindromic Repeats
Antigens, Differentiation

@article {pmid42457405,
year = {2026},
author = {Hong, JF and Zou, QL and Xie, XY and Jiang, YP and Wang, SY and Ling, X and Zhou, C and Cai, X and Yang, YX and Chen, Y and Sun, W and Chen, B and Qiao, L},
title = {Engineered promoter system enables high-efficiency transgenic CRISPR editing in Malaria transmitting mosquito Anopheles sinensis.},
journal = {Zoological research},
volume = {47},
number = {4},
pages = {1045-1058},
doi = {10.24272/j.issn.2095-8137.2026.040},
pmid = {42457405},
issn = {2095-8137},
mesh = {Animals ; *Anopheles/genetics ; *Promoter Regions, Genetic/genetics ; *CRISPR-Cas Systems ; Animals, Genetically Modified ; Female ; *Gene Editing/methods ; Malaria/transmission ; *Mosquito Vectors/genetics ; },
abstract = {The binary CRISPR/Cas9 system deployed through crosses of transgenic lines facilitates efficient mutagenesis, but its application in non-model insects remains limited by the scarcity of validated species-specific regulatory elements. In the malaria vector Anopheles sinensis, we screened three germline-biased promoters (Asvasa2, Aszpg, Asnanos) for Cas9 expression, and found that Asvasa2 drove the highest editing efficiency with respect to target site mutagenesis. For gRNA transcription, comparative analysis identified AsU6-1 as the most active of four endogenous U6 promoters. Crossing stable transgenic lines harboring these components yielded F 1 progeny with complete germline editing penetrance at the Aswhite locus, a phenotype inherited in the F 2 generation. Quantitative sequencing of F 1 ovaries confirmed near-saturation (>99%) targeted mutagenesis using the optimal Asvasa2/ AsU6-1 combination, whereas alternative promoters showed markedly lower mutagenesis efficiency. Functional validation through knockout of Asdsx- F, a key sex differentiation regulator, efficiently induced complete female-to-male sexual reversal and sterility. This study provides a foundational genetic toolkit for genome engineering in this vector species, as well as an effective reference for binary transgenic manipulation in non-model insects.},
}

Animals
*Anopheles/genetics
*Promoter Regions, Genetic/genetics
*CRISPR-Cas Systems
Animals, Genetically Modified
Female
*Gene Editing/methods
Malaria/transmission
*Mosquito Vectors/genetics

@article {pmid41307817,
year = {2026},
author = {Zhang, F and Lu, Q and Qian, X and Xing, Y and Wang, W},
title = {Development of Knockout Cardiac Muscle Cell Lines Using Integrase-Deficient Lentivirus-Mediated CRISPR/Cas9 Gene Editing.},
journal = {Biochemical genetics},
volume = {64},
number = {4},
pages = {5394-5413},
pmid = {41307817},
issn = {1573-4927},
support = {82101314 to Y. X.; 81772559 to W. W//National Natural Science Foundation of China (NSFC) grants/ ; },
mesh = {*Lentivirus/genetics ; *CRISPR-Cas Systems ; *Myocytes, Cardiac/metabolism/cytology ; Animals ; *Gene Editing/methods ; *Gene Knockout Techniques/methods ; *Integrases/genetics/deficiency ; Cell Line ; Humans ; TRPM Cation Channels/genetics ; Rats ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genetic Vectors ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing technology is a highly efficient genome editing tool that can genetically disrupt genes and genetic elements, making it a timely, cost-effective, and powerful tool for studying gene function. The success of gene editing depends on the ability to introduce CRISPR components, including guide RNA (gRNA) and Cas9 nuclease, into the target cell, which is challenging in numerous difficult-to-transfect cell types, such as cardiomyocytes. Lentiviral vectors (LVs) are among the primary delivery methods for the CRISPR/Cas9 system as they can stably maintain robust expression in various dividing and non-dividing cells. However, stably integrated LVs consistently express CRISPR/Cas9 components at high levels, rendering them susceptible to off-target effects. New-generation integrase-deficient LV (IDLV) offers an attractive alternative approach for delivering CRISPR/Cas9 components. This study constructed transient receptor potential cation channel mucolipin subfamily member 1 gene knockout models in H9C2 cell lines using IDLVs. Strategies for gRNA design and screening, the IDLV packaging process, CRISPR delivery, and knockout validation are outlined. These protocols will assist researchers in the application of CRISPR technology to study gene function in mammalian cells.},
}

*Lentivirus/genetics
*CRISPR-Cas Systems
*Myocytes, Cardiac/metabolism/cytology
Animals
*Gene Editing/methods
*Gene Knockout Techniques/methods
*Integrases/genetics/deficiency
Cell Line
Humans
TRPM Cation Channels/genetics
Rats
RNA, Guide, CRISPR-Cas Systems/genetics
Genetic Vectors

@article {pmid42020342,
year = {2026},
author = {He, Z and Liu, M and Zhang, N and Yan, J and Li, F and Zhao, P and Guo, C},
title = {A CRISPR-Cas9 screen identifies LAPTM4A (lysosomal protein transmembrane 4 alpha) as a key host barrier against PRRSV infection.},
journal = {Autophagy},
volume = {22},
number = {8},
pages = {1882-1902},
doi = {10.1080/15548627.2026.2664607},
pmid = {42020342},
issn = {1554-8635},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Autophagy/genetics ; Lysosomes/metabolism ; *Porcine respiratory and reproductive syndrome virus/physiology ; Swine ; *Porcine Reproductive and Respiratory Syndrome/virology/genetics/metabolism ; Humans ; Host-Pathogen Interactions/genetics ; Virus Replication ; Signal Transduction ; Ubiquitination ; HEK293 Cells ; Sequestosome-1 Protein/metabolism ; },
abstract = {Porcine reproductive and respiratory syndrome virus (PRRSV) manipulates host intracellular processes, particularly macroautophagy/autophagy and lysosomal function, to facilitate its replication and spread. However, the precise host factors and molecular mechanisms by which PRRSV remodels the autophagy-lysosome axis remain poorly defined. Here, we performed a CRISPR-Cas9 knockout screen targeting 1,332 genes involved in protein degradation, metabolism, and vesicular trafficking, and identified LAPTM4A (lysosomal protein transmembrane 4 alpha) as a critical antiviral factor involved in the lysosomal pathway. A yeast two-hybrid screen identified LAPTM4A as an interactor of PRRSV GP5 (glycoprotein 5). Mechanistically, GP5 recruits the E3 ubiquitin ligase NEDD4 and the autophagy receptor SQSTM1/p62 to promote K63-linked polyubiquitination of LAPTM4A, leading to its autophagic degradation. This selective degradation activates the AMPK-ULK1-MAP1LC3/LC3 signaling cascade, initiating autophagy while facilitating MTOR-lysosome colocalization, thereby suppressing TFEB nuclear translocation and transcription of lysosome-related genes. The resulting incomplete autophagic flux enhances viral replication. Additionally, in terms of host defense, LAPTM4A maintains lysosomal homeostasis by restraining excessive autophagy through AMPK-ULK1-LC3 signaling and promoting TFEB-dependent lysosomal gene expression by impairing the binding of RPTOR/raptor to MTOR, thus providing broad antiviral protection against multiple RNA viruses. Collectively, our findings identify LAPTM4A as a central regulator of lysosome-autophagy homeostasis and reveal a viral strategy that dismantles this defense axis to facilitate infection.Abbreviations: ATG5: autophagy related 5; AMPK: adenosine 5'-monophosphate (AMP)-activated protein kinase; Baf A1: bafilomycin A1; CHX: cycloheximide; Co-IP: co-immunoprecipitation; DMVT library: protein degradation, metabolism, and vesicular trafficking library; LAPTM4A: lysosomal protein transmembrane 4 alpha; MAGeCK: model-based analysis of genome-wide CRISPR-Cas9 knockout; MOI: multiplicity of infection; MTOR: mechanistic target of rapamycin kinase; NC: negative control; PAMs: porcine alveolar macrophages; PRKAA/AMPKα: protein kinase AMP-activated catalytic subunit alpha; PRRSV: porcine reproductive and respiratory syndrome virus; qRT-PCR: quantitative real-time PCR; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; TCID50: 50% tissue culture infective dose; TFEB: transcription factor EB; Ub: ubiquitin; ULK1: unc-51 like autophagy activating kinase 1; WT: wild type.},
}

Animals
*CRISPR-Cas Systems/genetics
Autophagy/genetics
Lysosomes/metabolism
*Porcine respiratory and reproductive syndrome virus/physiology
Swine
*Porcine Reproductive and Respiratory Syndrome/virology/genetics/metabolism
Humans
Host-Pathogen Interactions/genetics
Virus Replication
Signal Transduction
Ubiquitination
HEK293 Cells
Sequestosome-1 Protein/metabolism

@article {pmid42283463,
year = {2026},
author = {Sugiokto, FG and Liu, Y and Li, R},
title = {Proteomic screening identifies HNRNPA2B1 as an epigenetic repressor of Epstein-Barr virus reactivation.},
journal = {Journal of virology},
volume = {100},
number = {7},
pages = {e0061326},
doi = {10.1128/jvi.00613-26},
pmid = {42283463},
issn = {1098-5514},
mesh = {Humans ; *Herpesvirus 4, Human/physiology/genetics ; *Heterogeneous-Nuclear Ribonucleoprotein Group A-B/metabolism/genetics ; *Epigenesis, Genetic ; Proteomics/methods ; *Virus Activation/genetics ; Histone Demethylases/metabolism/genetics ; Promoter Regions, Genetic ; Gene Expression Regulation, Viral ; Trans-Activators/genetics/metabolism ; Virus Latency/genetics ; *Epstein-Barr Virus Infections/virology/genetics/metabolism ; Histones/metabolism ; Immediate-Early Proteins/genetics/metabolism ; Chromatin Immunoprecipitation ; CRISPR-Cas Systems ; Host-Pathogen Interactions ; Cell Line, Tumor ; },
abstract = {Epstein-Barr virus (EBV) establishes lifelong persistent infection in over 90% of the world's population. The virus persists as an episome in the host cells during latency and periodically reactivates through transcriptional activation of the immediate-early (IE) genes. While epigenetic regulation is central to maintaining viral latency, the host factors that enforce repression at these promoters remain incompletely defined. Here, we employed a novel Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-based engineered DNA-binding molecule-mediated chromatin immunoprecipitation coupled with mass spectrometry (enChIP-MS) approach to identify proteins associated with the promoter of EBV IE gene ZTA. This approach revealed an enrichment of multiple heterogeneous nuclear ribonucleoproteins and identified HNRNPA2B1 as a potential regulator of EBV ZTA gene expression. Functional analyses across multiple EBV+ cancer cell models demonstrated that HNRNPA2B1 acts as a restriction factor for EBV lytic reactivation. Depletion of HNRNPA2B1 led to increased expression of IE and downstream lytic genes, enhanced RNA polymerase II recruitment to the ZTA and RTA promoters, and elevated the proportion of cells entering the lytic cycle. Conversely, enforced expression of HNRNPA2B1 suppressed EBV lytic reactivation. Mechanistically, HNRNPA2B1 enhances repressive viral chromatin states by facilitating recruitment of the histone demethylase LSD1 to EBV IE gene promoters, thereby limiting the activating histone H3 lysine 4 trimethylation. Together, these findings identify HNRNPA2B1 as a key epigenetic regulator of EBV latency and link RNA-binding proteins to epigenetic control of viral reactivation.IMPORTANCEThis study identifies HNRNPA2B1 as a previously unrecognized host factor that promotes Epstein-Barr virus (EBV) latency through direct regulation of viral chromatin at immediate-early gene promoters. By integrating locus-specific chromatin proteomics with functional and mechanistic analyses, our work reveals how an RNA-binding protein HNRNPA2B1 recruits a histone-modifying enzyme to control EBV reactivation. These findings provide new insights into host-virus interactions that control EBV latency and reactivation and highlight the role of RNA-binding proteins in chromatin regulation that may be broadly relevant to other latent DNA viruses.},
}

Humans
*Herpesvirus 4, Human/physiology/genetics
*Heterogeneous-Nuclear Ribonucleoprotein Group A-B/metabolism/genetics
*Epigenesis, Genetic
Proteomics/methods
*Virus Activation/genetics
Histone Demethylases/metabolism/genetics
Promoter Regions, Genetic
Gene Expression Regulation, Viral
Trans-Activators/genetics/metabolism
Virus Latency/genetics
*Epstein-Barr Virus Infections/virology/genetics/metabolism
Histones/metabolism
Immediate-Early Proteins/genetics/metabolism
Chromatin Immunoprecipitation
CRISPR-Cas Systems
Host-Pathogen Interactions
Cell Line, Tumor

@article {pmid42313514,
year = {2026},
author = {Xu, Y and Kou, S and Huang, X and Cui, D and Guo, Y and Sun, F},
title = {CRISPR-Based Programmable RNA-Responsive Protein Materials.},
journal = {ACS macro letters},
volume = {15},
number = {7},
pages = {1005-1012},
doi = {10.1021/acsmacrolett.6c00237},
pmid = {42313514},
issn = {2161-1653},
mesh = {Pseudomonas aeruginosa ; *CRISPR-Cas Systems ; Hydrogels/chemistry ; *RNA, Viral/analysis/genetics ; Biofilms ; },
abstract = {With the rapid expansion of RNA biology and associated biotechnologies, smart materials with programmable RNA responsiveness offer immense opportunities for biosensing, diagnostics, and therapeutics. Here, we present a programmable RNA-responsive protein material system leveraging CRISPR-Cas7-11, an RNA-guided protease complex. By immobilizing the protease complex and cleavable payload proteins onto protein scaffolds via SpyTag/SpyCatcher chemistry, we developed two platforms: (1) synthetic spider-silk fibers and (2) protein hydrogels. These materials enable sequence-specific RNA detection, triggering the controlled release of payloads such as GFP or the biofilm-degrading enzyme PslG. Applications demonstrated include viral RNA sensing and Pseudomonas aeruginosa detection with targeted biofilm degradation.},
}

Pseudomonas aeruginosa
*CRISPR-Cas Systems
Hydrogels/chemistry
*RNA, Viral/analysis/genetics
Biofilms

@article {pmid42424186,
year = {2026},
author = {Yang, Y and Pan, Q and Liu, M and Zeng, L and Zou, H and Liu, S and Fu, S and Xu, Y and Dai, Z and Fang, S and Pan, Y},
title = {Pan-Cancer Liquid Biopsy and Treatment Monitoring via a Split crRNA-Activated Label-Free CRISPR/Cas12a Platform for Ultrasensitive MicroRNA Detection.},
journal = {Analytical chemistry},
volume = {98},
number = {28},
pages = {20968-20977},
doi = {10.1021/acs.analchem.6c02601},
pmid = {42424186},
issn = {1520-6882},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *MicroRNAs/blood/genetics/analysis ; Liquid Biopsy/methods ; Limit of Detection ; *Neoplasms/diagnosis ; Biosensing Techniques/methods ; Cell Line, Tumor ; },
abstract = {Liquid biopsy based on circulating microRNAs (miRNAs) holds great promise for cancer diagnosis and treatment monitoring. However, the development of detection methods that are sensitive, specific, cost-effective, and compatible with diverse biofluids remains a challenge. Here, we report a sensitive and label-free detection platform, termed SCAN (Split crRNA-Activated CRISPR/Cas12a and Amplification Network), that integrates split CRISPR/Cas12a with catalytic hairpin assembly (CHA) for isothermal miRNA analysis. In this design, the target miRNA, serving as an alterable spacer RNA (sRNA), assembles with a conserved repeat RNA (rRNA) to reconstitute a functional full-length crRNA, activating the trans-cleavage activity of Cas12a. This cleaves a blocker probe and releases an initiator strand, which subsequently triggers a CHA cascade. The CHA reaction generates abundant G-quadruplex (G4) structures that bind specifically to N-methylmesoporphyrin IX (NMM), yielding a strong turn-on fluorescence signal. The optimized "signal-on" model achieved a detection limit of 2 fM for miR-21, offering approximately 5 orders of magnitude higher sensitivity than the basic split CRISPR/Cas12a system. The platform exhibited excellent specificity, capable of single-base mismatch discrimination, and could be readily adapted for detecting miR-128, miR-27a, and miR-155 through simple exchange of the double-stranded DNA activator. Importantly, by employing the label-free G4/NMM reporter, the cost of the signaling module was reduced by more than 45-fold compared to conventional dual-labeled probes. The SCAN platform reliably quantified miR-21 overexpression in colon cancer cell lines and robustly differentiated plasma samples from patients with multiple cancer types (colorectal, lung, cervical, breast, and thyroid cancers) from healthy individuals. Furthermore, it demonstrated utility in tracking treatment response through noninvasive urine analysis in prostate cancer and bladder cancer. This work establishes a sensitive, specific, low-cost, and versatile biosensing platform for miRNA-based liquid biopsy, holding strong potential for clinical diagnostic applications.},
}

Humans
*CRISPR-Cas Systems/genetics
*MicroRNAs/blood/genetics/analysis
Liquid Biopsy/methods
Limit of Detection
*Neoplasms/diagnosis
Biosensing Techniques/methods
Cell Line, Tumor

@article {pmid42424600,
year = {2026},
author = {Li, X and Gao, X and Gu, T and Li, Q and Wang, L and Deng, F and Guo, M and Huo, D and Hou, C},
title = {Tribos: A Modular Hairpin-Enhanced CRISPR/Cas12a Biosensor for Ultrasensitive Detection of HER2 Protein.},
journal = {Analytical chemistry},
volume = {98},
number = {28},
pages = {21055-21063},
doi = {10.1021/acs.analchem.6c03581},
pmid = {42424600},
issn = {1520-6882},
mesh = {Humans ; *Erb-b2 Receptor Tyrosine Kinases/analysis/genetics ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Female ; Breast Neoplasms/diagnosis ; Limit of Detection ; Aptamers, Nucleotide/chemistry/genetics ; Molecular Docking Simulation ; *Bacterial Proteins/genetics ; *Endodeoxyribonucleases/genetics/metabolism ; Inverted Repeat Sequences ; CRISPR-Associated Proteins ; },
abstract = {Accurate detection of human epidermal growth factor receptor 2 (HER2) is critical for early breast cancer screening and personalized therapy. This study constructed a target-triggered, hairpin-enhanced CRISPR/Cas12a biosensor named "Tribos" for ultrasensitive HER2 detection. The system integrates an aptamer hairpin switch (HAS), HAS-allosterically triggered rolling circle amplification (RCA), and a hairpin-enhanced CRISPR/Cas12a fluorescence reporter module. Taking advantage of Cas12a's high affinity for stem-loop structures, we designed a double-stem-loop reporter probe (DS-FQ) and validated its trans-cleavage enhancement mechanism via molecular docking. Under optimal conditions, Tribos exhibited a linear range from 10 fg/mL to 10 ng/mL, with a limit of detection as low as 1.08 fg/mL. In clinical validation with 29 breast cancer patients and 13 healthy controls, the sensor achieved a sensitivity of 82.76% and a specificity of 100%, which were highly consistent with clinical diagnoses and ELISA results, and it effectively distinguished different HER2 expression levels. The modular design of Tribos offers a new strategy for high-performance CRISPR diagnostics and lays a foundation for next-generation molecular diagnostic technologies based on nucleic acid conformational regulation.},
}

