@article {pmid40962493,
year = {2026},
author = {Ferreira, A},
title = {The future of reproduction in Eve Smith's Off Target and Kira Peikoff's Baby X.},
journal = {Medical humanities},
volume = {52},
number = {2},
pages = {190-198},
doi = {10.1136/medhum-2025-013321},
pmid = {40962493},
issn = {1473-4265},
mesh = {Humans ; *Gene Editing/ethics ; Germ Cells ; *Gametogenesis ; *Reproduction ; *Reproductive Techniques, Assisted/ethics ; CRISPR-Cas Systems ; *Genetic Engineering/ethics ; Genome, Human ; },
abstract = {This essay analyses two novels that explore reproductive scenarios which may be implemented in the near future: human germline genome editing and in vitro gametogenesis (IVG), dramatised in Eve Smith's Off-Target (2022) and Kira Peikoff's Baby X (2024), respectively.Smith's Off Target considers the plethora of medical, ethical, social and familial implications of germline editing, which is used to modify an embryo's genome and enhance it with traits deemed optimal that will be passed on to future generations, in this way effectively creating designer babies and radically interfering with the laws of evolution and genetic inheritance. The novel deals with the potential unintended consequences of genetic engineering, in particular CRISPR/Cas9. Genetic edits, if insufficiently tested, can have off-target effects, alluded to in the name of the novel. Peikoff's Baby X takes the premise of Off Target a step further by introducing IVG, exploring the multiple ramifications of this as yet undeveloped reproductive technology which would radically change the reproductive landscape as we know it.Meanwhile, the science that will enable germline editing and IVG is advancing rapidly, making it imperative to consider the far-reaching repercussions of these technologies, not only in medical terms but specifically with regard to ethical implications for families and society in general. These novels are powerful reminders that the debate about the future applications of genome editing and IVG needs to happen now. The issues raised by the novels will be analysed mainly through a gender studies and bioethics lens.},
}

Humans
*Gene Editing/ethics
Germ Cells
*Gametogenesis
*Reproduction
*Reproductive Techniques, Assisted/ethics
CRISPR-Cas Systems
*Genetic Engineering/ethics
Genome, Human

@article {pmid40973816,
year = {2026},
author = {Janjuha, S and Haenggi, T and Chamberlain, TC and Rothgangl, T and Kissling, L and Wilhelm, M and Mathis, N and Boeck, D and Marquart, K and Ioannidi, E and Moon, WJ and Muramatsu, H and Vadovics, M and Pardi, N and Semple, SC and Tam, YK and Schwank, G},
title = {Spatial profiling of gene editing by in situ sequencing in mice and macaques.},
journal = {Nature biomedical engineering},
volume = {10},
number = {5},
pages = {968-979},
pmid = {40973816},
issn = {2157-846X},
support = {185293/SNSF_/Swiss National Science Foundation/Switzerland ; P01 AI158571/AI/NIAID NIH HHS/United States ; P01 AI172531/AI/NIAID NIH HHS/United States ; R01 AI153064/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; *Gene Editing/methods ; Mice ; Liver/metabolism ; Nanoparticles/chemistry ; Brain/metabolism ; Macaca ; RNA, Messenger/genetics ; Dependovirus/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Mice, Inbred C57BL ; Male ; CRISPR-Cas Systems ; Lipids/chemistry ; },
abstract = {Base and prime editing technologies precisely install defined nucleotide edits in both dividing and non-dividing cells, offering potential for correcting pathogenic mutations directly in organisms. However, to fully leverage their therapeutic potential, accurately measuring editing rates with high spatial resolution is crucial. Here we use imaging-based in situ sequencing (ISS) to map base and prime editing events within native tissues. We establish and validate this technology in mouse brains treated with intein-split adenine base editors or prime editors delivered via adeno-associated viral vectors. We next apply ISS in the liver of mice and macaques treated with adenine base editors encoded on lipid nanoparticle-encapsulated mRNA and guide RNA (RNA-LNP). Effective editing was observed across all metabolic zones of liver lobules. Moreover, in experiments where repeated doses of RNA-LNP are administered, the initial dose does not affect the editing efficiency and distribution of the subsequent dose. Our results demonstrate how ISS can visualize gene editing events in vivo and suggest that base editor delivery using RNA-LNP could be used to address a wide spectrum of metabolic liver diseases.},
}

Animals
*Gene Editing/methods
Mice
Liver/metabolism
Nanoparticles/chemistry
Brain/metabolism
Macaca
RNA, Messenger/genetics
Dependovirus/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Mice, Inbred C57BL
Male
CRISPR-Cas Systems
Lipids/chemistry