Humans
*Erb-b2 Receptor Tyrosine Kinases/analysis/genetics
*Biosensing Techniques/methods
*CRISPR-Cas Systems/genetics
Female
Breast Neoplasms/diagnosis
Limit of Detection
Aptamers, Nucleotide/chemistry/genetics
Molecular Docking Simulation
*Bacterial Proteins/genetics
*Endodeoxyribonucleases/genetics/metabolism
Inverted Repeat Sequences
CRISPR-Associated Proteins

@article {pmid42451761,
year = {2026},
author = {Yilmaz, I and Yoğurtçu, BM and Aisida, S and Ezer, EB},
title = {Next-Generation Strategies to Encounter Antimicrobial Resistance (AMR): From Lariocidin to Gene Editing and Nanotechnology-Based Approaches.},
journal = {Molecules (Basel, Switzerland)},
volume = {31},
number = {13},
pages = {},
pmid = {42451761},
issn = {1420-3049},
mesh = {*Gene Editing/methods ; *Anti-Bacterial Agents/pharmacology/chemistry ; *Nanotechnology/methods ; *Drug Resistance, Bacterial/drug effects/genetics ; Humans ; Biofilms/drug effects ; CRISPR-Cas Systems ; Nanoparticles/chemistry ; },
abstract = {The escalation of antimicrobial resistance (AMR) represents a serious global threat to public health, with AMR-associated mortality estimated to increase by 70% by 2050. As pathogens evolve through enzymatic inactivation, target modification, efflux-mediated clearance, biofilm formation, and broader genetic adaptation, conventional therapies are increasingly compromised, while the antibiotic development pipeline remains critically constrained by high discovery and development costs, weak commercial incentives, and the escalating complexity of resistance mechanisms. This review comprehensively synthesizes advanced pharmacological and biotechnological innovations designed to circumvent these entrenched resistance mechanisms. We highlight the development of novel therapeutic classes, particularly lariocidin, which disrupts bacterial protein synthesis via a previously unexploited ribosomal-binding site. Moreover, we critically evaluate molecular interventions, emphasizing CRISPR/Cas-based gene silencing and genome editing as precise tools to neutralize specific resistance determinants, such as the mecA gene in methicillin-resistant Staphylococcus aureus (MRSA). Concurrently, we explore the integration of engineered nanoparticles to revitalize existing antimicrobials by overcoming biofilm barriers, improving drug solubility, and enabling targeted delivery. Collectively, mastering the evolving AMR landscape requires a multidimensional framework that seamlessly integrates these novel molecular targets with advanced rapid diagnostics and robust international governance.},
}

*Gene Editing/methods
*Anti-Bacterial Agents/pharmacology/chemistry
*Nanotechnology/methods
*Drug Resistance, Bacterial/drug effects/genetics
Humans
Biofilms/drug effects
CRISPR-Cas Systems
Nanoparticles/chemistry

@article {pmid42452230,
year = {2026},
author = {Song, S and Fan, X and Zhang, N and Lin, N and Wang, G},
title = {Molecular Crosstalk Between Flowering Time and Drought Adaptation in Cereal Crops.},
journal = {Plants (Basel, Switzerland)},
volume = {15},
number = {13},
pages = {},
pmid = {42452230},
issn = {2223-7747},
support = {32501990//National Natural Science Foundation of China/ ; 242300421572//Natural Science Foundation of Henan Province/ ; 242300421571//Natural Science Foundation of Henan Province/ ; },
abstract = {Increasingly frequent and severe drought events restrict global agricultural productivity. As sessile organisms, cereal crops have evolved phenotypic plasticity, drawing on drought escape (DE) and drought avoidance (DA) strategies to balance survival and reproduction. While the mechanisms governing photoperiodic flowering and drought responses are well characterized individually, their molecular intersection remains poorly understood. This review summarizes recent advances in the crosstalk between these two pathways. We highlight the divergent roles of core genetic hubs, such as florigen regulation, GIGANTEA (GI), DELLA proteins, and dual-function transcription factors (e.g., ZmCCT, Ghd7, Ppd-H1), and the breeding-selected alleles, including Green Revolution variants, that can partly uncouple stress tolerance from developmental penalties, though trade-offs often remain. Furthermore, we examine the internal networks driving this crosstalk, including circadian clock phase shifts, sugar and energy signaling through the trehalose-6-phosphate (T6P)-SNF1-related protein kinase 1 (SnRK1) module, and the antagonistic balance within phytohormone networks centered on abscisic acid (ABA). Finally, we propose that integrating epigenetic stress memory, systemic root-to-shoot signaling, and targeted CRISPR/Cas promoter engineering provides a useful conceptual framework for breeding climate-resilient, yield-stable crops.},
}

@article {pmid42452277,
year = {2026},
author = {Kim, HJ and Chae, J and Han, SJ and Kim, JH and Chung, YS and Karthik, S and Heo, JB},
title = {AI-Guided DNA-Free and Genotype-Independent Genome Editing for Soybean Improvement.},
journal = {Plants (Basel, Switzerland)},
volume = {15},
number = {13},
pages = {},
pmid = {42452277},
issn = {2223-7747},
abstract = {Soybean is a strategic crop for global protein and vegetable oil supply chains; however, genetic improvement remains constrained by genotype-dependent regeneration, variable transformation efficiency, and regulatory concerns regarding stable transgene integration. This review synthesizes emerging DNA-free and genotype-independent genome-editing frameworks for soybean, where genotype independence is defined as the ability to recover fertile, non-chimeric edited plants across elite germplasm. We critically examine the soybean genome-editing toolbox, including CRISPR-Cas9, Cas12a, multiplex editing systems, base editing, and prime editing, and discuss persistent bottlenecks associated with target selection, off-target assessment, editability, and plant recovery. Particular emphasis is placed on artificial intelligence (AI)-assisted approaches that integrate genomic, epigenomic, chromatin-accessibility, and multi-omics datasets to improve target prioritization, guide RNA design, off-target prediction, and locus- and genotype-specific editability assessment. We further evaluate DNA-free genome-editing technologies, including CRISPR-Cas ribonucleoproteins, transient RNA-based systems, and nanocarrier-mediated delivery platforms, highlighting their potential to generate non-integrative edits while reducing prolonged nuclease exposure. In addition, we discuss regeneration reprogramming strategies based on developmental regulators and morphogenic modules, including BBM-WUS, GRF-GIF, de novo meristem induction, and somatic embryogenesis, as enabling technologies for overcoming cultivar-dependent regeneration barriers. Importantly, this review proposes an integrated AI-to-field framework that connects target discovery, editability prediction, DNA-free editing, regeneration reprogramming, phenotypic validation, and breeding deployment into a unified soybean improvement pipeline. We further highlight emerging opportunities in multi-omics-guided target discovery, genotype-aware prediction models, regeneration-aware editing strategies, and closed-loop machine-learning systems that continuously improve editing decisions through experimental feedback. Collectively, these convergent innovations provide a practical foundation for accelerating the development of climate-resilient, nutritionally enhanced, and industry-ready soybean cultivars.},
}

@article {pmid41872727,
year = {2026},
author = {Sivaprakasam, M and Jeanpierre, AR and Mohammed, S and Srinivasan, R and Mohanty, AK},
title = {Zebrafish and CRISPR-A synergistic approach to decipher and cure human diseases.},
journal = {Animal models and experimental medicine},
volume = {9},
number = {6},
pages = {1167-1179},
doi = {10.1002/ame2.70141},
pmid = {41872727},
issn = {2576-2095},
mesh = {Animals ; *Zebrafish/genetics ; Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Disease Models, Animal ; Genetic Diseases, Inborn/therapy/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Rapidly emerging infectious and genetic diseases demand robust vertebrate models to investigate pathogenesis and accelerate therapeutic discovery. Zebrafish (Danio rerio) offer substantial translational value owing to their conserved physiology, optical transparency, rapid reproduction, and the presence of orthologs for approximately 70% of human genes and approximately 82% of disease-associated genes. The integration of CRISPR/Cas9 technology has transformed zebrafish research, enabling efficient generation of targeted knockouts, knockins, and high-throughput mutagenesis screens. This synergy supports mechanistic dissection and modeling of cardiovascular, oncologic, viral, and other genetic disorders. Despite these advantages, rigorous allele validation, consideration of paralog redundancy, maternal contribution, and off-target analysis remain essential to ensure translational accuracy. This review summarizes current applications, methodological advances, limitations, and best-practice recommendations for combining zebrafish models with genome editing to improve understanding and treatment of human diseases.},
}

Animals
*Zebrafish/genetics
Humans
*Gene Editing/methods
*CRISPR-Cas Systems
*Disease Models, Animal
Genetic Diseases, Inborn/therapy/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42185265,
year = {2026},
author = {Li, XQ and Li, XY and Chen, WF and Xu, ZY and Liu, ZQ and Wang, Y and Zhang, JY and Gu, YY and Yao, L and Tan, YF and Chen, XJ and Deng, B and Wang, KH and Xu, JQ and He, MJ and Geng, ZH and Fan, KY and Zhang, ZC and Wang, L and Xiang, AY and Pan, HT and Hu, ZB and Xie, YL and Wang, C and Zhou, PH and Li, QL},
title = {A frameshift variant in FAM129C contributes to achalasia through B cell responses against the GABAA receptor.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42185265},
issn = {2041-1723},
support = {82570629//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82500618//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Animals ; *Esophageal Achalasia/genetics/immunology/pathology ; *Frameshift Mutation ; Mice ; *B-Lymphocytes/immunology/metabolism ; Disease Models, Animal ; Humans ; Female ; Esophageal Sphincter, Lower/immunology/pathology ; Male ; CRISPR-Cas Systems ; Autoantibodies/immunology ; Lymphocyte Activation ; Mice, Inbred C57BL ; },
abstract = {Achalasia is a rare esophageal motility disorder of poorly understood etiology. Here, we perform a large trio-based whole-genome sequencing study of achalasia and identify a recessively inherited frameshift variant in FAM129C (p.Ala454fs). A CRISPR/Cas9-engineered Fam129c-mutant mouse model recapitulating key features of achalasia was established, including growth retardation, elevated lower esophageal sphincter (LES) pressure, and selective loss of inhibitory neurons. Multi-omic analyses revealed substantial B cell expansion and activation within the LES, accompanied by enhanced humoral immune responses. Time-course experiments demonstrated that B cell accumulation preceded overt neuronal loss, while B cell depletion via anti-CD20 antibodies or intravenous immunoglobulin treatment partially rescued the phenotypes. Further protein profiling and cell-based assays suggested that the GABAA receptor may represent one potential neuronal antigen targeted by circulating autoantibodies. Together, these findings identify FAM129C as a genetic contributor to achalasia and support a neuroimmune mechanism in which B cell activation and autoantibody-mediated responses contribute to inhibitory neuronal injury. These results provide important insights into achalasia pathogenesis and highlight the potential of immunomodulatory strategies for disease intervention in the early stage.},
}

Animals
*Esophageal Achalasia/genetics/immunology/pathology
*Frameshift Mutation
Mice
*B-Lymphocytes/immunology/metabolism
Disease Models, Animal
Humans
Female
Esophageal Sphincter, Lower/immunology/pathology
Male
CRISPR-Cas Systems
Autoantibodies/immunology
Lymphocyte Activation
Mice, Inbred C57BL

@article {pmid42225068,
year = {2026},
author = {Fenoglio, S and Yu, Y and Tepper, J and Grove, L and Bejnood, A and Meier, SR and Choi, AH and Wu, HJ and Devault, A and Liu, S and Shen, B and Khendu, T and Stowe, H and Uijttewaal, ECH and Zhang, M and Haines, BB and Wilker, E and Huang, A and Schramek, D and Elling, U and Pan, X and Andersen, JN and Teng, T},
title = {Temporal control of sgRNA library activation unlocks large-scale in vivo CRISPR screens.},
journal = {Cell reports methods},
volume = {6},
number = {7},
pages = {101470},
doi = {10.1016/j.crmeth.2026.101470},
pmid = {42225068},
issn = {2667-2375},
mesh = {Humans ; Animals ; *Gene Library ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cell Line, Tumor ; *CRISPR-Cas Systems/genetics ; Mice ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Reproducibility of Results ; },
abstract = {CRISPR-StAR (stochastic activation by recombination) is an inducible pooled screening system that activates gene knockout after tumor engraftment and provides matched internal controls for guide-level normalization. In this study, we explore the scalability and reproducibility of this approach for in vivo cancer screens. Through barcode-embedded sequencing and the development of a Bayesian analysis pipeline, we screened a 30,000-sgRNA library in A549 xenografts, achieving reproducible dropout and enrichment phenotypes using just ∼30 tumors. Across additional xenograft models, single tumors yielded reliable functional annotation for ∼1,000 genes. Comparing in vivo and in vitro screens uncovered tumor suppressor effects detectable only in vivo; for example, KMT2C and KMT2D knockouts produced contrasting growth and transcriptional programs. Together with our R analysis package, we show that CRISPR-StAR enables scalable in vivo dependency mapping that complements in vitro resources and reduces animal use by up to 7-fold versus conventional dropout screens, improving methodological rigor at genome-scale clonal resolution.},
}

Humans
Animals
*Gene Library
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Cell Line, Tumor
*CRISPR-Cas Systems/genetics
Mice
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
Reproducibility of Results

@article {pmid42391025,
year = {2026},
author = {Bakhtiyari, N and Masoudi-Sobhanzadeh, Y and Farajnia, S and Kumar, S},
title = {An interpretable deep learning framework uncovers features governing CRISPR-Cas9 genome-editing efficiency.},
journal = {Bioinformatics (Oxford, England)},
volume = {42},
number = {7},
pages = {},
doi = {10.1093/bioinformatics/btag483},
pmid = {42391025},
issn = {1367-4811},
support = {//Princess Margaret Cancer Foundation, Canada Research Chair Program/ ; //Terry Fox Research Institute/ ; 7130//Drug Applied Research Center, Tabriz University of Medical Sciences/ ; },
mesh = {*Deep Learning ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genome ; *Computational Biology/methods ; *Gene Editing/methods ; },
abstract = {MOTIVATION: CRISPR-Cas9 genome-editing efficiency is strongly influenced by the sequence composition and positional context of single-guide RNAs (sgRNAs). Although numerous deep learning-based models have been developed to predict Cas9 efficiency from sgRNA sequences, most operate as black boxes, offering limited insight into the sequence determinants underlying Cas9 activity. In addition, previous studies often overlook how the positional context of sequence motifs within sgRNAs influences their effects on Cas9 binding or cleavage.

RESULTS: We introduce DeepCC9, an interpretable machine learning framework that combines explicit sequence feature extraction with a residual block-based deep architecture to improve interpretability and identify composition- and position-based motifs governing Cas9 genome-editing efficiency. We applied this method to multiple Cas9 variant datasets, achieving superior predictive performance compared with existing methods while enabling direct interpretation of sequence motifs and their positional effects. Our analysis uncovered 74 sequence motifs enriched or depleted at specific positions within sgRNAs and strongly associated with Cas9 efficiency, providing mechanistic insight into sequence features that influence guide performance. Together, these results establish DeepCC9 as a generalizable and interpretable framework for modeling sequence-function relationships and advancing the understanding of the sequence determinants underlying CRISPR-Cas9 genome editing.