@article {pmid40983646,
year = {2026},
author = {Siddiqui, SM and Welch, NL and Nguyen, TG and Razmi, A and Chang, T and Senft, R and Arizti-Sanz, J and Mirhashemi, ME and Stirling, DR and Ackerman, CM and Cimini, BA and Blainey, PC and Sabeti, PC and Myhrvold, C},
title = {Bead-based approaches for increased sensitivity and multiplexing of CRISPR diagnostics.},
journal = {Nature biomedical engineering},
volume = {10},
number = {5},
pages = {939-951},
pmid = {40983646},
issn = {2157-846X},
support = {D18AC00006//United States Department of Defense | Defense Advanced Research Projects Agency (DARPA)/ ; 75D30122C15113//U.S. Department of Health & Human Services | Centers for Disease Control and Prevention (CDC)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Sensitivity and Specificity ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genes, Reporter ; },
abstract = {CRISPR-based diagnostics have emerged as a promising tool for fast, accurate and portable pathogen detection. There has been rapid progress in pre-amplification processes and CRISPR-related enzymes used in these approaches, but the development of reporter systems and reaction platforms has lagged behind. In this paper, we develop bead-based techniques to address these gaps. First, we develop a novel bead-based split-luciferase reporter system with up to 20× sensitivity compared with standard fluorescence-based reporter design in CRISPR diagnostics. Second, we develop a highly deployable, bead-based platform capable of detecting nine distinct viral targets in parallelized, droplet-based reactions, with sensitivity reaching as low as 2.5 copies per µl of input RNA. We demonstrate the enhanced performance of both approaches on synthetic and clinical sample sensitivity, speed, multiplexing and deployability.},
}

*CRISPR-Cas Systems/genetics
Humans
Sensitivity and Specificity
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Genes, Reporter

@article {pmid41904145,
year = {2026},
author = {Zhao, DS and Wang, S and Xu, YC and Chen, YT and Farooq, MA and Lin, S and Cao, P and Li, J and Hu, ZW and Li, N and Scarsini, M and Si, C and Li, SM and Yan, X and Pang, Q and Bowler, C and Zou, HX},
title = {Tryptophanol enhances nitrogen assimilation in marine diatoms.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41904145},
issn = {2041-1723},
support = {LY23D060001//Natural Science Foundation of Zhejiang Province (Zhejiang Provincial Natural Science Foundation)/ ; 31670402//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Diatoms/metabolism/genetics/growth & development ; *Nitrogen/metabolism ; *Tryptophan/metabolism/analogs & derivatives ; Peptide Synthases/metabolism/genetics ; CRISPR-Cas Systems ; },
abstract = {Diatoms exhibit high competitive capacity in nitrogen assimilation, but the underlying mechanisms remain unclear. Here, we identify a non-ribosomal peptide synthase-like gene (PtNRPS1) with an atypical domain structure (A-T-R1-R2) in the marine diatom Phaeodactylum tricornutum, crucial for short-term nitrogen assimilation. In vitro enzyme assays show PtNRPS1 catalyzes conversion of L-tryptophan to tryptophanol, a tryptophan-derived indole compound that promotes diatom growth at concentrations far lower than indole-3-acetic acid. Transcriptomic, metabolomic analyses, and stable-isotope analyses indicate tryptophanol enhances short-term nitrogen assimilation. CRISPR-Cas9 knockout of PtNRPS1 abolishes tryptophanol biosynthesis and reduces nitrogen-assimilation enzymes activities, which are restored by exogenous tryptophanol. PtNRPS1 overexpression results in delayed but sustained enzyme elevation. Global distribution of PtNRPS1 homologues in stramenopiles positively correlates with nitrogen-assimilation gene abundance. Our findings suggest tryptophanol, synthesized by a diatom NRPS, accelerates nitrogen assimilation, providing a competitive edge in oceanic nitrogen acquisition.},
}

*Diatoms/metabolism/genetics/growth & development
*Nitrogen/metabolism
*Tryptophan/metabolism/analogs & derivatives
Peptide Synthases/metabolism/genetics
CRISPR-Cas Systems

@article {pmid41920952,
year = {2026},
author = {Nicolia, A and Cuccurullo, A and Tamada, K and Yoneyama, K and Rambla, JL and Granell, A and Camerlengo, F and Festa, G and Francese, G and Contaldi, F and D'Alessandro, A and Rigano, MM and Principio, L and D'Agostino, N and Cardi, T},
title = {Editing strigolactone biosynthesis genes in tomato reveals novel phenotypic effects and highlights D27 as a breeding target for parasitic weed resistance.},
journal = {Plant & cell physiology},
volume = {67},
number = {4},
pages = {667-684},
doi = {10.1093/pcp/pcag042},
pmid = {41920952},
issn = {1471-9053},
support = {DM 15924//Italian Ministry of Agriculture, Food Sovereignty and Forests/ ; 18-05-2018//Italian Ministry of Agriculture, Food Sovereignty and Forests/ ; IJC2020-045612-I//Spanish Ministry of Science and Innovation/ ; PID2022-141438OB-I00//Spanish Ministry/ ; 101000716//EU for Harnesstom/ ; JPMJFR220F//Japan Science and Technology Agency/ ; },
mesh = {*Solanum lycopersicum/genetics/metabolism/parasitology ; *Lactones/metabolism ; *Plant Weeds/physiology ; *Plant Proteins/genetics/metabolism ; *Gene Editing ; Phenotype ; Germination ; Plant Breeding ; CRISPR-Cas Systems ; Seeds ; Plant Growth Regulators/metabolism/biosynthesis ; Gene Expression Regulation, Plant ; Gene Knockout Techniques ; Genes, Plant ; *Heterocyclic Compounds, 3-Ring/metabolism ; },
abstract = {Parasitic weed infestations represent an increasing threat to agriculture worldwide, especially in the Mediterranean region. Phelipanche ramosa (L.) and Phelipanche aegyptiaca (Pers.) (broomrapes) cause severe yield losses in field-grown tomato (Solanum lycopersicum L.). Strigolactones (SLs) are apocarotenoid phytohormones that not only play a critical role in plant physiology and development but also act as the primary germination signals for parasitic weed seeds. In this study, we generated CRISPR/Cas9 tomato knock-out (KO) lines targeting the SlD27 gene and three other key genes involved in SL biosynthesis (SlCCD7, SlCCD8, and SlMAX1), all in the same genetic background. All the edited lines exhibited undetectable SL levels in root exudates, leading to a strong reduction in the in vitro germination of Phelipanche spp. seeds. Consistently, reduced parasitism was also observed in vivo when Sld27 lines were tested. A comprehensive evaluation of morphological, reproductive, and fruit-related traits revealed gene-specific phenotypic effects, including changes in vegetative growth, fruit set, fruit development, and volatilome. Specifically, KO of two carotenoid cleavage dioxygenases and SlMAX1 affected shoot architecture, fruit development, and the production of volatile organic compounds during fruit ripening. In contrast, the newly developed Sld27 lines in this study displayed a mild phenotype generally comparable to nonedited control plants and likely due to the expression of SlD27 paralogues. Overall, our results indicate that SlD27 represents a promising breeding target for enhancing resistance to parasitic weeds in tomato while minimizing negative impacts on plant development and fruit quality.},
}

*Solanum lycopersicum/genetics/metabolism/parasitology
*Lactones/metabolism
*Plant Weeds/physiology
*Plant Proteins/genetics/metabolism
*Gene Editing
Phenotype
Germination
Plant Breeding
CRISPR-Cas Systems
Seeds
Plant Growth Regulators/metabolism/biosynthesis
Gene Expression Regulation, Plant
Gene Knockout Techniques
Genes, Plant
*Heterocyclic Compounds, 3-Ring/metabolism

@article {pmid41997156,
year = {2026},
author = {Peng, J and Chan, DCT and Chu, HY and Fong, JHC and Lam, YK and Cheung, MSH and Leung, W and Choi, GCG and Wong, ASL},
title = {Rapid customization of base editors via machine learning-powered combinatorial mutagenesis.},
journal = {Molecular cell},
volume = {86},
number = {10},
pages = {1839-1855.e10},
doi = {10.1016/j.molcel.2026.03.030},
pmid = {41997156},
issn = {1097-4164},
mesh = {Humans ; *Machine Learning ; *Gene Editing/methods ; *Mutagenesis ; Mutation ; *DNA/genetics/metabolism ; CRISPR-Cas Systems ; Deep Learning ; Nucleotide Motifs ; Cytosine/metabolism ; },
abstract = {Being able to control the complementarity and hindrance between target DNA and base editor proteins enables precise, bystander-free editing. Here, we combined combinatorial mutagenesis with machine learning to analyze and engineer these interactions at scale. By profiling DNA motif preferences across 160,000 evoAPOBEC1 and 64 million TadA variants in human cells, we used as little as 0.004% of the mutational landscape to make predictions. This identified variants with motif-specific activity and eliminated residual adenine editing in cytosine base editors. In correcting >800 disease-associated mutations, our variants outperformed previous versions in precluding unintended edits at purine motifs, achieving undetectable bystander edits in 50% of cases. Additionally, a pre-trained, structure-based deep learning model predicted functional TadA variants with 63% success across 20[26] variants spanning 26 amino acid sites, without experimental data and in a single prediction round. These approaches streamline the re-engineering of base editors for enhanced precision tailored to specific targets.},
}