The authors have implemented their algorithm in the Python programming language (version 3.X), which is accessible using (https://zenodo.org/records/20073890).},
}

*Deep Learning
*CRISPR-Cas Systems
RNA, Guide, CRISPR-Cas Systems/genetics
Genome
*Computational Biology/methods
*Gene Editing/methods

@article {pmid42450220,
year = {2026},
author = {Haratau, JIC and Niculescu, LS and Barbalata, T and Sanda, GM and Fuior, EV and Sasson, S and Sima, AV and Stancu, CS and Toma, L},
title = {Longstanding Transcriptional Activation of APOA1 and PON1 in Human Hepatocytes by CRISPR/dCas9 Technology: Transcriptomic Profile and Crosstalk with Endothelial Cells.},
journal = {International journal of molecular sciences},
volume = {27},
number = {13},
pages = {},
pmid = {42450220},
issn = {1422-0067},
support = {PNRR-III-C9-2022-I8-197//EU - PNRR Programme/ ; PN-IV-P6-6.1-CoEx-2024-0029//Ministry of Education and Research, CCCDI - UEFISCDI/ ; },
mesh = {Humans ; *Apolipoprotein A-I/genetics/metabolism ; *Aryldialkylphosphatase/genetics/metabolism ; *Hepatocytes/metabolism ; Human Umbilical Vein Endothelial Cells/metabolism ; *Transcriptional Activation ; *Transcriptome ; *CRISPR-Cas Systems ; Oxidative Stress ; Tumor Necrosis Factor-alpha ; Endothelial Cells/metabolism ; Gene Expression Profiling ; },
abstract = {Apolipoprotein A1 (APOA1) and paraoxonase 1 (PON1) are key proteins of high-density lipoproteins (HDL). The aim of the present study was to obtain and characterize an in vitro model for endogenous APOA1 and PON1 longstanding upregulation in hepatocytes that can be further used to decipher the mechanisms of their protective action. Cultured human hepatocytes (HuH-7 cell line) were transfected with CRISPR/dCas9 activation plasmids targeting APOA1/PON1 genes. Following selection with specific antibiotics, RNA sequencing was used for the transcriptomic characterization of the transfected hepatocytes. The functionality of the secreted APOA1/PON1 was evaluated as the capacity of the conditioned medium (CM) from transfected HuH-7 to modulate the oxidative and inflammatory stress in TNFα-activated primary human umbilical endothelial cells (HUVEC). The results showed that: (1) a robust, longstanding upregulation (46 days) of endogenous APOA1/PON1 was obtained after CRISPR/dCas9 transfection and antibiotics selection; (2) APOA1/PON1 upregulation led to a modified transcriptomic profile and increased the expression of several antioxidant genes in transfected hepatocytes as demonstrated by RNAseq analysis; (3) secreted APOA1/PON1 were functional as demonstrated by the CM ability to reduce the levels of reactive oxygen species and inflammatory markers (VCAM-1, MCP-1) in TNFα-activated HUVEC. In conclusion, we achieved an experimental model of successful longstanding upregulation of endogenous APOA1 and PON1 in human hepatocytes. The targeted proteins are secreted in a functional form and can be used for deciphering their complex mechanism of protective action in various pathological conditions.},
}

Humans
*Apolipoprotein A-I/genetics/metabolism
*Aryldialkylphosphatase/genetics/metabolism
*Hepatocytes/metabolism
Human Umbilical Vein Endothelial Cells/metabolism
*Transcriptional Activation
*Transcriptome
*CRISPR-Cas Systems
Oxidative Stress
Tumor Necrosis Factor-alpha
Endothelial Cells/metabolism
Gene Expression Profiling

@article {pmid42450255,
year = {2026},
author = {Mais, R and Kumar, A and Ahmetaj, A and Burgos-Crespo, G and Sanchez, MM and Roxas, DC and Dcosta, C and Ilyas, A and Hadjiargyrou, M and Zanganeh, S},
title = {Nanoengineering Systems for Gene Therapy: Mechanisms, Modalities, and Future Directions.},
journal = {International journal of molecular sciences},
volume = {27},
number = {13},
pages = {},
pmid = {42450255},
issn = {1422-0067},
mesh = {Humans ; *Genetic Therapy/methods ; *Gene Editing/methods ; *Nanotechnology/methods ; Animals ; CRISPR-Cas Systems ; Nanostructures/chemistry ; Gene Transfer Techniques ; },
abstract = {Nanotechnology has become an important platform in the fields of gene therapy and genome editing, providing delivery strategies that address persistent therapeutic challenges by improving the precision, efficiency, and safety of genetic modifications. This review highlights the central role of nanomaterials in overcoming persistent barriers to genetic interventions, including inefficient delivery, instability of genetic cargo, and off-target effects. Specifically, we emphasize the combined use of nanomaterials with clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) systems, which can improve editing specificity and therapeutic efficacy. Beyond the classical CRISPR/Cas9 platform, this review also discusses next-generation modalities such as base editors, Cas13, prime editing, and the recently described Tandem Interspaced Guide RNA and TIGR-associated protein (TIGR-Tas) system, while considering their therapeutic potential and distinct delivery challenges. By using nanomaterials, the stability and intracellular delivery of genome-editing systems are improved, enabling more effective treatments for genetic disorders and acquired diseases such as cancer and infectious diseases. In addition, nanocarriers provide controlled release, protection from degradation, and better biocompatibility, thereby improving the safety and reliability of gene-editing therapies. Despite these advances, important translational challenges remain, including immunotoxicity, large-scale manufacturing, and regulatory integration. Overall, the continued convergence of nanotechnology and genome engineering may support the development of personalized medicine strategies that adapt genetic engineering tools for patient-specific applications.},
}

Humans
*Genetic Therapy/methods
*Gene Editing/methods
*Nanotechnology/methods
Animals
CRISPR-Cas Systems
Nanostructures/chemistry
Gene Transfer Techniques

@article {pmid42450278,
year = {2026},
author = {Podralska, M and Górska, A and Kaczmarek, M},
title = {Genome Editing Approaches in Flax (Linum usitatissimum L.): From Tools to Trait Improvement.},
journal = {International journal of molecular sciences},
volume = {27},
number = {13},
pages = {},
pmid = {42450278},
issn = {1422-0067},
support = {DHR.hn.070.1.2026//Ministry of Agriculture and Rural Development/ ; },
mesh = {*Flax/genetics ; *Gene Editing/methods ; CRISPR-Cas Systems ; *Genome, Plant ; Plant Breeding/methods ; Plants, Genetically Modified/genetics ; },
abstract = {Genome editing, particularly CRISPR/Cas-based systems, has emerged as a key tool for functional genomics and trait improvement in flax (Linum usitatissimum L.), an important fiber and oilseed crop. This review focuses specifically on flax as an emerging target species and distinguishes experimentally validated applications from approaches adapted from model plants. Recent progress includes the characterization of endogenous U6 promoters, which improved guide RNA expression and contributed to enhanced genome editing performance under optimized conditions. Reported studies demonstrate efficient targeted mutagenesis in flax; however, editing outcomes remain strongly dependent on genotype, construct design, and regeneration capacity, and stable homozygous edited lines are still limited. Target genes include pathways involved in lignin and cellulose biosynthesis, fatty acid metabolism, and stress responses, influencing fiber quality, oil composition, and stress adaptation. Despite current bottlenecks such as low homologous recombination efficiency and regeneration constraints, base editing, prime editing, and multiplex CRISPR systems provide promising avenues for precision breeding in flax.},
}

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

@article {pmid42450173,
year = {2026},
author = {Effah, SN and Barrera, SC and Urturi Ortiz, N and Dampier, W and Nonnemacher, MR and Wigdahl, B},
title = {CRISPR/Cas9-Based Genome Editing: Understanding Differences in DNA Repair Pathways, Profiles, and Outcomes.},
journal = {International journal of molecular sciences},
volume = {27},
number = {13},
pages = {},
pmid = {42450173},
issn = {1422-0067},
support = {MH110360/MH/NIMH NIH HHS/United States ; MH092177/MH/NIMH NIH HHS/United States ; MH079785/MH/NIMH NIH HHS/United States ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *DNA Repair/genetics ; Animals ; Genetic Therapy/methods ; },
abstract = {Over a decade of advances in Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) and CRISPR-associated protein 9 (Cas9)-based technologies have culminated in the first-ever FDA-approved CRISPR/Cas-based therapy. Aside from this approved therapy for sickle cell anemia, several CRISPR/Cas-based therapies are currently under development or testing for a range of chronic diseases, including viral diseases like human immunodeficiency virus type 1 (HIV-1) infection, genetic diseases like familial hypercholesterolemia, and cancer. The success of these therapies hinges on the effective delivery of CRISPR/Cas9 components to target regions, efficient Cas endonuclease editing, repair profiles generated, and their resulting outcomes. Here, we discuss the factors that influence the generation of CRISPR/Cas9-generated repair edits, the overall profiles, and outcome prediction(s), as well as the analytical tools that have been developed to date. Finally, how this technology has been used towards a functional HIV-1 cure is discussed.},
}

Humans
*CRISPR-Cas Systems
*Gene Editing/methods
*DNA Repair/genetics
Animals
Genetic Therapy/methods

@article {pmid42447097,
year = {2026},
author = {Zhang, Y and Xing, J and Zhang, H and Kong, J and Nang, SC and Zhang, M and Pan, Y and Zhai, Y and Yuan, L and Zhao, J and Wu, H},
title = {Disruption of rcnB modulates colistin susceptibility in Acinetobacter baumannii AB5075.},
journal = {Virulence},
volume = {17},
number = {1},
pages = {2697100},
pmid = {42447097},
issn = {2150-5608},
mesh = {*Colistin/pharmacology ; *Acinetobacter baumannii/drug effects/genetics/metabolism ; *Anti-Bacterial Agents/pharmacology ; *Bacterial Proteins/genetics/metabolism ; Microbial Sensitivity Tests ; Drug Resistance, Bacterial ; Drug Resistance, Multiple, Bacterial/genetics ; Oxidative Stress ; CRISPR-Cas Systems ; },
abstract = {Acinetobacter baumannii AB5075 is a clinically relevant multidrug-resistant (MDR) isolate that poses a major therapeutic challenge. Although colistin has been reinstated as a last-resort antibiotic against MDR Gram-negative infections, the rapid emergence of colistin resistance threatens its clinical utility. Here, we employed a CRISPR-Cas9-based genome editing system to generate an A. baumannii AB5075 ΔrcnB mutant and uncovered a previously underappreciated role of rcnB in modulating colistin susceptibility. Loss of rcnB markedly potentiated colistin-mediated killing through multiple associated changes, including compromised membrane integrity, impaired oxidative stress defenses, and reduced efflux pump activity. Transcriptomic profiling further revealed that rcnB deletion reshaped global stress-response networks, including suppression of fatty acid biosynthesis and reactive oxygen species (ROS)-detoxifying pathways, alongside altered metal ion and sulfur metabolism during colistin exposure. Collectively, our findings suggest that rcnB may contribute to colistin susceptibility of colistin resistance and provide mechanistic insights that may inform the development of targeted strategies to enhance colistin efficacy against MDR A. baumannii.},
}

*Colistin/pharmacology
*Acinetobacter baumannii/drug effects/genetics/metabolism
*Anti-Bacterial Agents/pharmacology
*Bacterial Proteins/genetics/metabolism
Microbial Sensitivity Tests
Drug Resistance, Bacterial
Drug Resistance, Multiple, Bacterial/genetics
Oxidative Stress
CRISPR-Cas Systems

@article {pmid42449928,
year = {2026},
author = {Poonooru, R and Park, KE and Schmelzle, A and Telugu, BP},
title = {Functional Inactivation of PAX4 Results in Disrupted Endocrine Pancreas Development and Neonatal Diabetes in Pigs.},
journal = {International journal of molecular sciences},
volume = {27},
number = {13},
pages = {},
pmid = {42449928},
issn = {1422-0067},
support = {W81XWH-22-1-0017//Congressionally Directed Medical Research Programs/ ; },
mesh = {Animals ; *Paired Box Transcription Factors/genetics/metabolism ; *Islets of Langerhans/metabolism/pathology/growth & development ; Swine ; *Homeodomain Proteins/genetics/metabolism ; Animals, Newborn ; CRISPR-Cas Systems ; *Diabetes Mellitus/genetics/metabolism/pathology ; Humans ; },
abstract = {Variants in the human PAX4 gene are associated with both monogenic and complex forms of diabetes, yet their pathogenic effects remain difficult to define in models that accurately mimic human islet architecture and neonatal metabolic transitions. Here, we created a porcine PAX4 loss-of-function model using CRISPR/Cas9 cytidine deaminase base editing to introduce a premature stop codon in the PAX4 coding sequence. PAX4 knockout piglets developed severe hyperglycemia within 24 h of birth, followed by rapid postnatal clinical deterioration and uniform death by day 3. Biochemical analysis showed significant diabetic decompensation, including electrolyte imbalances, hyperosmolality, azotemia, dyslipidemia, and metabolic acidosis. Gross and histological examinations revealed notable pancreatic hypoplasia with preservation of exocrine tissue. Single-nucleus RNA sequencing and immunohistochemistry demonstrated an almost complete loss of insulin- and somatostatin-producing β- and δ-cells, respectively, with relative preservation of glucagon-expressing α-cells. Overall, these results establish PAX4 as a crucial factor in pancreatic endocrine development and postnatal glucose regulation in a large-animal model. This platform offers a human-relevant system for studying diabetes-associated PAX4 variants and for testing regenerative and gene-based therapies for insulin-deficient diabetes.},
}

Animals
*Paired Box Transcription Factors/genetics/metabolism
*Islets of Langerhans/metabolism/pathology/growth & development
Swine
*Homeodomain Proteins/genetics/metabolism
Animals, Newborn
CRISPR-Cas Systems
*Diabetes Mellitus/genetics/metabolism/pathology
Humans

@article {pmid42450131,
year = {2026},
author = {Samoń, M and Przyborowski, M},
title = {Wheat's Up with CRISPR-Cas-Current Advances, Obstacles and Perspectives.},
journal = {International journal of molecular sciences},
volume = {27},
number = {13},
pages = {},
pmid = {42450131},
issn = {1422-0067},
support = {Dotacja Celowa task 4.1//Ministry of Agriculture and Rural Development/ ; },
mesh = {*Triticum/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Plants, Genetically Modified/genetics ; Genome, Plant ; Genetic Engineering/methods ; },
abstract = {The emergence of CRISPR-Cas editing systems-comprising clustered regularly interspaced short palindromic repeats and associated Cas proteins-marked a breakthrough in genetic engineering, owing to the simplicity, efficiency, and adaptability of the method. Despite continuous improvements and the incorporation of innovative discoveries to develop reliable, fine-tuned tools, the effective application of CRISPR-Cas technology in cereals remains challenging. This review provides a technically oriented overview of CRISPR-Cas-mediated genome editing in wheat (Triticum aestivum L.), one of the world's fundamental crops. While focusing on established solutions and progressive methodological modifications, we also discuss pertinent topics, including plant genetic transformation, prospective innovations, and compliance considerations.},
}

*Triticum/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
Plants, Genetically Modified/genetics
Genome, Plant
Genetic Engineering/methods

@article {pmid41168295,
year = {2026},
author = {Yeo, JH and Lee, S and Kim, S and Min, JG and Gopalappa, R and Oh, HC and Kim, HK and Nam, EJ and Kim, HH},
title = {High-throughput evaluation of in vitro CRISPR activities enables optimized large-scale multiplex enrichment of rare variants.},
journal = {Nature biomedical engineering},
volume = {10},
number = {7},
pages = {1410-1430},
pmid = {41168295},
issn = {2157-846X},
support = {RS-2022-NR070713, 2018R1A5A2025079, RS-2022-NR067326, RS-2022-NR067345, RS-2023-00260968//National Research Foundation of Korea (NRF)/ ; 2021R1I1A1A01047269//National Research Foundation of Korea (NRF)/ ; RS-2023-NR076625//National Research Foundation of Korea (NRF)/ ; 2024-22-0165//Yonsei University/ ; 22B-000-0101//Seoul National University Hospital (SNUH)/ ; RS-2024-00467177//Korea Drug Development Fund (KDDF)/ ; 1730158, 1540112, 1541349, 1826967, 2138811, 2112167, 2100237, 2120019, 1419152, 1743354, 2027170//National Science Foundation (NSF)/ ; 6-2019-0166//Yonsei University | Yonsei University College of Medicine (YUCM)/ ; },
mesh = {RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems/genetics ; Humans ; *High-Throughput Nucleotide Sequencing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Base Sequence ; Genetic Variation ; },
abstract = {Previous high-throughput evaluations of CRISPR activities for a large number of target and guide RNA sequences were based on measuring insertion-deletion frequencies rather than cleavage efficiencies. Here we develop two high-throughput in vitro methods, Cut-seq1 and Cut-seq2, to evaluate Cas9 cleavage efficiency for tens of thousands, or even hundreds of thousands, of guide RNA-target pairs. These methods reveal low correlations between in vitro cleavage efficiencies and insertion-deletion frequencies in cells, yet high concordances in protospacer adjacent motif compatibility. Using the resulting large datasets of in vitro cleavage efficiencies, we develop DeepCut, a set of deep learning models that can identify optimized single-guide RNAs that can selectively cleave specific sequences, even in the presence of similar noise sequences. Using these optimized single-guide RNAs, we develop a method, CLOVE-seq (which stands for cleavage for large-scale optimized variant enrichment sequencing), to enrich rare variants in a multiplexed manner by Cas9-mediated specific cleavage of noise or rare variant sequences. Our methods can enhance the understanding of CRISPR nuclease activities and could be used to detect a large number of rare variants in various biomedical contexts.},
}

RNA, Guide, CRISPR-Cas Systems/genetics
*CRISPR-Cas Systems/genetics
Humans
*High-Throughput Nucleotide Sequencing/methods
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Base Sequence
Genetic Variation

@article {pmid41947506,
year = {2026},
author = {Park, JC and Song, Y and Choi, HW and Sung, J and Jin, H and Gwak, W and Yoo, K and Lee, S and Park, J and Kim, J and Jo, HJ and Koo, J and Jeong, Y and Lee, KH and Kee, SJ and Kim, H},
title = {Viral Infection-Inspired Autonomous Detection of Fusion-Competent Viruses for Screening and Environmental Surveillance.},
journal = {Advanced materials (Deerfield Beach, Fla.)},
volume = {38},
number = {40},
pages = {e21241},
doi = {10.1002/adma.202521241},
pmid = {41947506},
issn = {1521-4095},
support = {2021R1A2C2013961//The National Research Foundation of Korea (NRF) grant funded by the Ministry of Science and ICT (MSIT)/ ; 2022R1C1C2007002//The National Research Foundation of Korea (NRF) grant funded by the Ministry of Science and ICT (MSIT)/ ; RS-2024-00345402//The National Research Foundation of Korea (NRF) grant funded by the Ministry of Science and ICT (MSIT)/ ; RS-2023-00209955//The National Research Foundation of Korea (NRF) grant funded by the Ministry of Science and ICT (MSIT)/ ; Institutional 2E33111//Korea Institute of Science and Technology (KIST)/ ; RS-2024-00396818//Korea Institute of Planning and Evaluation for Technology in Food, Agricultural and Forestry (IPET) through High-Risk Animal Infectious Disease Control Technlogy Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA)/ ; 6634-332//The Korea Disease Control and Prevention Agency (KDCA)/ ; RS-2025-25409767//The Materials & Components Technology Development Program funded by the Ministry of Trade, Industry and Energy (MOTIE, Korea)/ ; },
mesh = {*Environmental Monitoring/methods ; Humans ; RNA, Viral/analysis ; SARS-CoV-2/isolation & purification/genetics ; CRISPR-Cas Systems ; Virus Internalization ; Respiratory Syncytial Viruses/isolation & purification ; Influenza A virus/isolation & purification/genetics ; Liposomes/chemistry ; },
abstract = {The persistent burden of respiratory viruses requires rapid, simple, and robust screening and environmental surveillance technologies that enable widespread and frequent testing. Importantly, these technologies should be based on infectivity-relevant signals, as RNA detection alone has limited correlation with transmission risk. Here, we present a membrane fusion-mediated platform that autonomously detects viruses by recapitulating the native viral entry mechanism. Fusogenic vesicles selectively fuse with fusion-competent viral particles, triggering encapsulated CRISPR-Cas13a components to generate fluorescent signals upon recognition of the released viral RNA. Through an autonomous workflow and accelerated signal generation within a confined vesicle, our platform achieves one-step detection of viruses within 2 min. The assay robustly detects three major respiratory viruses, with analytical sensitivities down to 5 TCID50/mL for RSV and 50 TCID50/mL for SARS-CoV-2 and IAV. Clinical validation with 100 nasopharyngeal samples achieved 91.7% sensitivity. Remarkably, the sprayable format enables large-area surveillance of surface contamination-like luminol revealing hidden bloodstains, it makes invisible viral threats visible. This approach establishes an intuitive real-time detection platform, extending beyond clinical specimens to encompass environmental threats.},
}