Humans
*Machine Learning
*Gene Editing/methods
*Mutagenesis
Mutation
*DNA/genetics/metabolism
CRISPR-Cas Systems
Deep Learning
Nucleotide Motifs
Cytosine/metabolism

@article {pmid42017790,
year = {2026},
author = {Gopinath, A and Shen, L and Ouellette, SP},
title = {CRISPRi and beyond: studying essential gene function in the obligate intracellular bacterium Chlamydia trachomatis.},
journal = {Journal of bacteriology},
volume = {208},
number = {5},
pages = {e0005926},
pmid = {42017790},
issn = {1098-5530},
support = {R35 GM151971/GM/NIGMS NIH HHS/United States ; R21AI175651/NH/NIH HHS/United States ; R35GM151971/NH/NIH HHS/United States ; },
mesh = {*Chlamydia trachomatis/genetics/metabolism ; *Genes, Essential ; Gene Expression Regulation, Bacterial ; *CRISPR-Cas Systems ; Bacterial Proteins/genetics/metabolism ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Chlamydia Infections/microbiology ; },
abstract = {Chlamydia trachomatis is an obligate intracellular bacterium that is the leading cause of bacterial sexually transmitted infections (STIs) and preventable infectious blindness. Its unique biphasic developmental cycle comprises an infectious but non-dividing elementary body and a replicative but non-infectious reticulate body. C. trachomatis possesses a reduced genome where more than half of the open reading frames (ORFs) are predicted to code for essential genes, abrogation of which with traditional chromosomal disruption methods is expected to block bacterial growth and developmental cycle progression. However, understanding the function of such genes is critical to expand our knowledge of chlamydial biology and reveal new therapeutic targets. This review aims to compare and contrast four systems developed in the past 5 years for studying essential genes in Chlamydia. These include systems to conditionally knock down or knockout a target gene product using CRISPR interference (CRISPRi), inducible small RNAs (sRNA), fluorescence-reported allelic exchange mutagenesis (FRAEM) with inducible complementation of the target gene, and dependence on plasmid expression (DOPE).},
}

*Chlamydia trachomatis/genetics/metabolism
*Genes, Essential
Gene Expression Regulation, Bacterial
*CRISPR-Cas Systems
Bacterial Proteins/genetics/metabolism
Humans
*Clustered Regularly Interspaced Short Palindromic Repeats
Chlamydia Infections/microbiology

@article {pmid42070443,
year = {2026},
author = {Awais, M and Chen, Y and Bibi, S and Wu, J and Zhang, W and Tlili, I and Hu, J},
title = {Breaking boundaries for PFAS surveillance in water reservoirs: CRISPR-electrochemical synergies from MXene to microfluidics- A review.},
journal = {Biosensors & bioelectronics},
volume = {307},
number = {},
pages = {118756},
doi = {10.1016/j.bios.2026.118756},
pmid = {42070443},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods/instrumentation ; CRISPR-Cas Systems ; *Water Pollutants, Chemical/analysis/isolation & purification ; *Fluorocarbons/analysis/isolation & purification ; Lab-On-A-Chip Devices ; Environmental Monitoring/methods ; Electrochemical Techniques/methods ; Nitrites ; Transition Elements ; },
abstract = {The hydro-spherical contamination of water resources by per- and polyfluoroalkyl substances (PFAS) demands analytical technologies that transcend the limitations of current methods, which struggle to simultaneously achieve ultra-sensitivity, specificity, portability, and low cost. Therefore, this review advances next-generation PFAS surveillance by proposing a framework built on the synergistic convergence of molecularly engineered sulfonate-MXenes, CRISPR-Cas12a, and microfluidic automation. The manuscript critically analyzes how sulfonate-terminated Ti3C2Tx MXenes achieve picomolar affinity and rapid preconcentration of PFAS through biomimetic binding architectures. It also details the mechanism by which CRISPR-Cas12a, guided by PFAS-specific aptamers, enables single-molecule discrimination with attomolar sensitivity. Finally, this review paper demonstrates how microfluidic networks orchestrate this synergy, miniaturizing the entire assay into a portable, multiplexed platform that reduces analysis time from hours to minutes. Synergistically unifying breakthroughs in nanomaterials, synthetic biology, and lab-on-a-chip design, this work provides both a methodological blueprint for ultrasensitive PFAS sensors and a relevant roadmap for implementing proactive, decentralized water quality monitoring.},
}

*Biosensing Techniques/methods/instrumentation
CRISPR-Cas Systems
*Water Pollutants, Chemical/analysis/isolation & purification
*Fluorocarbons/analysis/isolation & purification
Lab-On-A-Chip Devices
Environmental Monitoring/methods
Electrochemical Techniques/methods
Nitrites
Transition Elements