*Environmental Monitoring/methods
Humans
RNA, Viral/analysis
SARS-CoV-2/isolation & purification/genetics
CRISPR-Cas Systems
Virus Internalization
Respiratory Syncytial Viruses/isolation & purification
Influenza A virus/isolation & purification/genetics
Liposomes/chemistry

@article {pmid42086608,
year = {2026},
author = {Cao, J and Liu, Z and Chen, X and Lan, Z and Liu, L and Zhai, Y and Wang, W and Xue, C and Cheng, H and Yao, Y and Cheng, T and Rao, S},
title = {Engineered dCas12f1-SAM enables robust transcriptional activation and gain-of-function screening in primary human cells.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42086608},
issn = {2041-1723},
support = {82370118, 82470240 and 82370117//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Humans ; *Transcriptional Activation/genetics ; T-Lymphocytes/metabolism/immunology ; *CRISPR-Cas Systems/genetics ; Transcription Factors/genetics/metabolism ; Hematopoietic Stem Cells/metabolism ; Interleukin-2/genetics/metabolism ; Kruppel-Like Transcription Factors/genetics/metabolism ; LIM-Homeodomain Proteins/genetics/metabolism ; Single-Cell Gene Expression Analysis ; Receptors, Chimeric Antigen/metabolism/genetics ; },
abstract = {Despite considerable powers, the application of CRISPR activation (CRISPRa) screens in primary human cells remains a formidable challenge. Here, we develop dCas12f1-SAM, a compact SAM-based transcriptional activation platform, that outperforms existing systems in both immortalized cell lines and primary human T cells and hematopoietic stem/progenitor cells (HSPCs). Using dCas12f1-SAM, we perform a pooled CRISPRa screen targeting 1559 human transcription factors (TFs) in primary human T cells and identify multiple positive regulators of IL-2 expression. We further implement a single-cell CRISPRa screen via our miCROP-seq construct, resolving how these genetic perturbations reshape T cell activation dynamics and drive functionally distinct cellular states. Among the top-ranking genes, we spotlight KLF12 and LHX5, whose overexpression significantly improves antigen-specific responses of chimeric antigen receptor T (CAR-T) cells. Collectively, these findings establish dCas12f1-SAM as a robust transcriptional activation tool, highlighting its potential to advance applications in cellular engineering and immunotherapy.},
}

Humans
*Transcriptional Activation/genetics
T-Lymphocytes/metabolism/immunology
*CRISPR-Cas Systems/genetics
Transcription Factors/genetics/metabolism
Hematopoietic Stem Cells/metabolism
Interleukin-2/genetics/metabolism
Kruppel-Like Transcription Factors/genetics/metabolism
LIM-Homeodomain Proteins/genetics/metabolism
Single-Cell Gene Expression Analysis
Receptors, Chimeric Antigen/metabolism/genetics

@article {pmid42103719,
year = {2026},
author = {Bourgeois, W and Rice, HE and Wenge, DV and Perner, F and Yue, H and Regalado, BD and Wan, G and Schroeder, JC and Sommerschield, A and Hatton, C and Singh, S and Singh, S and Bijpuria, S and McKeever, BM and Miller, WH and Safer, JF and Iqbal, S and Perry, JA and Fischer, ES and Doench, JG and McGeehan, GM and Cutler, JA and Armstrong, SA},
title = {CRISPR base editor screening identifies spectrum of MEN1 mutations impacting menin inhibitors in clinical trials.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42103719},
issn = {2041-1723},
support = {Chromatin Collaborative award//St. Jude Children's Research Hospital/ ; P50 CA206963/CA/NCI NIH HHS/United States ; R01 CA259273/CA/NCI NIH HHS/United States ; NIH 5 P30 CA06516//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; P01 CA066996/CA/NCI NIH HHS/United States ; CA206963//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; 511811315//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 2021-EKEA.111//Else Kröner-Fresenius-Stiftung (Else Kroner-Fresenius Foundation)/ ; CA066996//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; CA259273//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; PE 3217/4-1, PN: 517204983//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
mesh = {*Proto-Oncogene Proteins/genetics/antagonists & inhibitors/chemistry/metabolism ; Humans ; Histone-Lysine N-Methyltransferase/genetics/metabolism/antagonists & inhibitors ; *Mutation ; Nucleophosmin ; Myeloid-Lymphoid Leukemia Protein/genetics/metabolism ; Animals ; Drug Resistance, Neoplasm/genetics ; Nuclear Proteins/genetics ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Mice ; },
abstract = {Menin inhibitors have entered clinical trials for histone lysine methyltransferase 2 A (KMT2A)-rearranged and nucleophosmin 1 (NPM1)-mutant acute leukemias and are demonstrating promising activity. CRISPR base editor screening previously predicted several MEN1 (menin) mutations that have arisen in patients receiving SNDX-5613 and confer resistance. The extent to which MEN1 mutations will impact each menin inhibitor is mostly unknown. Here we show that CRISPR base editor screens can be leveraged to profile the MEN1 mutations that may impact five different menin inhibitors in clinical trials. We identify shared (M327I/V/T, G331D) and inhibitor-specific (C334R, E368K/V, V372A) resistance mutations. Co-crystal structures of menin bound to each menin inhibitor suggest resistance mechanisms related to how each inhibitor engages the KMT2A binding pocket of menin. Orthogonal in vitro and in vivo MEN1 mutation generation under therapeutic pressure suggest the MEN1 mutations identified with CRISPR base editor screening are likely to arise and impact all menin inhibitors.},
}

*Proto-Oncogene Proteins/genetics/antagonists & inhibitors/chemistry/metabolism
Humans
Histone-Lysine N-Methyltransferase/genetics/metabolism/antagonists & inhibitors
*Mutation
Nucleophosmin
Myeloid-Lymphoid Leukemia Protein/genetics/metabolism
Animals
Drug Resistance, Neoplasm/genetics
Nuclear Proteins/genetics
CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
Mice

@article {pmid42103787,
year = {2026},
author = {Cipria, D and Baccega, T and Rizzo, M and Quarato, P and Reschigna, A and El Khoury, R and Cappelluti, MA and Ammann, S and Poeta, VM and Conti, M and Valsoni, S and Spinelli, P and Merelli, I and Cathomen, T and Casucci, M and Lombardo, A},
title = {Simultaneous orthogonal cell engineering by a single CRISPR-Cas9 polyfunctional editor.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42103787},
issn = {2041-1723},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Cell Engineering/methods ; *Gene Editing/methods ; T-Lymphocytes/metabolism ; Epigenome Editing ; RNA, Guide, CRISPR-Cas Systems/genetics ; Epigenesis, Genetic ; Receptors, Antigen, T-Cell/genetics ; Transgenes ; Gene Silencing ; Receptors, Chimeric Antigen/genetics ; },
abstract = {The parallel disruption of multiple genes coupled with targeted transgene insertion offers a powerful strategy for more effective and precise cell engineering. However, such orthogonal editing involves the induction of multiple DNA breaks, raising safety concerns related to the risks of chromosomal translocations. Here, we present a polyfunctional CRISPR-Cas9-based strategy that enables both transgene insertion and epigenetic silencing at distinct genomic loci in a single treatment without inducing reciprocal chromosomal translocations. This is accomplished through an optimized all-in-one epigenome editor equipped with a catalytically active Cas9, whose endonuclease activity is selectively disabled at epigenetically silenced loci using truncated gRNAs. As a proof of concept, we demonstrate that this platform enables efficient multi-locus editing, including functional replacement of the endogenous TCR with a tumor-selective one, targeted insertion of a prototypic CAR with either a selectable marker or an immunomodulatory receptor into a TCR locus or a ubiquitously expressed gene, and durable, multiplexed epigenetic silencing of clinically relevant genes in primary human T cells. Polyfunctional editing establishes a versatile and safe framework for orthogonal editing, broadening the scope of genome and epigenome engineering in cancer immunotherapy and beyond.},
}

Humans
*CRISPR-Cas Systems/genetics
*Cell Engineering/methods
*Gene Editing/methods
T-Lymphocytes/metabolism
Epigenome Editing
RNA, Guide, CRISPR-Cas Systems/genetics
Epigenesis, Genetic
Receptors, Antigen, T-Cell/genetics
Transgenes
Gene Silencing
Receptors, Chimeric Antigen/genetics

@article {pmid42120395,
year = {2026},
author = {Morency, C and Rousseau, GM and Morneau, Z and Moineau, S},
title = {Phage satellites induced by virulent phages are mobilized by natural competence leading to phage resistance in a new host.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42120395},
issn = {2041-1723},
mesh = {*Streptococcus thermophilus/virology/genetics ; *Bacteriophages/genetics/pathogenicity/physiology ; CRISPR-Cas Systems ; Chromosomes, Bacterial/genetics ; Virulence ; Genome, Viral ; Prophages/genetics ; },
abstract = {A phage satellite (PS) typically resides within repeat regions (attL and attR sites) of a bacterial genome. Its genome ranges from 7 to 20-kb and includes genes encoding an integrase along with regulatory and DNA replication functions. However, it lacks genes associated with viral structural proteins. Streptococcus thermophilus (S.t.) is extensively used to produce yogurt and specialty cheeses. Intriguingly, the majority of S.t. strains harbor a PS while very few possess a complete prophage, suggesting that PSs may confer advantages to their hosts. In this study, we showed that PSs of S.t. can excise from the bacterial chromosome, at a very low rate, without any phage interaction. Furthermore, we found that they can also be induced by virulent phages. By leveraging CRISPR-Cas9, we selected S.t. cells devoid of any PS (delta-PS strain). Then, we mobilized a PS from one strain to a delta-PS strain, using only natural competence, bypassing the need for a helper phage. The resulting strain exhibited increased resistance to virulent phages. Through the isolation of phage mutants escaping the resistance phenotype, we pinpointed a specific phage protein responsible for the induction of a PS. Lastly, we demonstrated that a PS can be significantly induced by a virulent phage, which, in turn, greatly promotes its transfer and specific integration into new cells through natural competence. Our study introduces a novel natural approach to develop phage-resistant strains.},
}

*Streptococcus thermophilus/virology/genetics
*Bacteriophages/genetics/pathogenicity/physiology
CRISPR-Cas Systems
Chromosomes, Bacterial/genetics
Virulence
Genome, Viral
Prophages/genetics

@article {pmid42140952,
year = {2026},
author = {Wang, S and Hou, S and Luo, C and Zhang, H and Jin, Y and Zhang, R and Zhao, Y and Xiong, X and Guo, R and Wang, C and Bao, Y and Wen, L and Pan, D and Ye, Y and Zeng, Z and Gao, Z},
title = {Arid3b suppresses CD8 + T cell infiltration and function in microsatellite-stable colorectal cancer via Runx3.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42140952},
issn = {2041-1723},
support = {92478117//National Natural Science Foundation of China (National Science Foundation of China)/ ; 92374116//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32470664//National Natural Science Foundation of China (National Science Foundation of China)/ ; T2321001//National Natural Science Foundation of China (National Science Foundation of China)/ ; L248043//Natural Science Foundation of Beijing Municipality (Beijing Natural Science Foundation)/ ; },
mesh = {*Core Binding Factor Alpha 3 Subunit/genetics/metabolism/immunology ; Animals ; *Colorectal Neoplasms/genetics/immunology/pathology/metabolism ; *CD8-Positive T-Lymphocytes/immunology/metabolism ; Tumor Microenvironment/immunology/genetics ; Humans ; Mice ; *DNA-Binding Proteins/genetics/metabolism ; Cell Line, Tumor ; Lymphocytes, Tumor-Infiltrating/immunology ; CRISPR-Cas Systems ; Microsatellite Repeats ; *Transcription Factors/genetics/metabolism ; },
abstract = {Microsatellite-stable/proficient mismatch repair (MSS/pMMR) colorectal cancer (CRC) is characterized by a cold tumor microenvironment, with limited CD8[+] T cell infiltration and poor responsiveness to immune checkpoint inhibitors (ICIs). Here, using an in vivo CRISPR/Cas9 screen in a CMT93 cell-derived murine tumor model, we identify Arid3b as a key negative regulator of CD8[+] T cell infiltration and antitumor activity. Genetic ablation of Arid3b in CD8[+] T cells significantly enhances their intratumoral accumulation and promotes robust tumor control. Mechanistically, Arid3b deficiency upregulates Runx3, driving a tissue-resident memory-like phenotype and effector function. Notably, the benefits conferred by Arid3b deficiency are abrogated upon Runx3 deletion, indicating a RUNX3-dependent mechanism. Together, targeting ARID3B could offer a promising strategy to reshape the tumor microenvironment and sensitize MSS CRC to immunotherapy.},
}

*Core Binding Factor Alpha 3 Subunit/genetics/metabolism/immunology
Animals
*Colorectal Neoplasms/genetics/immunology/pathology/metabolism
*CD8-Positive T-Lymphocytes/immunology/metabolism
Tumor Microenvironment/immunology/genetics
Humans
Mice
*DNA-Binding Proteins/genetics/metabolism
Cell Line, Tumor
Lymphocytes, Tumor-Infiltrating/immunology
CRISPR-Cas Systems
Microsatellite Repeats
*Transcription Factors/genetics/metabolism

@article {pmid42264060,
year = {2026},
author = {Zaki, HF and Bishri, J and Abdul Muqtadir, M and Abu-Zaid, A},
title = {Correcting photoreceptor diseases at their source: CRISPR strategies for cone-rod dystrophy and achromatopsia.},
journal = {Experimental eye research},
volume = {270},
number = {},
pages = {111119},
doi = {10.1016/j.exer.2026.111119},
pmid = {42264060},
issn = {1096-0007},
mesh = {*Color Vision Defects/therapy/genetics ; Humans ; Animals ; *Genetic Therapy/methods ; *Cone-Rod Dystrophies/therapy/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Disease Models, Animal ; },
abstract = {BACKGROUND: Cone-rod dystrophy (CORD) and achromatopsia (ACHM) are inherited retinal dystrophies for which conventional adeno-associated virus (AAV) gene augmentation has important limitations, particularly in autosomal-dominant gain-of-function CORD and recessive ACHM. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) offers the potential for one-time, mutation-specific gene correction or allele ablation. This systematic review summarizes preclinical evidence on CRISPR/Cas9-based approaches for CORD and ACHM, focusing on editing efficiency, phenotypic rescue, and safety.

METHODS: This review followed PRISMA guidelines. PubMed, Google Scholar, and ScienceDirect were searched through June 2025 for original experimental studies using CRISPR/Cas9 in CORD or ACHM animal models or human-derived cell lines. Dual independent screening and data extraction were performed. Outcomes related to editing efficiency, structural or functional rescue, and safety were synthesized narratively.

RESULTS: Four studies were included: three targeting CORD and one targeting ACHM. In vivo studies used AAV-delivered SaCas9 to disrupt GUCY2D (or murine orthologs) in mouse and macaque photoreceptors, achieving approximately 8-45% on-target editing in mice and approximately 13% in macaques. Although ablation alone reduced retGC1 expression, it did not improve retinal function; however, a dual-AAV "ablate-and-replace" strategy preserved outer nuclear layer thickness for up to 24 weeks in CORD6 mice. In vitro, PROM1 correction in patient-derived iPSCs restored CD133 expression, and SpCas9-HiFi-mediated PDE6C correction in ACHM iPSCs achieved approximately 80% editing efficiency while preserving pluripotency and showing no detectable off-target effects. Safety data were limited, with immune responses assessed in only one primate study.