@article {pmid42090813,
year = {2026},
author = {Hyeon, LS and Yang, JW and Bae, S and Yoo, JS and Kim, E and Lee, SS and Kang, S},
title = {Rapid and sensitive detection of blaKPC and blaNDM in carbapenemase-producing Enterobacterales using a one-pot recombinase polymerase amplification-CRISPR/Cas12a system with modified protospacer adjacent motifs.},
journal = {Biosensors & bioelectronics},
volume = {307},
number = {},
pages = {118725},
doi = {10.1016/j.bios.2026.118725},
pmid = {42090813},
issn = {1873-4235},
mesh = {*beta-Lactamases/genetics/isolation & purification ; *Bacterial Proteins/genetics/isolation & purification ; Humans ; *Biosensing Techniques ; CRISPR-Cas Systems/genetics ; *Enterobacteriaceae/genetics/enzymology/isolation & purification ; Nucleic Acid Amplification Techniques ; Limit of Detection ; Point-of-Care Systems ; *Carbapenem-Resistant Enterobacteriaceae/genetics/isolation & purification ; },
abstract = {Carbapenem-resistant Enterobacterales pose a critical global health threat because of their rapid transmission and resistance to last-line antibiotics. To address this threat, rapid and user-friendly point-of-care (POC) detection is essential. Quantitative polymerase chain reaction (qPCR), while providing high sensitivity, relies on expensive equipment and skilled personnel, hindering its practical use in resource-limited settings. Here, we aimed to develop an optimized one-pot RPA-CRISPR/Cas12a (RCCS) system with enhanced diagnostic performance to provide a reliable and user-friendly platform for rapid carbapenemase gene screening in both clinical and resource-limited settings. The developed assay was used for the detection of blaKPC and blaNDM genes and was integrated into a portable diagnostic device. By using suboptimal protospacer adjacent motif sequences, the assay provided a streamlined workflow and substantially enhanced detection sensitivity. This platform achieved a limit of detection of 10[-17] M for blaKPC and 10[-16] M for blaNDM within 30 min. Validation with 44 clinical samples demonstrated that the assay had 100% sensitivity and specificity, matching the effectiveness of qPCR. The one-pot RCCS platform offers a robust and highly sensitive POC solution for on-site testing.},
}

*beta-Lactamases/genetics/isolation & purification
*Bacterial Proteins/genetics/isolation & purification
Humans
*Biosensing Techniques
CRISPR-Cas Systems/genetics
*Enterobacteriaceae/genetics/enzymology/isolation & purification
Nucleic Acid Amplification Techniques
Limit of Detection
Point-of-Care Systems
*Carbapenem-Resistant Enterobacteriaceae/genetics/isolation & purification

@article {pmid42106044,
year = {2026},
author = {Dutta, TK and Ray, S and Akhil, VS and Rupinikrishna, K and Chauhan, U and Dutta, A and Vijayan, J and Iquebal, MA and Chinnusamy, V},
title = {A synthetic guide RNA scaffold enhanced CRISPR/Cas9 editing efficiency in plants across multiple gene targets.},
journal = {International journal of biological macromolecules},
volume = {365},
number = {},
pages = {152456},
doi = {10.1016/j.ijbiomac.2026.152456},
pmid = {42106044},
issn = {1879-0003},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics/chemistry ; Arabidopsis/genetics ; Oryza/genetics ; *Plants/genetics ; },
abstract = {CRISPR/Cas9 mediated genome editing is a highly powerful and versatile tool for accelerating crop improvement. The editing efficiency of CRISPR/Cas9 system in planta has been highly variable owing to the variable binding affinity between native CRISPR RNA and Cas9 protein in vivo. In plant systems, systematic, large-scale engineering and benchmarking of guide RNA (gRNA) scaffold variants is still relatively limited compared with work in mammalian systems, despite several important studies demonstrating that scaffold and expression-cassette engineering can substantially improve CRISPR/Cas9 efficacy. Current study addresses the limitations of commonly used gRNA scaffold architecture by incorporating a stabilized stem-loop RAR (tetra loop) extension and a transcription-termination site mutation, resulting in improved RNA folding, increased Cas9 binding affinity, and enhanced in vivo editing outcomes. The synthesized scaffold boosted CRISPR/Cas9 efficiency in monocot or dicot plants across the 19 diverse target sites in Arabidopsis, rice and tomato. Furthermore, the synthetic scaffold is compatible with multiplex genome editing architectures, including polycistronic tRNA-gRNA (PTG) expression systems, enabling efficient simultaneous targeting of multiple genomic loci. The findings of this study have broad applications in precision plant breeding, functional genomics, and agricultural biotechnology, facilitating reliable gene modification across diverse plant species and transformation platforms.},
}