CONCLUSIONS: CRISPR/Cas9 shows promising preclinical efficacy for CORD and ACHM, particularly allele-specific ablate-and-replace strategies for CORD and precise HDR-based correction for ACHM. However, the available evidence remains limited, underscoring the need for expanded safety assessment, non-human primate studies, and standardized functional outcomes measures before clinical translation.},
}

*Color Vision Defects/therapy/genetics
Humans
Animals
*Genetic Therapy/methods
*Cone-Rod Dystrophies/therapy/genetics
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Disease Models, Animal

@article {pmid42284763,
year = {2026},
author = {Liu, L and Wang, H and Shi, L and Zhang, C and Zhang, L and Lv, L and Wang, Y},
title = {Development of super Vδ2 T cells for relapsed/refractory acute myeloid Leukemia via non-viral site-specific integration.},
journal = {International immunopharmacology},
volume = {185},
number = {},
pages = {116981},
doi = {10.1016/j.intimp.2026.116981},
pmid = {42284763},
issn = {1878-1705},
mesh = {Humans ; *Leukemia, Myeloid, Acute/therapy/immunology ; Animals ; *Immunotherapy, Adoptive/methods ; *Receptors, Antigen, T-Cell, gamma-delta/genetics/immunology/metabolism ; Receptors, Chimeric Antigen/genetics ; *T-Lymphocytes/immunology/transplantation ; NK Cell Lectin-Like Receptor Subfamily K/genetics/metabolism ; Mice ; Cell Line, Tumor ; CD3 Complex/genetics ; CRISPR-Cas Systems ; Cytotoxicity, Immunologic ; Mice, SCID ; },
abstract = {The efficacy of chimeric antigen receptor (CAR)-T cell therapy in relapsed/refractory acute myeloid leukemia (R/R AML) is limited by tumor heterogeneity, antigen evasion, and treatment-related toxicities. Gamma delta (γδ) T cells mediate antitumor activity independent of MHC by sensing stress-induced ligands. A prominent mechanism involves NKG2D ligand (NKG2DL) recognition, which is highly upregulated in malignancies but generally low or restricted expression in healthy tissues under homeostatic conditions. In human peripheral blood, the Vδ2 subset represents the predominant population. Vδ2 T cells transduced with the NKG2D-CD3ζ construct, which incorporates into the natural γδ TCR/CD3 complex, preserve innate phosphoantigen recognition while acquiring potent NKG2DL-directed cytotoxicity, enabling dual-pathway tumor recognition. These cells are termed "Super Vδ2 T cells." We successfully generated TRAC-specific integrated Super Vδ2 T cells using CRISPR/Cas9 technology, achieving 90-93% CAR[+] expression. In vitro assays demonstrated that the engineered "Super Vδ2 T cells" exhibited potent cytotoxic activity against multiple AML targets, including cell lines and primary R/R AML blasts, in contrast to their negligible toxicity on monocytes. In vivo, Super Vδ2 T cells demonstrated substantial tumor reduction without graft-versus-host disease (GvHD) reaction. Collectively, our data demonstrated that Super Vδ2 T cells represent a viable allogeneic therapy for AML.},
}

Humans
*Leukemia, Myeloid, Acute/therapy/immunology
Animals
*Immunotherapy, Adoptive/methods
*Receptors, Antigen, T-Cell, gamma-delta/genetics/immunology/metabolism
Receptors, Chimeric Antigen/genetics
*T-Lymphocytes/immunology/transplantation
NK Cell Lectin-Like Receptor Subfamily K/genetics/metabolism
Mice
Cell Line, Tumor
CD3 Complex/genetics
CRISPR-Cas Systems
Cytotoxicity, Immunologic
Mice, SCID

@article {pmid42439627,
year = {2026},
author = {Park, SH and Hong, J and Hwang, W and Kim, M and Yu, HJ and Bae, T and Lee, HK and Lee, JY and Lee, YC and Park, CK and Hur, JK},
title = {CRISPRi-Mediated Epigenetic Suppression of TERT Reduces Cell Growth in Non-Small-Cell Lung Cancer Cells.},
journal = {Cells},
volume = {15},
number = {13},
pages = {},
pmid = {42439627},
issn = {2073-4409},
support = {RS-2021-NR056589; RS-2023-00261114; RS-2025-02218918//Ministry of Science and ICT/ ; RS-2025-16063805//the Korea US Collaborative Research Fund/ ; 2023R1A6C101A009//the Ministry of Education/ ; RS-2023-00260529, RS-2026-25493418//National Research Foundation of Korea/ ; },
mesh = {Humans ; *Telomerase/genetics/metabolism ; *Carcinoma, Non-Small-Cell Lung/genetics/pathology ; *Lung Neoplasms/genetics/pathology ; Cell Line, Tumor ; Cell Proliferation/genetics ; *Epigenesis, Genetic ; *CRISPR-Cas Systems/genetics ; Gene Expression Regulation, Neoplastic ; Cell Survival/genetics ; Epigenome Editing ; },
abstract = {TERT, the catalytic subunit of telomerase, is aberrantly activated in most cancers and represents an attractive therapeutic target. However, conventional TERT-targeting strategies, including chemical inhibitors and siRNA, are limited by several issues, such as insufficient efficacy and off-target effects. In this study, we investigated whether dCas9-KRAB-mediated CRISPR interference (CRISPRi) could overcome the limitations by transcriptional repression of TERT without DNA cleavage. We first assessed the efficacy of the dCas9-KRAB system by applying it to H1299 non-small-cell lung cancer cells and observed reduction in TERT expression up to approximately 80% and significant decreases in cell viability and growth. Transcriptome-wide analysis showed limited detectable changes in non-target-gene expression under the conditions tested. Together, the results suggest that dCas9-KRAB-mediated CRISPRi could serve as a proof-of-principle approach for targeted repression of TERT in cancer cells with limited detectable effects on non-target-gene expression.},
}

Humans
*Telomerase/genetics/metabolism
*Carcinoma, Non-Small-Cell Lung/genetics/pathology
*Lung Neoplasms/genetics/pathology
Cell Line, Tumor
Cell Proliferation/genetics
*Epigenesis, Genetic
*CRISPR-Cas Systems/genetics
Gene Expression Regulation, Neoplastic
Cell Survival/genetics
Epigenome Editing

@article {pmid42439976,
year = {2026},
author = {Mundada, AR and Badikol, AR and Mangu, K},
title = {CRISPR-Cas9-based therapies for Huntington's disease and Friedreich's ataxia: mechanisms, advances, and future perspectives.},
journal = {Neurogenetics},
volume = {27},
number = {1},
pages = {},
pmid = {42439976},
issn = {1364-6753},
mesh = {*Friedreich Ataxia/therapy/genetics ; Humans ; *Huntington Disease/therapy/genetics ; *CRISPR-Cas Systems ; *Genetic Therapy/methods/trends ; *Gene Editing/methods ; Animals ; Trinucleotide Repeat Expansion ; },
abstract = {Huntington's disease (HD) and Friedreich's ataxia (FRDA) are progressive inherited neurodegenerative disorders caused by trinucleotide repeat expansions but characterized by distinct pathogenic mechanisms. HD arises from a coding-region CAG expansion in the HTT gene that produces toxic gain-of-function effects of mutant huntingtin (mHTT), whereas FRDA results primarily from intronic GAA repeat expansion in FXN, leading to epigenetic repression and frataxin deficiency. The emergence of CRISPR-based genome engineering has created new opportunities to address these diseases at their genetic origin. This review examines current CRISPR therapeutic strategies for HD and FRDA, including allele-specific editing, transcriptional suppression, repeat excision, epigenetic reactivation, and emerging precision editing approaches such as base editing and prime editing. We compare the molecular rationale, preclinical outcomes, and translational limitations associated with each approach while highlighting how disease architecture influences therapeutic design. Although preclinical studies demonstrate promising restoration of cellular phenotypes and functional improvement, significant barriers remain. Efficient delivery to the central nervous system and cardiac tissue, control of editing duration, immune responses, off-target activity, and emerging concerns regarding on-target genomic instability continue to limit clinical translation. Recent advances in delivery engineering, non-viral systems, and programmable editing platforms suggest that future therapeutic success will depend on integrating disease-specific biology with increasingly precise and controllable genome engineering technologies. Ethical and regulatory concerns remain substantial, particularly regarding informed consent in the context of cognitive decline and the irreversibility of genomic modification.},
}

*Friedreich Ataxia/therapy/genetics
Humans
*Huntington Disease/therapy/genetics
*CRISPR-Cas Systems
*Genetic Therapy/methods/trends
*Gene Editing/methods
Animals
Trinucleotide Repeat Expansion

@article {pmid42441240,
year = {2026},
author = {Yu, W and Huang, X and Hu, Y and Chen, S},
title = {Beyond adaptive immunity: Functional diversity of the type III-A CRISPR-Cas system in Mycobacterium tuberculosis.},
journal = {Cell insight},
volume = {5},
number = {4},
pages = {100342},
pmid = {42441240},
issn = {2772-8927},
abstract = {CRISPR-Cas systems are best known as prokaryotic adaptive immune pathways that defend against invading genetic elements. Mycobacterium tuberculosis (Mtb) harbors a type III-A CRISPR-Cas system that is structurally conserved yet exhibits little evidence of ongoing spacer acquisition. Nevertheless, its interference machinery remains functional, and increasing evidence suggests that this system has evolved roles beyond canonical adaptive immunity. Accumulating studies indicate that this system is deeply integrated into cellular regulatory networks by governing stress responses, metabolic adaptation, and host-pathogen interactions. Mechanistically, the Mtb type III-A CRISPR-Cas system operates through transcription-dependent target recognition and cyclic oligoadenylate (cOA)-mediated signal amplification, in which the ancillary ribonuclease Csm6 serves as a key effector. Functionally, CRISPR-associated proteins influence antibiotic susceptibility, oxidative stress resistance and host immune responses, and may even act as secreted immunomodulatory factors. In this review, we summarize current understanding of the genomic organization, regulatory mechanisms, and non-canonical functions of the Mtb type III-A CRISPR-Cas system, with particular emphasis on its emerging roles in stress adaptation and host immune regulation.},
}

@article {pmid42442865,
year = {2026},
author = {Zheng, J and Zhang, W and Conrad, M},
title = {Ferroptosis induction via genetic approaches - CRISPR/Cas9-based disruption on key anti-ferroptotic genes.},
journal = {Methods in cell biology},
volume = {209},
number = {},
pages = {91-103},
doi = {10.1016/bs.mcb.2026.05.002},
pmid = {42442865},
issn = {0091-679X},
mesh = {*Ferroptosis/genetics/drug effects ; Humans ; *CRISPR-Cas Systems/genetics ; *Phospholipid Hydroperoxide Glutathione Peroxidase/genetics ; Amino Acid Transport System y+/genetics ; Cell Line, Tumor ; Gene Knockout Techniques/methods ; },
abstract = {Unlike apoptosis, necroptosis, or pyroptosis which are executed by dedicated proteins, ferroptosis is a distinct form of regulated cell death driven by lipid peroxidation downstream of metabolic dysfunction. In most physiological settings, the cyst(e)ine/glutathione/glutathione peroxidase 4 (GPX4) axis constitutes the central anti-ferroptotic machinery, and disruption of this axis is usually sufficient to trigger ferroptosis. For in vitro studies, commonly employed ferroptosis inducers include erastin, which blocks cystine uptake by targeting system xc[-], and (1S,3R)-RSL3, which inhibits GPX4 activity. However, both compounds exhibit off-target effects - erastin can activate voltage-dependent anion channels in mitochondria, whereas (1S,3R)-RSL3 affects other selenoproteins in addition to GPX4. Thus, genetic approaches to induce ferroptosis provide a valuable complement to chemical inducers by excluding off-target concerns. Here, we describe an efficient CRISPR/Cas9-based strategy to generate SLC7A11- and GPX4-knockout HT1080 cells. These knockout lines require routine culture in medium supplemented with β-mercaptoethanol or liproxstatin-1, while withdrawal of these supplements readily induces ferroptosis.},
}

*Ferroptosis/genetics/drug effects
Humans
*CRISPR-Cas Systems/genetics
*Phospholipid Hydroperoxide Glutathione Peroxidase/genetics
Amino Acid Transport System y+/genetics
Cell Line, Tumor
Gene Knockout Techniques/methods

@article {pmid42444979,
year = {2026},
author = {Yang, M and Song, Y and Wang, Z and Chao, K and Li, L and Zhang, X and Duan, X and Yu, C and Xue, R and Zhao, J},
title = {Delivery Systems for Therapeutic Genome Editing: Challenges, Innovations, and Future Perspectives.},
journal = {MedComm},
volume = {7},
number = {7},
pages = {e70791},
pmid = {42444979},
issn = {2688-2663},
abstract = {Therapeutic genome editing has advanced rapidly with the development of diverse programmable nucleases, from zinc-finger nucleases and transcription activator-like effector nucleases to clustered regularly interspaced short palindromic repeats (CRISPR)-based systems such as base and prime editors. Despite these breakthroughs, clinical translation remains constrained by the challenge of achieving safe, efficient, and tissue-specific delivery. Viral vectors, particularly adeno-associated viruses, have enabled durable editing in selected organs but are limited by their restricted cargo capacity, immunogenicity, and complex manufacturing. Nonviral platforms, most notably ionizable lipid nanoparticles, have demonstrated remarkable efficacy for hepatic targets, with clinical trials reporting up to 93% protein knockdown after a single dose. An expanding set of emerging modalities, including virus-mimicking nanosystems, cell-derived extracellular vesicles, cell-penetrating peptides, and intelligent-responsive multifunctional scaffolds, further enriches the delivery toolbox by supporting transient expression and programmable targeting across diverse editors and tissues. Parallel advances in high-throughput barcoded screening and machine learning are accelerating vector optimization, while rational chemical modification of payloads improves in vivo stability and specificity. This review provides a comprehensive overview of current and emerging delivery systems for genome editing, highlighting key innovations, unresolved challenges, and interdisciplinary strategies poised to unlock broader therapeutic potential.},
}

@article {pmid42445839,
year = {2026},
author = {Kruglova, NA and Borovikova, SE and Shepelev, MV},
title = {Production of virus-like particles with AsCas12a nuclease and CMV-driven crRNA for mammalian genome editing.},
journal = {Frontiers in genome editing},
volume = {8},
number = {},
pages = {1735339},
pmid = {42445839},
issn = {2673-3439},
abstract = {CRISPR/Cas genome editing tools represent a promising technology for biomedicine with significant therapeutic potential for numerous human diseases. However, efficient delivery of these tools into primary cells, particularly in the form of ribonucleoprotein (RNP) complexes, remains a critical bottleneck that limits clinical translation. Virus-like particles (VLPs) derived from human immunodeficiency virus type 1 (HIV-1) or murine leukemia virus (MLV) have emerged as promising delivery vehicles for RNP complexes, yet their activity is limited by suboptimal nuclease and guide RNA packaging. Previously, we generated NanoMEDIC VLPs incorporating the AsCas12a nuclease with CMV-driven crRNA, which demonstrated substantially enhanced editing efficiency over SpCas9-VLPs with U6-driven gRNA. Here, we describe a detailed protocol for a small-scale production of AsCas12a-VLPs using three distinct transfection methods [cationic lipids, polyethyleneimine (PEI), and calcium-phosphate] and a large-scale production of VLPs using calcium-phosphate transfection. We show that both production scales yield comparable nuclease loading into VLPs and similar editing efficiencies, reaching up to 60% of CXCR4 knockout in Jurkat T cells.},
}

@article {pmid42120643,
year = {2026},
author = {Cui, H and Peng, J and Song, J and Yang, Y and Hao, X},
title = {An ultrasensitive CRISPR-strand displacement amplification biosensor achieves piRNA-54265 detection and imaging in colorectal cancer cells.},
journal = {Analytical and bioanalytical chemistry},
volume = {418},
number = {14},
pages = {4613-4621},
pmid = {42120643},
issn = {1618-2650},
support = {20252BAC250153//Natural Science Foundation of Jiangxi Province/ ; 22277047//National Natural Science Foundation of China/ ; 82160631//National Natural Science Foundation of China/ ; },
mesh = {Humans ; *Biosensing Techniques/methods ; *Colorectal Neoplasms/genetics/diagnosis ; *Piwi-Interacting RNA/analysis/genetics ; *CRISPR-Cas Systems ; Limit of Detection ; *Nucleic Acid Amplification Techniques/methods ; Cell Line, Tumor ; *RNA, Small Interfering/analysis/genetics ; },
abstract = {PIWI-interacting RNAs (piRNAs) are well-recognized as promising diagnostic biomarkers for cancer, yet their quantitative detection remains a great challenge owing to their short sequences, low cellular abundance, high degradation susceptibility, and significant sequence homology among family members. Herein, we developed an ultrasensitive and highly specific biosensor for the detection of piRNA-54265-a colorectal cancer (CRC)-associated piRNA-by integrating strand displacement amplification (SDA) with the CRISPR/Cas12a system. After systematic optimization, the biosensor exhibited remarkably enhanced amplification efficiency and target specificity, achieving an ultra-low limit of detection (LOD) of 57.54 aM for piRNA-54265. Notably, this CRISPR-SDA platform enabled accurate discrimination of CRC cells from other cancer cells via high-fidelity intracellular imaging of piRNA-54265 and also realized reliable detection of the target in complex biological matrices with favorable recovery. Benefiting from its simple sequence design, user-friendly operation, and isothermal reaction conditions, the developed biosensor not only overcomes the inherent technical bottlenecks in piRNA detection but also shows great potential for applications in cellular imaging and early clinical diagnosis of CRC.},
}

Humans
*Biosensing Techniques/methods
*Colorectal Neoplasms/genetics/diagnosis
*Piwi-Interacting RNA/analysis/genetics
*CRISPR-Cas Systems
Limit of Detection
*Nucleic Acid Amplification Techniques/methods
Cell Line, Tumor
*RNA, Small Interfering/analysis/genetics

@article {pmid42130163,
year = {2026},
author = {Nomura, C and Kanzaki, H and Kanzaki, E and Shimizu, M and Oikawa, K and Utsushi, H and Ito, K and Sugimura, Y and Terauchi, R and Abe, A},
title = {Fine-tuning quantitative agronomic traits by manipulating gene copy number in rice.},
journal = {The New phytologist},
volume = {251},
number = {4},
pages = {1609-1616},
doi = {10.1111/nph.71258},
pmid = {42130163},
issn = {1469-8137},
support = {KAKENHI JP22K20584//Japan Society for the Promotion of Science/ ; KAKENHI JP23K26882//Japan Society for the Promotion of Science/ ; JPJ007097//Bio-oriented Technology Research Advancement Institution/ ; },
mesh = {*Oryza/genetics ; *Gene Dosage ; *Quantitative Trait, Heritable ; Plants, Genetically Modified ; Gene Expression Regulation, Plant ; Genes, Plant ; Phenotype ; Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems/genetics ; },
abstract = {Although plant pan-genome studies have revealed extensive copy number variations, their phenotypic consequences remain poorly understood. Here, we manipulated the copy number of OsMADS18 in rice (Oryza sativa) cv 'Hitomebore' using the CRISPR/Cas9 system. We established rice lines harboring one to three tandem copies of OsMADS18, as identified by quantitative PCR and sequencing. The presence of one to three OsMADS18 tandem copies was reflected in stepwise increases in transcript levels and concomitant agronomic trait values. These results demonstrate that manipulating gene copy number can fine-tune important quantitative traits, providing a novel breeding strategy for crop improvement.},
}

*Oryza/genetics
*Gene Dosage
*Quantitative Trait, Heritable
Plants, Genetically Modified
Gene Expression Regulation, Plant
Genes, Plant
Phenotype
Plant Proteins/genetics/metabolism
CRISPR-Cas Systems/genetics

@article {pmid42155537,
year = {2026},
author = {Albeladi, HA and Al-Zahrani, MH and Alghamdi, RA},
title = {Modulating claudin-2 with CRISPR-Cas9 to improve photodynamic therapy outcomes in colorectal cancer.},
journal = {Tissue & cell},
volume = {102},
number = {},
pages = {103586},
doi = {10.1016/j.tice.2026.103586},
pmid = {42155537},
issn = {1532-3072},
mesh = {Humans ; *Colorectal Neoplasms/genetics/pathology/drug therapy/therapy ; *Photochemotherapy ; Apoptosis/drug effects/genetics ; *CRISPR-Cas Systems/genetics ; Reactive Oxygen Species/metabolism ; *Claudin-2/metabolism/genetics ; Cell Movement/drug effects/genetics ; Chlorophyllides ; HCT116 Cells ; Gene Expression Regulation, Neoplastic/drug effects ; Porphyrins/pharmacology ; Claudins ; },
abstract = {Claudin-2 (CLDN2) is a tight junction protein that is overexpressed in colorectal cancer (CRC) and is associated with chemoresistance. Photodynamic therapy (PDT) is an emerging treatment that utilizes a photosensitizer (in this case, chlorin e6 [Ce6]) and light to generate cytotoxic reactive oxygen species (ROS). This paper investigated the influence of the combination of Ce6-PDT and CRISPR-Cas9-mediated CLDN2 knockout (KO) on the relative metabolic activity of the CRC cell line. CRISPR-Cas9 was used to produce HCT116 cells with CLDN2 KO. Ce6 was placed on the cells, and the red laser (659 nm, 6 J/cm[2]) was used to illuminate the cells. The relative metabolic activity, migration, Apoptosis, cell cycle, and ROS generation, gene expression, protein expression were measured by MTT assay, wound healing assay, flow cytometry, DCFH-DA method, RT-PCR, western blot and bioinformatics, respectively. PDT significantly decreased the relative metabolic activity and/or migration, more in CLDN2KO cells (p < 0.0001) than in the WT. The CLDN2KO cells had a high level of Apoptosis (46.56 ± 2.05%), compared to the WT (26.03 ± 6.72%), the p = 0.0072. The production of ROS was also increased to 779.51 % in CLDN2KO cells, which is higher than the production in WT cells at 767.10 %. Upregulation of P53 and BAX following PDT was greatly enhanced, and BCL2 expression was significantly reduced as compared to wild-type groups. Coexistence of Ce6-PDT with CLDN2 KO enhances the Apoptosis of the CRC cell line. The Western blot results showed a decrease in ZO-1 and Occludin proteins after Ce6-PDT. Bioinformatics analysis demonstrated that increased CLDN2 expression in CRC, associated with multiple genes and implicated in various cellular pathways. Although the initial results are encouraging regarding the possibility of synergy, further studies are needed to determine its effectiveness and safety in clinical practice. The results of this research suggest the possibility of a therapeutic approach aimed at enhancing the effectiveness of CRC treatment through genetic regulation with the aid of PDT.},
}

Humans
*Colorectal Neoplasms/genetics/pathology/drug therapy/therapy
*Photochemotherapy
Apoptosis/drug effects/genetics
*CRISPR-Cas Systems/genetics
Reactive Oxygen Species/metabolism
*Claudin-2/metabolism/genetics
Cell Movement/drug effects/genetics
Chlorophyllides
HCT116 Cells
Gene Expression Regulation, Neoplastic/drug effects
Porphyrins/pharmacology
Claudins

@article {pmid42284917,
year = {2026},
author = {Zhao, D and Peng, W and Liu, Z and Zhou, Q and Wu, L and Zhou, Y and Ran, F},
title = {A sensitive detection of C-reactive protein based on the combination of CRISPR/Cas13a, MNPs and RNase H.},
journal = {Journal of pharmaceutical and biomedical analysis},
volume = {280},
number = {},
pages = {117609},
doi = {10.1016/j.jpba.2026.117609},
pmid = {42284917},
issn = {1873-264X},
mesh = {*C-Reactive Protein/analysis ; Humans ; *Biosensing Techniques/methods ; *Ribonuclease H/chemistry/metabolism ; *CRISPR-Cas Systems/genetics ; Limit of Detection ; Aptamers, Nucleotide/chemistry ; *Magnetite Nanoparticles/chemistry ; },
abstract = {C-reactive protein (CRP) is a potential risk factor for disease. Here, developed a rapid and accurate fluorescence biosensor for detecting CRP, which contributes to early diagnosis and timely treatment of diseases. The CRP binds with the aptamer resulting in the probe 1 (P1) releasing from the complex of aptamer/P1/magnetic nanoparticles (MNPs). After magnetic separation, the free P1 hybridized with the RNA (P2) modified on the MNPs, leading to the P2 being multiple-turnover cut by ribonuclease H (RNase H). The formed free RNA can specifically bind with the crRNA and the tans-cleavage activity of CRISPR/Cas13a was triggered, leading to the RNA reporter containing a dye and quencher pair being cleaved and generating the fluorescence signal. This developed fluorescent biosensor takes full advantage of the synergy of aptamer, RNase H, MNPs and CRISPR/Cas13a. Here, the developed fluorescent biosensor exhibits excellent sensitivity and specificity towards the detection of CRP with a linear range from 10 pg/mL to 200 ng/mL. The detection limit is low down to 7.5 pg/mL. Additionally, this method successfully detected the CRP in human serum samples with satisfactory recoveries. Therefore, this developed biosensor will offer a valuable tool for the rapid diagnosis of CRP-related diseases.},
}

*C-Reactive Protein/analysis
Humans
*Biosensing Techniques/methods
*Ribonuclease H/chemistry/metabolism
*CRISPR-Cas Systems/genetics
Limit of Detection
Aptamers, Nucleotide/chemistry
*Magnetite Nanoparticles/chemistry

@article {pmid42341421,
year = {2026},
author = {Le, HT and Nghi, NB and My, VD and Vu, HA and Thanh, MC and Tri, BM and Niem, VVT and Phuong, HA},
title = {Functional characterization of PIK3CA E545A mutation in MCF-7 breast cancer cells reveals enhanced proliferation and resistance to Alpelisib.},
journal = {Biochemical and biophysical research communications},
volume = {829},
number = {},
pages = {154189},
doi = {10.1016/j.bbrc.2026.154189},
pmid = {42341421},
issn = {1090-2104},
mesh = {Humans ; *Class I Phosphatidylinositol 3-Kinases/genetics/antagonists & inhibitors/metabolism ; Cell Proliferation/drug effects/genetics ; *Breast Neoplasms/genetics/drug therapy/pathology ; Female ; *Drug Resistance, Neoplasm/genetics/drug effects ; MCF-7 Cells ; *Mutation ; *Thiazoles/pharmacology ; CRISPR-Cas Systems ; },
abstract = {PIK3CA mutations are central oncogenic drivers in hormone receptor-positive, HER2-negative breast cancer; however, the functional and therapeutic relevance of noncanonical variants remains incompletely defined. The E545A mutation, increasingly reported in specific patient populations, has not been systematically investigated. We generated an isogenic MCF-7 cell model harboring the PIK3CA E545A mutation using CRISPR/Cas9-mediated homology-directed repair to delineate its phenotypic and pharmacological consequences. E545A induced a robust gain-of-function phenotype, characterized by a mesenchymal-like morphological transition with reduced circularity and decreased cell size. This structural shift was accompanied by enhanced tumor cell fitness, including accelerated proliferation kinetics, increased metabolic activity, and significantly elevated clonogenic capacity compared with wild-type controls. Notably, growth trajectories showed sustained divergence between mutant and control cells across all time points, indicating a stable proliferative advantage. Importantly, E545A conferred diminished sensitivity to the PI3Kα inhibitor Alpelisib. Mutant cells retained migratory capacity under treatment and exhibited a pronounced, time-dependent increase in IC50, consistent with adaptive resistance. Collectively, these findings identify E545A as a functionally active and therapeutically consequential PIK3CA variant. Our study expands the current understanding of PIK3CA-driven oncogenic diversity beyond canonical hotspot mutations and underscores the need for variant-resolved stratification to improve the efficacy of PI3K-targeted therapies.},
}

Humans
*Class I Phosphatidylinositol 3-Kinases/genetics/antagonists & inhibitors/metabolism
Cell Proliferation/drug effects/genetics
*Breast Neoplasms/genetics/drug therapy/pathology
Female
*Drug Resistance, Neoplasm/genetics/drug effects
MCF-7 Cells
*Mutation
*Thiazoles/pharmacology
CRISPR-Cas Systems

@article {pmid42342973,
year = {2026},
author = {Duan, M and Meng, B and Zhou, L and Wu, L and Tong, X and Huang, D and Yin, H and Liu, ZJ and Zhang, Y},
title = {Structural basis of AtCas9 recognition of PAM mutants in underwound DNA topology.},
journal = {Nature structural & molecular biology},
volume = {33},
number = {7},
pages = {1062-1074},
pmid = {42342973},
issn = {1545-9985},
mesh = {Cryoelectron Microscopy ; *DNA/chemistry/metabolism/genetics ; Models, Molecular ; Mutation ; *Alicyclobacillus/enzymology/genetics ; Nucleic Acid Conformation ; CRISPR-Cas Systems ; *CRISPR-Associated Proteins/chemistry/metabolism ; },
abstract = {The CRISPR-Cas9 system locates targets through guide RNA pairing and recognition of a protospacer-adjacent motif (PAM). Although PAM specificity is sequence-determined, DNA topology can relax PAM requirements and enable near-PAMless cleavage by the type II-C Alicyclobacillus tengchongensis Cas9 (AtCas9). However, the structural mechanism underlying this regulation remains unknown. Here we report cryogenic-electron microscopy (cryo-EM) structures of AtCas9 bound to B-form DNA or a 340 bp underwound minicircle DNA containing wild-type or mutant PAMs. Despite PAM sequences differences, all three underwound complexes adopt an almost identical architecture distinct from the B-form DNA-bound state. On B-form DNA, AtCas9 recognizes the PAM through base-specific hydrogen bonds and steric exclusion, conferring preference for N4CNNN and N4RNNA (R = A/G). By contrast, underwound DNA widens the PAM major groove and promotes sequence-independent backbone contacts, explaining the near-PAMless cleavage. These findings uncover a topology-dependent mechanism of PAM recognition and establish a cryo-EM platform using underwound minicircle DNA for structural studies under native-like topological states.},
}

Cryoelectron Microscopy
*DNA/chemistry/metabolism/genetics
Models, Molecular
Mutation
*Alicyclobacillus/enzymology/genetics
Nucleic Acid Conformation
CRISPR-Cas Systems
*CRISPR-Associated Proteins/chemistry/metabolism

@article {pmid42432276,
year = {2026},
author = {Tian, Y and Li, M and Liu, C and Liu, T and Zhang, X and Liu, Q},
title = {Simultaneous detection of multiple foodborne pathogens using a CRISPR/Cas12a-based pump-free microfluidic chip.},
journal = {Analytical and bioanalytical chemistry},
volume = {},
number = {},
pages = {},
pmid = {42432276},
issn = {1618-2650},
abstract = {The development of microfluidic chips for nucleic acid detection provides efficient technical support for monitoring food safety. With the increasing maturity of CRISPR technology, it has the advantages of high specificity and high sensitivity in the detection of single or multiple nucleic acids. In this study, a microfluidic biosensor based on the CRISPR/Cas12a system was constructed using a pump-free microfluidic chip as the carrier, with a focus on the rapid, simultaneous detection of Listeria monocytogenes, Staphylococcus aureus, Escherichia coli O157:H7, and Cronobacter sakazakii. For each target pathogenic bacterium, two replicate channels for each pathogen were established, along with corresponding negative and positive controls, which effectively ensured the reliability and repeatability of the detected results and successfully achieved the simultaneous high-sensitivity, high-specificity, and high-accuracy detection of multiple foodborne pathogens. The detection sensitivity of the sensor for S. aureus, E. coli O157:H7, L. monocytogenes, and C. sakazakii was as low as 10[3] CFU/mL, 10[3] CFU/mL, 10[2] CFU/mL, and 10[3] CFU/mL, respectively. This integrated CRISPR/Cas12a sensor chip has the advantages of the simultaneous efficient detection of multiple pathogens, parallel verification, and control settings, and the detection results can be visualized by fluorescence, indicating broad application prospects in the field of on-site rapid nucleic acid analysis.},
}

@article {pmid42434562,
year = {2026},
author = {Liang, Z and Li, Z and Li, C and Zhao, Y and Liu, J and Zhang, J},
title = {Advances in gene editing tools for four typical Gram-positive bacteria.},
journal = {Frontiers in microbiology},
volume = {17},
number = {},
pages = {1882312},
pmid = {42434562},
issn = {1664-302X},
abstract = {Gram-positive bacteria serve as important chassis microorganisms in synthetic biology, industrial fermentation, and probiotic development. The rapid advancement of gene editing technologies has provided critical technical support for the iterative construction and functional validation of engineered strains. However, due to factors such as cell wall structure, differences in genetic backgrounds, and tool compatibility, the development and editing efficiency of gene editing systems for Gram-positive bacteria still face many challenges. This review focuses on four representative Gram-positive bacterial species-Lactobacillus plantarum, Lactococcus lactis, Bacillus subtilis, and Corynebacterium glutamicum-and traces the evolution and current state of their editing tools, from traditional homologous recombination to CRISPR-Cas9, base editors, and large-fragment integration tools. On this basis, we summarize the common challenges and corresponding strategies concerning host repair capacity, tool compatibility, and inherent limitations of editors in these four bacterial species, and propose recommendations for tool selection based on different application scenarios. This review aims to provide a technical reference for gene editing studies of the above-mentioned bacterial species. Although the conclusions cannot be directly extended to all Gram-positive bacteria, the common issues summarized here may inform the development of gene editing tools for other Gram-positive bacteria.},
}

@article {pmid42434939,
year = {2026},
author = {Wang, S and Hasan, R},
title = {CRISPR Biosensing for Environmental Monitoring: Workflow Design and Performance Benchmarking.},
journal = {Environmental science & technology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.est.6c02006},
pmid = {42434939},
issn = {1520-5851},
abstract = {CRISPR-based biosensing has rapidly emerged as a promising platform for environmental monitoring due to its high specificity, programmability, and compatibility with portable readouts. However, translation from biomedical diagnostics to environmental matrices remains challenging because of diverse sample types, complex inhibitors, and the breadth of biological and chemical targets. This Review provides a comprehensive analysis of CRISPR-based sensing technologies tailored for environmental contaminant detection, spanning both biological and chemical targets. We systematically evaluate published studies across target classes, Cas effectors, recognition mediators, sample matrices, pretreatment strategies, preamplification or signal-gain approaches, readout modalities, and reported performance metrics. To support practical implementation, we summarize a five-step experimental framework for environmental CRISPR sensing. We then propose a decision-guided design flowchart that links monitoring goals and matrix constraints to the selection of effectors, mediator-enabled transduction routes, pretreatment modules, amplification strategies, readouts, and validation controls. We further benchmark reported detection limits by normalizing units and comparing trends across preamplification-aided versus preamplification-free designs and by contextualizing performance against relevant regulatory or guideline thresholds when available. Across the literature, most studies rely on spiked-matrix validation, highlighting the need for broader nonspiked real environmental sample testing and more transparent reporting of sampling, pretreatment, and performance evaluation. Finally, we advocate standardized data reporting, including consistent units, workflow metadata, and matrix-matched validation, to enable cross-study comparison and accelerate the deployment of CRISPR-based sensors for real-world environmental monitoring.},
}

@article {pmid42435220,
year = {2026},
author = {Khan, MA and Durand, A and Skouri-Panet, F and Benzerara, K and Cassier-Chauvat, C and Chauvat, F and Ouchane, S},
title = {Targeted genome editing of the non-model cyanobacterium Cyanothece PCC 7425 via CRISPR/Cas12a.},
journal = {Applied microbiology and biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1007/s00253-026-13959-y},
pmid = {42435220},
issn = {1432-0614},
support = {ANR-19-CE44-0017//Agence Nationale de la Recherche/ ; ANR-19-CE44-0017//Agence Nationale de la Recherche/ ; },
abstract = {Cyanobacteria are diverse photosynthetic microorganisms of great interest for fundamental science and sustainable biotechnological applications. However, their polyploidy makes genetic manipulation challenging and time-consuming. The development of CRISPR/Cas tools has greatly accelerated genome editing and metabolic engineering of some cyanobacterial model species. In this work, we extend the CRISPR/Cas12a system for targeted gene deletion in the non-model cyanobacterium Cyanothece sp. PCC 7425, interesting for its ability to perform intracellular calcium carbonate (CaCO3) biomineralization, nitrogen fixation, etc. We demonstrate for the first time its tractability to gene knockout by generating deletion mutants of four genes (cax3-cax4, gor, and sodB) acting in metabolism and/or response to stresses, using Cas12a-mediated homologous recombination. Importantly, full chromosome segregation was rapidly achieved after a single round of selection in all cases. All mutants were genotypically and phenotypically characterised. Moreover, biochemical analysis in the case of the ΔsodB mutant further confirmed its targeted deletion. Overall, CRISPR/Cas12a provides a rapid and efficient system for genome editing in Cyanothece sp. PCC 7425, establishing this organism as a versatile model for studying oxidative stress pathways, metal toxicity, and moreover, the still poorly known mechanism(s) of intracellular CaCO3 biomineralization. KEY POINTS: • Rapid and efficient CRISPR/Cas12a editing established in Cyanothece sp. PCC 7425. • Fully segregated knockout mutants obtained after a single selection round. • Platform for exploring the biotechnological potential of Cyanothece sp. PCC 7425.},
}

@article {pmid42436385,
year = {2026},
author = {Vásquez-Herrera, L and Vallejos, OP and Acevedo-López, J and Campos-Gajardo, S and Piña-Iturbe, A and Tichy-Navarro, D and Seeram, D and Uhlemann, AC and González, PA and Kalergis, AM and Moreno-Switt, AI and Bueno, SM},
title = {Whole genome sequence-based comparative genomics reveals preliminary genomic features of Salmonella enterica subsp. enterica serovar Enteritidis phage type 1 and phage type 4 strains from EnteroBase.},
journal = {BMC microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1186/s12866-026-05329-5},
pmid = {42436385},
issn = {1471-2180},
support = {N° 21251772//ANID National Doctoral Fellowship/ ; N° 1240971//Fondo Nacional de Desarrollo Científico y Tecnológico/ ; N° 1231851//Fondo Nacional de Desarrollo Científico y Tecnológico/ ; N° 1231905//Fondo Nacional de Desarrollo Científico y Tecnológico/ ; ICN 2021_045//Millennium Institute of Immunology and Immunotherapy/ ; ECOS220027//ECOS-ANID/ ; },
abstract = {BACKGROUND: Whole Genome Sequencing (WGS) enables detailed characterization of circulating and emerging bacterial strains. Although tens of thousands of Salmonella genomes have been acquired over the years, analyses of the genomic differences between strains of different phage types are scarce.