*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics/chemistry
Arabidopsis/genetics
Oryza/genetics
*Plants/genetics

@article {pmid42120728,
year = {2026},
author = {DiPersio, JF and Koehne, G and Shah, NN and Bernard, L and Suh, HC and Koura, D and Tamari, R and Mushtaq, MU and Maakaron, J and Rimando, J and Kennedy, VE and Patel, SS and Hudson, C and Loken, MR and Slapak, CA and Lloyd, DM and Stanizzi, DA and Lee-Sundlov, MM and Thosar, S and Mundelboim, G and Guo, G and Ge, HG and Li, BE and Xavier-Ferrucio, J and Hyzy, SL and Lin, MI and Raffel, GD and Cooper, BW},
title = {CRISPR-Cas9 CD33-deleted allogeneic hematopoietic cell transplantation with gemtuzumab ozogamicin maintenance in AML: a phase 1/2 trial.},
journal = {Nature medicine},
volume = {32},
number = {5},
pages = {1763-1772},
pmid = {42120728},
issn = {1546-170X},
mesh = {Humans ; *Hematopoietic Stem Cell Transplantation/methods/adverse effects ; *Gemtuzumab/therapeutic use/administration & dosage/adverse effects ; *Leukemia, Myeloid, Acute/therapy/genetics/drug therapy ; *Sialic Acid Binding Ig-like Lectin 3/genetics ; Middle Aged ; Female ; Male ; Adult ; Aged ; *CRISPR-Cas Systems/genetics ; Transplantation, Homologous ; Graft vs Host Disease ; Myelodysplastic Syndromes/therapy/genetics ; },
abstract = {Patients with high-risk acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) are likely to relapse despite allogenic hematopoietic cell transplantation (HCT). Post-HCT preventative maintenance can be limited by toxicity toward the normal donor cells. Tremtelectogene empogeditemcel (trem-cel) is a CRISPR-Cas9 gene-edited allogeneic HCT product lacking CD33, designed to shield the donor graft from cytotoxicity of subsequent CD33-targeted therapies such as gemtuzumab ozogamicin (GO). In this multicenter, phase 1/2a, open-label study, adult patients with AML/MDS with high relapse risk received trem-cel after myeloablative conditioning followed by GO maintenance (0.5-2.0 mg m[-][2] day 1 per 28-day cycles). Patients receiving trem-cel were assessed for the primary safety endpoint of neutrophil engraftment by day 28 and secondary endpoints including time to neutrophil engraftment, incidence of graft-versus-host disease and graft failure, transplant-related mortality, percentage of CD33-negative myeloid cells and survival. Patients receiving trem-cel and GO were assessed for the additional secondary endpoints of safety of maintenance GO with trem-cel and pharmacokinetics of GO after trem-cel transplant. All 30 patients receiving trem-cel achieved the primary safety endpoint of neutrophil engraftment by day 28 with a median engraftment time of 10 days (95% confidence interval: 9-10). Nineteen patients received GO maintenance in phase 1 dose escalation (n = 15) and in phase 2 dose expansion (n = 4). The trial was stopped early, and this is the final report on the trial including the completed phase 1 portion. GO treatment was safely tolerated up to the recommended phase 2 dose of 2 mg m[-][2], and no prolonged high-grade cytopenias were observed. The most common adverse events were cytopenias and infections. Three cases of transplant-related mortality were observed due to renal failure, sepsis and sinusoidal obstruction syndrome, respectively. In summary, trem-cel demonstrated safe, rapid, robust engraftment, and GO maintenance was administered without prolonged hematologic toxicity. ClinicalTrials.gov identifier: NCT04849910 .},
}

Humans
*Hematopoietic Stem Cell Transplantation/methods/adverse effects
*Gemtuzumab/therapeutic use/administration & dosage/adverse effects
*Leukemia, Myeloid, Acute/therapy/genetics/drug therapy
*Sialic Acid Binding Ig-like Lectin 3/genetics
Middle Aged
Female
Male
Adult
Aged
*CRISPR-Cas Systems/genetics
Transplantation, Homologous
Graft vs Host Disease
Myelodysplastic Syndromes/therapy/genetics

@article {pmid42159719,
year = {2026},
author = {Wu, B and Luo, H and Xie, H and Zhao, C and Xie, P and Yang, X and Shi, J and Huang, W and Fang, Z},
title = {Cis-regulatory editing of SD1 promoter enhances TCP19-mediated repression to optimize plant height in Kam sweet rice.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {139},
number = {6},
pages = {},
pmid = {42159719},
issn = {1432-2242},
mesh = {*Oryza/genetics/growth & development ; *Gene Editing ; *Promoter Regions, Genetic ; Gene Expression Regulation, Plant ; *Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *Transcription Factors/genetics/metabolism ; Gibberellins/metabolism ; Phenotype ; Plants, Genetically Modified ; Plant Breeding ; },
abstract = {This study demonstrates that CRISPR-mediated cis-regulatory element editing (CRE editing) of the SD1 promoter effectively reduces plant height in Kam sweet rice, without compromising yield or grain quality, offering a precise strategy for crop improvement. Kam sweet rice, a unique aromatic variety, faces challenges with excessive plant height and suboptimal yield. This study explores a precision breeding approach by strengthening an endogenous TCP19-SD1 repression module through CRISPR-Cas9-mediated CRE editing to modulate the expression of the SD1 gene, a key regulator of gibberellin biosynthesis and stem elongation. By introducing an adenine insertion in the GGCCCCCC cis-regulatory element in the SD1 promoter, we enhanced the binding affinity of the transcription factor TCP19, resulting in down-regulated SD1 expression. This led to a reduction in gibberellin levels, shortening internodes, and reducing plant height. Phenotypic evaluations revealed that the edited lines exhibited significantly shorter plant height while maintaining grain yield and nitrogen utilization efficiency compared to wild-type plants. Microscopic analysis of the internodes confirmed that the reduced plant height correlated with decreased cell length. Transcriptomic studies indicated that CRE editing modulated a network of genes involved in both gibberellin and auxin signaling pathways, critical for plant growth. Importantly, the genetic modification did not adversely affect grain quality. This study demonstrates the potential of strengthening endogenous transcriptional repression via CRE editing as a precise alternative to conventional gene knockout techniques, offering a powerful strategy for optimizing complex agronomic traits in rice, with applications in modern crop breeding strategies.},
}

*Oryza/genetics/growth & development
*Gene Editing
*Promoter Regions, Genetic
Gene Expression Regulation, Plant
*Plant Proteins/genetics/metabolism
CRISPR-Cas Systems
*Transcription Factors/genetics/metabolism
Gibberellins/metabolism
Phenotype
Plants, Genetically Modified
Plant Breeding

@article {pmid42162502,
year = {2026},
author = {Makhijani, S and Alasiri, G and Quadri, MSA and Ingle, RG},
title = {From mechanism to medicine: CRISPR‒Cas9 delivery strategies, therapeutic applications and translation challenges.},
journal = {Discover nano},
volume = {21},
number = {1},
pages = {},
pmid = {42162502},
issn = {2731-9229},
abstract = {The advent of CRISPR‒Cas technology has revolutionized genome editing by providing unprecedented precision, efficiency, and versatility in genetic manipulation. This revolutionary system originated from bacterial adaptive immunity. It allows the programmed modification of DNA sequences in a highly specific way, as long as the desired modification site contains an enzyme-compatible protospacer adjacent motif (PAM) sequence. Recent innovations with CRISPR‒Cas systems have focused on optimizing delivery methods, addressing challenges and expanding their therapeutic applications. Delivery approaches, including viral vectors, lipid nanoparticles, and other nonviral vectors, have been improved to increase target specificity while minimizing off-target effects, ensuring safe and efficient In Vivo genome editing. The applications of CRISPR‒Cas technology include the correction of genetic disorders, engineering of immune cells for cancer therapy, and combating viral infections. However, significant challenges in the form of unintended edits, immune responses, and delivery to specific tissues or organs remain formidable barriers to clinical translation. New CRISPR variants, such as base editing, prime editing, and epigenome editing, provide solutions to improve accuracy and broaden intervention. This review covers recent progress in various CRISPR delivery methodologies, discusses broadening therapeutic applications, highlights ongoing challenges, and describes the future trajectory of this disruptive technology.},
}