RESULTS: We compared two Salmonella enterica subsp. enterica serovar Enteritidis (SEn) phage types, namely phage types 1 and 4 from available databases, using bioinformatic tools and nanopore sequencing of a Chilean PT1 strain. Comparisons between the two phage types show very low genomic divergence and high genomic sequence similarity. Single nucleotide polymorphism (SNP) searches identified SNPs specific to each phage type. Although a translocated region was identified in the Chilean PT1 strain analyzed in this study when compared to the genome of a PT4 strain, this was not present in the genomes of other PT1 strains, suggesting a local strain-specific rearrangement. Further analyses yielded no differences in the CRISPR-Cas locus, but a slight difference was observed in Gifsy-2 prophage detection and DNA modification systems between PT1 and PT4 strains.

CONCLUSIONS: Our findings provide insights into the genomic differences between SEn strains of two different phage types, serving as a basis for future genomic studies, yet further analyses with more diverse geographical locations collected over a longer time span are essential to validate these differences with the potential to establish molecular markers for strain identification and characterization in the context of epidemiological surveillance as a complement to WGS when this technique is not available.},
}

@article {pmid42437521,
year = {2026},
author = {Asemoloye, MD},
title = {Enhancing the Secretion Systems: Genetic Engineering of Super Bioagents for Effective Plant Disease Control.},
journal = {Biotechnology and bioengineering},
volume = {},
number = {},
pages = {},
doi = {10.1002/bit.70303},
pmid = {42437521},
issn = {1097-0290},
abstract = {The escalating threat of plant diseases to global agriculture and food security necessitates innovative and sustainable control strategies. Conventional biological control agents (BCAs), while environmentally friendly, often suffer environmental challenges and secretion of limited/poor antimicrobial compounds. Advances in CRISPR/Cas genome editing, protease engineering, and synthetic biology have enabled precise modifications that improve pathogen targeting and secretion efficiency. Interest should now be shifted on development of "Super Bioagents (SBs)" with enhanced secretion systems (SSs) for plant disease suppression against changing environmental factors. This will create sustainable ecofriendly alternative to chemical pesticides. This review explores a detailed overview of molecular mechanisms of microbial SSs and the potentials of SBs as a frontier in plant disease management. While there are still challenges in mass deployment of BCAs in sustainable agriculture, this review is guided by the hypothesis that rational, quantitative engineering of microbial SSs can transform conventional BCAs into integrated SBs. It synthesizes current advances within a systems‑level bioengineering framework linking secretion efficiency, regulation, and field performance. It further explores possible integration of SBs in plant-microbiome interactions to further enhance their adaptability and effectiveness. Finally, the review dives into recent breakthroughs, current challenges, and future directions for SBs development and application as next-generation plant disease control agents.},
}

@article {pmid42438474,
year = {2026},
author = {Ravendran, S and Fammé, S and Noer, MG and Skov, TW and Mikkelsen, NS and Lee Schneller, J and Dorset, SR and Wolff, JH and Møller, AMJ and Haslund, D and Revenfeld, ALS and Holm, M and Mogensen, TH and Møller, BK and Mikkelsen, JG and Bak, RO},
title = {AAV vector production in suspension cells using PEI transfection and sodium butyrate with orthogonal assessment of function and quality.},
journal = {Molecular therapy. Advances},
volume = {34},
number = {3},
pages = {201787},
pmid = {42438474},
issn = {3117-387X},
abstract = {Adeno-associated virus (AAV) vectors are widely used in gene therapy, yet academic in-house production remains dominated by labor-intensive adherent cell workflows with limited scalability. Here, we describe an AAV vector production platform using suspension cells in orbital shaking Erlenmeyer flasks, based on polyethyleneimine (PEI) transfection and sodium butyrate supplementation. Following systematic evaluation of transfection conditions, this approach yields vectors with performance comparable to a commercial production kit. Vector quality was interrogated using orthogonal methodologies, including two-dimensional ddPCR, mass photometry, and nanopore sequencing, enabling comparative assessment of genome packaging, capsid composition, and vector heterogeneity. Functional validation was performed by in vitro transduction of K562 cells and primary human CD34+ hematopoietic stem and progenitor cells, as well as in vivo gene delivery to mouse liver and heart. Across assays, vectors produced using this protocol demonstrated comparable genome integrity and transgene expression. Comparative purification analysis revealed that iodixanol density gradient purification resulted in higher proportions of full capsids and reduced producer-cell-derived impurities relative to PEG 8000 precipitation. Together, this work establishes a scalable suspension-based AAV production workflow and demonstrates the value of orthogonal quality assessment combined with functional validation for robust vector benchmarking.},
}

@article {pmid42438737,
year = {2026},
author = {Faleiros, CA and Gonçalves, OS and Nunes, AT and Pires, CS and Poleti, MD and Fukumasu, H},
title = {Host breed and geography shape the antiviral defense landscape of the bovine rumen microbiome.},
journal = {ISME communications},
volume = {6},
number = {1},
pages = {ycag162},
pmid = {42438737},
issn = {2730-6151},
abstract = {The rumen microbiome represents a complex, phage-rich ecosystem where microbial survival depends on both metabolic cooperation and antiviral defense. However, global and breed-associated variations in rumen prokaryotic immune systems remain poorly understood. Here, we performed the most comprehensive profile to date of antiviral defense systems (DS) in the rumen, analyzing 6530 microbial genomes and metagenome-assembled genomes (MAGs) from diverse cattle breeds and geographic regions. In this global dataset, we identified >90 000 DS, the most abundant of which were restriction-modification, PDC-S01, deoxyribonucleic acid modification systems (DMS_other), AbiE and SoFic, with variations influenced by both host the lineage and geographic region. A more in-depth analysis was performed using two complementary antiviral annotation frameworks for Nellore cattle (Bos indicus) from Brazil. Data exhibited a remarkably enriched antiviral defense repertoire, with over 15 632 DS encoded across 547 high-quality MAGs. These systems were densely clustered in dominant rumen lineages, such as Prevotella, and positively correlated with prophage abundance, consistent with virus-host coevolution. Notably, we also detected viral contigs encoding both antiviral defense and anti-defense genes, underscoring the arms race between the phages and their microbial hosts. Metatranscriptomic data from North America and Oceania revealed high expression levels of toxin-antitoxin modules, clustered regularly interspaced short palindromic repeats components, and restriction enzymes, suggesting a basal level of antiviral activity. These findings reveal the rumen as an antiviral innovation hotspot, highlighting microbiome resilience with implications for ecology, adaptation, and phage-based interventions.},
}

@article {pmid42439519,
year = {2026},
author = {Iyer, MS and Hagström, E and Näslund, K and Andersson, SGE},
title = {Harnessing endogenous CRISPR-Cas9 for inducible genetic engineering of Apilactobacillus kunkeei.},
journal = {Applied and environmental microbiology},
volume = {},
number = {},
pages = {e0072826},
doi = {10.1128/aem.00728-26},
pmid = {42439519},
issn = {1098-5336},
abstract = {UNLABELLED: Despite substantial advances in bacterial genome engineering, functional genetic analysis remains challenging in many non-model bacterial species, particularly among host-associated gram-positive bacteria. The fructophilic species Apilactobacillus kunkeei has been investigated for more than two decades and is a dominant member of the honeybee microbiome, where it contributes to pathogen resistance and colony fitness. Nevertheless, the mechanistic investigation of this ecologically important species has remained limited despite its growing probiotic relevance. To enable functional genomics in this organism, we developed an inducible genome-engineering platform that leverages its endogenous Type II-A CRISPR-Cas9 system. The system uses a sakacin-responsive dual-plasmid initiator-effector design in which phage-derived recombineering genes and a single-guide RNA are coordinately expressed, while DNA cleavage is mediated by natively expressed Cas9. Using this approach, we achieved scarless deletion of individual genes, including targets as large as ~25 kb, gene replacement with a fluorescent reporter, C-terminal epitope tagging, and precise nucleotide substitutions, with editing efficiencies approaching 100%. Both plasmids can be readily cured following modification, allowing recovery of clean mutant genotypes. We further demonstrate that endogenous Cas9 can be repurposed for CRISPR interference using a single, self-contained plasmid to enable targeted transcriptional repression. Together, this work establishes a robust strategy for genetic manipulation of A. kunkeei and expands the toolkit available for harnessing endogenous CRISPR-Cas systems in genetically recalcitrant, non-model gram-positive bacteria.

IMPORTANCE: Many ecologically and industrially important bacteria remain genetically recalcitrant, limiting functional genomic studies. As research increasingly extends beyond traditional model organisms, these limitations are especially apparent in non-model gram-positive bacteria from host-associated or environmental niches. Here, we establish an inducible genome-editing framework exploiting the endogenous Cas9 system of Apilactobacillus kunkeei, a key member of the honeybee microbiota. This approach enables reliable scarless gene deletions, precise nucleotide changes, large-scale genome modifications, and programmable transcriptional repression. By enabling genetic manipulation in A. kunkeei, this work facilitates experimental studies of its roles in honeybee health, microbial interactions, and host-associated adaptation, and highlights the potential of endogenous CRISPR-Cas systems for expanding genetic access in non-model bacteria.},
}

@article {pmid42135407,
year = {2026},
author = {Bazick, HO and James, LM and Zylka, MJ},
title = {Nickase NmCas9 unsilences paternal Ube3a in a mouse model of Angelman syndrome without causing AAV vector integration.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {42135407},
issn = {2045-2322},
support = {631904//Simons Foundation/ ; 1R01NS109304/NS/NINDS NIH HHS/United States ; 1R01NS109304/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; *Ubiquitin-Protein Ligases/genetics/metabolism ; Mice ; *Dependovirus/genetics ; *Angelman Syndrome/genetics/therapy/metabolism ; Genetic Vectors/genetics ; Disease Models, Animal ; Neurons/metabolism ; *Deoxyribonuclease I/metabolism/genetics ; Gene Editing/methods ; Male ; Virus Integration ; CRISPR-Cas Systems ; },
abstract = {Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by loss of maternal UBE3A. In neurons, the paternal (pat)UBE3A allele is silenced by a long non-coding antisense transcript called Ube3a-ATS. Previous genome-editing approaches used active nucleases to unsilence patUbe3a by disrupting Ube3a-ATS. However, these methods create DNA double-strand breaks (DSBs) and promote integration of adeno-associated virus (AAV) vector genomes, both of which raise potential safety concerns. Here, we found that a nickase Neisseria meningitidis Cas9 variant (nNmCas9-D15A) disrupted Ube3a-ATS transcription when targeted to the non-template strand and unsilenced patUbe3a in cultured mouse neurons without generating DSBs or causing AAV integration. Intracerebroventricular delivery of AAV9-nNmCas9-D15A in AS model mice potently and durably reduced Ube3a-ATS and elevated Ube3a throughout the cerebral cortex and hippocampus for at least 6 months. Further, this vector restored UBE3A expression in ~ 87% of cortical neurons, which compares favorably to previously reported efficiencies with active Cas9, dead Cas9, and zinc finger nuclease vectors. These results demonstrate that nNmCas9 is a highly effective and potentially safer genome editor for the treatment of AS.},
}

Animals
*Ubiquitin-Protein Ligases/genetics/metabolism
Mice
*Dependovirus/genetics
*Angelman Syndrome/genetics/therapy/metabolism
Genetic Vectors/genetics
Disease Models, Animal
Neurons/metabolism
*Deoxyribonuclease I/metabolism/genetics
Gene Editing/methods
Male
Virus Integration
CRISPR-Cas Systems

@article {pmid42303099,
year = {2026},
author = {Zhou, Z and Dong, S and Li, S and Tan, X and Zhou, Z and Fan, L and Liang, J and Li, G},
title = {CRISPR/Cas12a and nanocomposite-based electrochemical/ colorimetric parallel dual-channel aptasensor for highly sensitive LDL detection.},
journal = {Nanomedicine : nanotechnology, biology, and medicine},
volume = {75},
number = {},
pages = {102981},
doi = {10.1016/j.nano.2026.102981},
pmid = {42303099},
issn = {1549-9642},
mesh = {*Nanocomposites/chemistry ; *Aptamers, Nucleotide/chemistry ; *Biosensing Techniques/methods ; *Lipoproteins, LDL/analysis/blood ; Humans ; *Electrochemical Techniques/methods ; *CRISPR-Cas Systems/genetics ; *Colorimetry/methods ; Graphite/chemistry ; DNA, Single-Stranded/chemistry ; },
abstract = {Atherosclerotic cardiovascular disease (ASCVD) remains a leading global health threat, necessitating precise monitoring of low-density lipoprotein (LDL) as a key risk biomarker for assessing ASCVD risk. Herein, an electrochemical/colorimetric dual- channel aptasensor was developed by integrating nitrogen-doped reduced graphene oxide-Hemin-trimanganese tetroxide nanoparticles (NrGO-Hemin-Mn3O4 NPs) with the CRISPR/Cas12a system. The CRISPR/Cas12a system introduces a powerful signal amplification cascade: a single target binding event activates the trans-cleavage of numerous ssDNA probes, translating into a highly amplified electrical and optical response. The NrGO-Hemin-Mn3O4 NPs serves as a conductive redox probe and exhibits superior peroxidase-like activity through the synergistic effect between Hemin and Mn3O4. Mechanistically, surface-bound single-stranded DNA (ssDNA) initially induces steric hindrance, which obstructs electron transfer and suppresses the enzyme-mimicking performance of the NrGO-Hemin-Mn3O4 NPs. Upon the target LDL binding, the released activator DNA triggers the trans-cleavage activity of Cas12a to degrade the ssDNA, thereby restoring both the electroactivity and catalytic performance of the probe. Experimental results demonstrated that the dual-channel aptasensor achieved a wide linear range from 0.01 to 1000 nM with a detection limit of 0.01 nM, demonstrating that CRISPR integration is pivotal for achieving high sensitivity in complex biological matrices. This dual-channel strategy offers a sensitive, intuitive tool for early clinical screening of ASCVD diseases.},
}

*Nanocomposites/chemistry
*Aptamers, Nucleotide/chemistry
*Biosensing Techniques/methods
*Lipoproteins, LDL/analysis/blood
Humans
*Electrochemical Techniques/methods
*CRISPR-Cas Systems/genetics
*Colorimetry/methods
Graphite/chemistry
DNA, Single-Stranded/chemistry

@article {pmid42420735,
year = {2026},
author = {Rijal, S and Zhang, R and Tian, XJ},
title = {Harnessing CRISPRi Competition to Develop Multimodule Controllers for Resource-Aware Circuit Design.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3041},
number = {},
pages = {305-318},
pmid = {42420735},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *Gene Regulatory Networks ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Genetic Engineering/methods ; },
abstract = {Cellular resource limitations give rise to resource competition, undermining the modularity and predictability of engineered genetic circuits. In systems containing positive feedback, such competition can drive Winner-Takes-All (WTA) dynamics, resulting in severe imbalances in resource allocation across circuit modules. In this chapter, we present an experimental implementation of a Negatively Competitive Regulatory (NCR) controller based on CRISPR interference (CRISPRi) in dual self-activation (DSA) circuits. We describe a detailed workflow for chromosomal integration of a tunable dCas9 expression cassette, as well as the design of self-activation modules and module-specific guide RNAs that induce self-repression through competition for limiting dCas9. This architecture introduces effective negative feedback to the more active module while reallocating resources to the less active one, thereby promoting balanced module activity. Finally, we provide guidelines for quantitatively assessing the regulation of resource competition between DSA modules using the NCR strategy. Overall, these guidelines demonstrate how CRISPRi can be leveraged to implement NCR strategy in gene circuits, thereby enhancing circuit modularity and predictability.},
}

*CRISPR-Cas Systems
*Gene Regulatory Networks
RNA, Guide, CRISPR-Cas Systems/genetics
*Genetic Engineering/methods

@article {pmid42423795,
year = {2026},
author = {Ata, A and Topuz Ata, D},
title = {A comprehensive review of CRISPR-Cas9-mediated genome editing in Leishmania strains: methodologies, applications, challenges and future directions.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42423795},
issn = {1573-4978},
mesh = {*Leishmania/genetics ; *Gene Editing/methods/trends ; *CRISPR-Cas Systems/genetics ; Animals ; Genome, Protozoan ; Leishmaniasis/parasitology/genetics ; Humans ; },
abstract = {Genome editing employing CRISPR-Cas9 has rapidly transformed experimental research in Leishmania, providing opportunities to investigate the genetic factors responsible for parasite survival, response to drugs and pathogenic traits. This review provides a comprehensive synthesis of CRISPR-based systems implemented across Leishmania species, spanning Cas9-mediated gene deletion, precise genome editing, endogenous locus tagging and pooled screening strategies. Furthermore, we highlight the emergence of Cas variants and next-generation CRISPR systems which expand the range of targetable genomic regions, improve editing precision and reduce the need for generation of double-strand DNA breaks (DSBs). Particular emphasis is placed on conditional and inducible genome-editing platforms, cytosine base-editing technologies, and recently developed CRISPR-based approaches such as prime editing, CRISPR activation/interference and Cas12-associated implementations. This review also discusses the principal biological and technical constraints influencing CRISPR-based studies in Leishmania, including genome plasticity, multicopy gene families, required genes, guide RNA design limitations and off-target considerations. Notably, the review also addresses CRISPR-Cas implementations in sand-fly vector biology, drawing on a foundational study in Phlebotomus papatasi. Through systematic compilation of published studies into comparative tables, we evaluate the strengths, limitations, experimental utility, delivery strategies, experimental workflows and representative applications of major CRISPR platforms. Together, these advances highlight the transition of CRISPR-Cas systems from proof-of-concept tools to versatile platforms for functional genomics, target validation and translational research in Leishmania, while offering a consolidated guide for selecting suitable CRISPR-Cas technologies and underscoring important considerations for their continued development in leishmaniasis research.},
}

*Leishmania/genetics
*Gene Editing/methods/trends
*CRISPR-Cas Systems/genetics
Animals
Genome, Protozoan
Leishmaniasis/parasitology/genetics
Humans

@article {pmid42424815,
year = {2026},
author = {Tang, Q and Zhang, Y and Garza, DR and Ruan, C and Liu, B and Rocha, U and Shen, P and Wei, Y and Deng, Y and Zhang, J and Richnow, HH},
title = {Virus-mediated fate of antimicrobial resistance genes in livestock manure anaerobic digestion.},
journal = {Water research},
volume = {305},
number = {},
pages = {126401},
doi = {10.1016/j.watres.2026.126401},
pmid = {42424815},
issn = {1879-2448},
abstract = {Antimicrobial resistance (AMR) poses a critical global health challenge, with livestock manure acting as a significant environmental reservoir for antimicrobial resistance genes (ARGs). Anaerobic digestion (AD) is a pivotal process for mitigating ARG dissemination at the livestock-environment-human interface. This study aims to elucidate the global dynamics of ARGs in AD systems, focusing on virus-host interactions and arms race, to identify actionable strategies for AMR control. We analyzed 205 metagenomic (4.5 Tb) and 36 meta-transcriptomic (640 Gb) datasets, including 15 newly generated datasets, revealing that pig manure AD harbors the highest ARG abundance (0.668 ARGs/16S rRNA), while AD systems generally exhibit limited transcriptional activation of ARGs. We constructed a viral dataset for livestock manure AD (GVD_LMAD), comprising 59,316 DNA and 727 RNA viral operational taxonomic units (vOTUs). Virus-host interactions established by CRISPR-Cas spacer, tRNA and homology matches revealed 889 lytic infections of antimicrobial-resistant bacteria (ARB) compared to only 18 ARG transduction events. Further analysis showed that the relative abundance of vOTUs assigned to the reduction role (4.11% ± 3.19%) was substantially higher than that of reproduction (0.72% ± 0.64%) and transduction (0.19% ± 0.30%), demonstrating that, among viral processes, lysis outweighs transduction in contributing to ARG abundance reduction in AD. Furthermore, an antiviral defense system (ADS) catalogue (GADSC_LMAD), derived from 2760 high-quality metagenome-assembled genomes (MAGs) containing 39,307 ADS, with ADS prevalence in ARB (7.8 ± 6.0 per MAG), indicating an intensified virus-host arms race in AD that may shield ARB from phage lysis. The resulting CRISPR-Cas immune network with expressed spacers targets foreign ARG-carrying sequences (primarily plasmids and ICEs), suggesting a mechanism that restricts horizontal gene transfer (HGT) via conjugation and transformation, despite shielding ARB from phage lysis. Collectively, these findings highlight that viral communities significantly contribute to ARG reduction through phage lysis relative to transduction, while the ADS-mediated arms race, despite protecting ARB, constructs a biological firewall that potentially limits HGT of ARGs. This study provides novel insights into virus-host dynamics as a key mechanism for controlling ARG dissemination in AD systems.},
}

@article {pmid42424969,
year = {2026},
author = {Samad, MA and Ahmad, I and Jabir, NR and Rehan, M and Zaidi, SK and Al-Abbasi, F and Tabrez, S},
title = {Role of long non-coding RNAs in therapeutic resistance and clinical applications in cancer.},
journal = {European journal of medicinal chemistry},
volume = {317},
number = {},
pages = {119090},
doi = {10.1016/j.ejmech.2026.119090},
pmid = {42424969},
issn = {1768-3254},
abstract = {Cancer is one of the leading causes of mortality worldwide and is recognized as a complex, multifactorial disease with no clearly defined etiology for its onset and progression. Long non-coding RNAs (lncRNAs) are widely distributed across the human body and play varied roles in regulating cellular processes. In recent years, they have gained the attention of the scientific community as key regulators of cancer due to their diverse functional roles and complex regulatory mechanisms. Aberrant expression of lncRNAs contributes to tumor progression, functioning as oncogenes that modulate various pathways through different mechanisms. Early technologies could not study lncRNAs effectively and considered it as "junk" RNA. Studies using gene-expression analyses, functional experiments, and animal-based models have shown that dysregulated lncRNAs are implicated in the maintenance of cancer stem cells (CSCs) and in driving therapeutic resistance. Additionally, lncRNAs have shown promise as valuable biomarkers for cancer diagnosis, prognosis, predicting patient outcomes, and guiding treatment strategies. Moreover, therapeutic strategies targeting lncRNAs, such as antisense oligonucleotides (ASOs), RNA interference (RNAi), exosome-based delivery systems, nanomedicine, virus-mediated therapy, and CRISPR-Cas technologies, have opened new avenues for cancer treatment. This review highlights the diverse roles of lncRNAs in therapeutic resistance and emphasizes their clinical potential as diagnostic and prognostic tools and emerging therapeutic strategies.},
}

@article {pmid42425355,
year = {2026},
author = {V, MS and Chaudhary, N and Hasan, M and Kumar, A and Tripathi, MK},
title = {Filamentous fungi as microbial cell factories for lignocellulosic biomass valorization: A comprehensive review.},
journal = {International journal of biological macromolecules},
volume = {375},
number = {},
pages = {153415},
doi = {10.1016/j.ijbiomac.2026.153415},
pmid = {42425355},
issn = {1879-0003},
abstract = {The transition toward a sustainable bioeconomy requires efficient conversion of lignocellulosic biomass (LCB), the most abundant renewable biological macromolecular resource on Earth, into fuels, chemicals, and other high-value products. However, the complex architecture of cellulose, hemicellulose, and lignin imparts significant recalcitrance, limiting biomass deconstruction and industrial utilization. Although recent reviews have examined fungal biorefineries, lignocellulolytic enzymes, or fungal strain engineering separately, an integrated synthesis linking lignocellulosic biomass characteristics, fungal deconstruction mechanisms, hydrolysate utilization, and cell-factory engineering remains limited. This review presents an integrated framework for lignocellulosic biomass valorization using filamentous fungi as microbial cell factories. We examine biomass composition, recalcitrance, and pretreatment strategies, followed by the fungal macromolecular machinery responsible for biomass deconstruction, including cellulases, hemicellulases, lignin-active oxidoreductases, and auxiliary activity enzymes. Particular emphasis is placed on the regulatory networks and engineering strategies that govern fungal performance, including transcription factor engineering, promoter engineering, metabolic rewiring, heterologous pathway engineering, RNA interference, and CRISPR-Cas-based genome editing. The review further discusses the conversion of lignocellulose-derived hydrolysates into biofuels, organic acids, industrial enzymes, and other high-value compounds, together with emerging advances in co-culture fermentation, downstream processing, and integrated biorefinery design. Collectively, this review highlights how the integration of fungal enzymatic systems, strain engineering, and process-level innovations can overcome biomass recalcitrance and improve lignocellulosic bioconversion efficiency. These insights provide a framework for developing robust fungal platforms for the sustainable production of high-value bioproducts from renewable biomass.},
}

@article {pmid42426280,
year = {2026},
author = {Grigg, S and Shembrey, C and Fareh, M and Blombery, P and Corn, JE and Seymour, JF and Casan, JML},
title = {CRISPR in clinical oncology: translational advances from molecular diagnostics to therapeutics.},
journal = {Nature reviews. Clinical oncology},
volume = {},
number = {},
pages = {},
pmid = {42426280},
issn = {1759-4782},
abstract = {Cancer care is increasingly driven by molecular classification, yet many key oncogenic drivers remain undruggable, and intrinsic or acquired resistance to treatment frequently limits durable clinical benefit. CRISPR-Cas technologies provide a modular, programmable platform to interrogate and directly manipulate cancer biology via sequence-specific targeting of DNA or RNA and have advanced from experimental tools to the early stages of clinical translation. In this Review, we outline how CRISPR-enabled functional genomics approaches can reveal unexpected cancer dependencies and resistance mechanisms. We discuss emerging applications of CRISPR-based diagnostics in oncology that convert precise nucleic acid sequence recognition into rapid mutation detection. We also discuss applications of CRISPR in therapeutic strategies ranging from ex vivo immune cell engineering to nascent in vivo interventions that directly target tumour-related sequences such as fusion junctions or single-nucleotide variants. Finally, we highlight technological and regulatory challenges, including effective delivery of the editing machinery to cells in vivo, safety and platform-level regulatory frameworks, that will determine the clinical utility of CRISPR-based diagnostics and therapies in oncology.},
}

@article {pmid42427176,
year = {2026},
author = {Łakomy, W and Myślińska, M and Tarnawska, E and Rogóż, W and Kulig, K and Owczarzy, A and Maciążek-Jurczyk, M},
title = {Biotechnological strategies to combat antibiotic resistance.},
journal = {Polimery w medycynie},
volume = {56},
number = {1},
pages = {41-51},
doi = {10.17219/pim/218777},
pmid = {42427176},
issn = {0370-0747},
mesh = {Humans ; Antimicrobial Peptides/pharmacology ; Phage Therapy ; *Biotechnology ; CRISPR-Cas Systems ; Gene Editing ; *Drug Resistance, Microbial ; *Drug Resistance, Bacterial ; Anti-Bacterial Agents/pharmacology ; Nanoparticles ; Nanotechnology ; Animals ; },
abstract = {This article aims to present the current state of knowledge on four major biotechnological antimicrobial strategies and to evaluate their potential clinical applications in the context of increasing antibiotic resistance. Approaches such as phage therapy, CRISPR-Cas9 gene editing, nanoparticles, and antimicrobial peptides (AMPs) may significantly contribute to limiting the spread of resistance genes. Particular attention is given to advances in genetic engineering that enable precise targeting and elimination of resistance determinants, as well as to the therapeutic potential of the microbiome. A literature review of studies published between 2010 and 2025 was conducted using the following keywords: antimicrobial resistance, phage therapy, CRISPR-Cas9, AMPs, and nanotechnology. Both review articles and original studies, including preclinical and clinical data, were considered. Phage therapy demonstrates high efficacy against antibiotic-resistant pathogens, particularly in the form of phage cocktails and genetically engineered phages. Antimicrobial peptides exhibit broad-spectrum activity and can be structurally optimized to improve stability and selectivity. CRISPR-Cas9 systems enable targeted elimination of resistance genes or direct disruption of pathogen genomes, while nanotechnology facilitates drug delivery, biofilm penetration, and bactericidal activity, particularly through metal-based nanoparticles. Notably, all approaches show potential for synergistic use with conventional antibiotics. Biotechnological treatment strategies may become a key component in combating antibiotic resistance. However, their clinical implementation requires further research, comprehensive safety evaluation, regulatory development, and integration into medical practice. Advances in these areas could significantly reduce the global burden of infectious diseases.},
}

Humans
Antimicrobial Peptides/pharmacology
Phage Therapy
*Biotechnology
CRISPR-Cas Systems
Gene Editing
*Drug Resistance, Microbial
*Drug Resistance, Bacterial
Anti-Bacterial Agents/pharmacology
Nanoparticles
Nanotechnology
Animals

@article {pmid42428244,
year = {2026},
author = {Zhang, X and Shi, H and Yang, J and Du, L and Zhang, X and Li, X},
title = {The application of CRISPR gene-editing technology in influenza prevention and control.},
journal = {Frontiers in genome editing},
volume = {8},
number = {},
pages = {1844919},
pmid = {42428244},
issn = {2673-3439},
abstract = {Influenza A virus (IAV) and influenza B virus (IBV) remain major global public health threats because of their rapid antigenic evolution and efficient human-to-human transmission. In contrast, influenza C virus (ICV) and influenza D virus (IDV) generally exhibit narrower host ranges and milder pathogenicity, yet their potential for interspecies transmission and zoonotic spillover still warrants attention. Conventional prevention strategies, such as inactivated and live-attenuated vaccines, suffer from prolonged development timelines and diminished efficacy against rapidly evolving viral strains. However, antiviral drugs are increasingly limited by the rapid emergence of drug-resistant variants. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) gene-editing technology has emerged as a promising platform for influenza prevention and control owing to its programmability and precise targeting capability. In this paper, we summarize recent advances in CRISPR-based strategies for influenza prevention and control. The RNA-targeting CRISPR-associated protein 13 (Cas13) system can recognize conserved viral RNA sequences and suppress replication across influenza subtypes, whereas the DNA-targeting CRISPR-associated protein 9 (Cas9) system can edit host susceptibility genes and thereby reduce cellular permissiveness to infection. In addition, lipid nanoparticle (LNP)-based delivery systems have become important tools for improving the in vivo delivery and expression of CRISPR components by enhancing targeting efficiency and reducing immunogenicity. CRISPR-based diagnostics, such as Specific High-sensitivity Enzymatic Reporter unLOCKing (SHERLOCK), further expand the clinical utility of this technology by enabling rapid and sensitive detection of influenza viruses. Despite these advances, substantial challenges remain, including delivery inefficiency, off-target activity, long-term safety concerns, and the risk of viral escape. With continued technological refinement and careful translational development, CRISPR may become a versatile tool for influenza prevention, diagnosis, and therapy.},
}

@article {pmid42429865,
year = {2026},
author = {Preetam, S and Rath, P and Al-Enazi, NM and Sharaf, AAM and Jumah, JB and Govindarajan, RK and Goud, P and Thiruvengadam, M and Mathivanan, K},
title = {Engineering extracellular vesicle biogenesis for therapeutic gene delivery: emerging genetic programming strategies and translational prospects.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42429865},
issn = {1573-4978},
mesh = {Humans ; *Extracellular Vesicles/metabolism/genetics ; CRISPR-Cas Systems/genetics ; *Gene Transfer Techniques ; Gene Editing/methods ; *Genetic Therapy/methods ; *Genetic Engineering/methods ; Animals ; },
abstract = {Extracellular vesicles (EVs) have emerged as promising biological nanocarriers for gene therapy due to their intrinsic ability to transport nucleic acids, proteins, and lipids between cells. Advances in EV biology have revealed complex regulatory mechanisms governing vesicle biogenesis, cargo sorting, secretion, and uptake, offering multiple opportunities for therapeutic engineering. Concurrently, modern genetic technologies, including the CRISPR-Cas9 genome editing system and synthetic biology tools, have enabled precise manipulation of EV composition and functionality. This review integrates current knowledge of EV biogenesis with emerging genetic engineering strategies to transform EVs into programmable gene delivery systems. We discuss recent advances in genetic tools for studying EV dynamics, methods for engineering EV cargo and targeting specificity, and the application of EV platforms for RNA and genome-editing therapies. Furthermore, key challenges related to vesicle heterogeneity, large-scale production, and clinical translation are examined. Finally, we highlight future perspectives on programmable EV therapeutics and their potential role in next-generation precision medicine.},
}

Humans
*Extracellular Vesicles/metabolism/genetics
CRISPR-Cas Systems/genetics
*Gene Transfer Techniques
Gene Editing/methods
*Genetic Therapy/methods
*Genetic Engineering/methods
Animals

@article {pmid42429888,
year = {2026},
author = {Thevendran, R and Maheswaran, S and Lee, SY},
title = {Development of attenuated and inactivated Dengue strains using advanced gene editing tools.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42429888},
issn = {1573-4978},
support = {FRGS/1/2023/SKK06/AIMST/03/2//Ministry of Higher Education, Malaysia/ ; },
mesh = {Humans ; *Dengue Virus/genetics/immunology/pathogenicity ; *Dengue Vaccines/immunology/genetics ; *Dengue/prevention & control/immunology/virology/genetics ; *Gene Editing/methods ; Vaccines, Attenuated/immunology/genetics ; Animals ; CRISPR-Cas Systems ; Vaccine Development/methods ; },
abstract = {Dengue fever remains a persistent viral threat, affecting millions of families every year, turning a simple mosquito bite into a potentially life-threatening emergency. This disease remains a constant burden on our global healthcare system, demanding innovative solutions to protect worldwide communities. While many researchers discuss general treatments, preventions and modern medical interventions, there is often a lack of focus on how current, cutting-edge molecular and genetic tools are employed to engineer dengue strains as vaccine candidates. Therefore, in this paper, we explore the recent genetic strategies, such as targeted virulent gene deletions, CRISPR-Cas inactivation, and viral codon deoptimization approaches used to attenuate or inactivate Dengue viruses specifically. Assays and techniques used in validating Dengue viral attenuation or inactivation are also discussed in detail, highlighting the importance of the balance between safety and immunogenicity for Dengue vaccine uses. The article also briefly elaborates the complex biological challenges and safety concerns that centre on Dengue vaccine developments. By bridging the gap between advanced genetics and public health, this review provides readers with a comprehensive understanding of how modern genetics is paving the way for the next generation of safe and effective Dengue vaccines.},
}

Humans
*Dengue Virus/genetics/immunology/pathogenicity
*Dengue Vaccines/immunology/genetics
*Dengue/prevention & control/immunology/virology/genetics
*Gene Editing/methods
Vaccines, Attenuated/immunology/genetics
Animals
CRISPR-Cas Systems
Vaccine Development/methods

@article {pmid42431872,
year = {2026},
author = {Kaya, NH and Abukhalaf, M and Fuentes, G and Taubenheim, J and Hentschel, U and Tholey, A and Fraune, S},
title = {c-JUN controls microbial colonization via selective phagocytosis in the sea anemone Nematostella.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42431872},
issn = {2041-1723},
support = {CRC 1182, Project B1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; CRC 1182, Project B1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; CRC 1182, Project Z3//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; CRC 1182, Project B1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; CRC 1182, Project B1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; CRC 1182, Project Z3//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
mesh = {Animals ; *Sea Anemones/microbiology/immunology/genetics/metabolism ; *Phagocytosis/immunology ; Immunity, Innate ; Vibrio/immunology ; *Proto-Oncogene Proteins c-jun/genetics/metabolism/immunology ; Microbiota/immunology ; CRISPR-Cas Systems ; Lysosomes/metabolism ; },
abstract = {Innate immunity is traditionally viewed as a broad defense system with limited specificity. However, increasing evidence suggests that innate immune cells can discriminate between distinct microbial partners. How such specificity arises in early-diverging animals remains unclear. Here, we identify in the sea anemone Nematostella vectensis a selective host innate immune mechanism mediated by nematosomes, motile multicellular bodies that differentially process bacterial cells. Nematosomes preferentially engulf non-native Vibrio isolates while showing reduced uptake of native host-associated strains. We identify the transcription factor cJUN as a key regulator of this process. CRISPR/Cas9-mediated knockout of cJUN reduces nematosome abundance, impairs lysosomal response, alters microbiome assembly, and increases susceptibility to bacterial infection. These results link immune gene function to microbial selectivity and demonstrate that even early-diverging animals exhibit sophisticated innate immunity mechanisms for microbiome regulation. Our findings support the idea that immune specificity can arise through repurposing deeply conserved pathways and may have deep evolutionary origin.},
}

Animals
*Sea Anemones/microbiology/immunology/genetics/metabolism
*Phagocytosis/immunology
Immunity, Innate
Vibrio/immunology
*Proto-Oncogene Proteins c-jun/genetics/metabolism/immunology
Microbiota/immunology
CRISPR-Cas Systems
Lysosomes/metabolism