@article {pmid42164567,
year = {2025},
author = {Li, X and Zhang, Z and Zhu, J and Zhang, Y and Guo, Y},
title = {Innovative Applications and Challenges of Isothermal Amplification Technology in miRNA Detection.},
journal = {Current genomics},
volume = {26},
number = {6},
pages = {581-598},
pmid = {42164567},
issn = {1389-2029},
abstract = {A class of endogenous non-coding RNAs with a length of roughly 18-25 nucleotides is known as microRNAs (miRNAs). They have been established as the best biomarkers for early cancer diagnosis, molecular subtyping, and prognostic evaluation. They also play important roles in important biological processes like cell proliferation, differentiation, apoptosis, and tumorigenesis through post-transcriptional regulatory networks. Traditional detection technologies (such as quantitative Polymerase Chain Reaction (qPCR), Northern blot, and microarrays) exhibit high sensitivity, yet they rely on expensive thermocycling equipment, involve cumbersome procedures, and are time-consuming, making it difficult to meet the demands of clinical point-of-care testing. In recent years, isothermal amplification technologies, represented by loop-mediated isothermal amplification (LAMP), Rolling Circle Amplification (RCA), Strand Displacement Amplification (SDA), and Exponential Amplification Reaction (EXPAR), have emerged as a research frontier in the field of miRNA detection, by virtue of their advantages including elimination of thermal cycling requirements, fast reaction kinetics, high sensitivity, and simple operation. This study aims to systematically organize the core principles, latest advancements, and integrated innovation patterns of isothermal amplification-based miRNA detection technologies, and provide support for the development of miRNA detection platforms suitable for clinical point-of-care testing.},
}

@article {pmid42164670,
year = {2026},
author = {Islam, MS and Fan, J and Vabna, MA and Haque, N and Monir, SB and Li, Y and Nime, I and Acharjee, M and Pan, F},
title = {Phage enabled precision drug delivery: dual function platforms for therapeutics and genetic cargo transport.},
journal = {Frontiers in microbiology},
volume = {17},
number = {},
pages = {1812871},
pmid = {42164670},
issn = {1664-302X},
abstract = {The rising concern of antimicrobial resistance, coupled with the continually challenging management of complicated diseases such as cancer, has provided momentum toward precision molecular medicine. This review provides an overview of bacteriophage enabled strategies encompassing both conventional antibacterial applications and advanced bioengineered delivery systems. Recent advances in phage therapy include the use of tailored phage formulations, phage immobilization approaches and phage antibiotic combinations to achieve targeted bacterial lysis particularly against multidrug-resistant pathogens and biofilm-associated infections. Beyond their intrinsic antibacterial activity, phages can be genetically and chemically engineered as nanoscale scaffolds. Phage display technologies enable the incorporation of targeting ligands for selective binding to specific tissues including tumor cells. Furthermore, phage capsids can be modified to encapsulate and deliver diverse therapeutic payloads such as small-molecule drugs, nucleic acids and gene-editing systems such as CRISPR-Cas, thereby expanding their utility beyond infectious diseases. The integration of phage biology with nanobiotechnology positions these viral platforms at the forefront of next generation therapeutics. Engineered phages have demonstrated potential as precision delivery vectors for cytotoxic agents, immunomodulators and genetic material with improved specificity and reduced off-target effects. Emerging strategies including phage antibiotic conjugates and enzyme functionalized phages further enhance therapeutic efficacy and facilitate penetration of physiological barriers. Collectively, phage-based platforms represent a versatile and transformative approach with significant implications for the treatment of infectious, oncologic and genetic disorders, supporting the advancement of targeted and personalized medicine.},
}

@article {pmid42164832,
year = {2026},
author = {Kumar, A and Krishan, B and Dhiman, S and Sharma, A and Thadiyan, V and Azmi, W},
title = {Beyond antibiotics: innovative and translational strategies to overcome antimicrobial resistance.},
journal = {3 Biotech},
volume = {16},
number = {6},
pages = {201},
pmid = {42164832},
issn = {2190-572X},
abstract = {The rapid rise of antimicrobial resistance demands therapeutic strategies that extend beyond conventional antibiotics. However, most existing reviews describe emerging alternatives without systematically linking their mechanistic advances to translational readiness and clinical implementation barriers. This review addresses this gap by integrating evidence across multiple beyond-antibiotic approaches, including antimicrobial peptides, bacteriophage therapy, CRISPR-based antimicrobials, nanotechnology-enabled delivery systems, anti-virulence agents, host-directed immunotherapies, microbiome modulation (engineered probiotics and fecal microbiota transplantation), and drug-repurposing or combination therapies. The principal contribution of this synthesis is a comparative framework that maps mechanisms of action, engineering innovations, and translational evidence across these diverse strategies. Advances such as peptidomimetics, engineered phages, and nanoparticle carriers that enhance stability, targeting, and therapeutic efficacy are highlighted, along with synergistic strategies including phage-antibiotic and CRISPR-nanocarrier combinations. The review further identifies major barriers limiting clinical translation, including delivery efficiency, toxicity and ecological concerns, large-scale production challenges, cost, inconsistent clinical outcomes, and regulatory fragmentation for biologics and live therapeutics. To facilitate clinical implementation, the study proposes a translational roadmap emphasizing standardized evaluation assays, physiologically relevant infection models, integrated rapid diagnostics, and regulatory frameworks tailored for emerging antimicrobial platforms, thereby supporting the development of sustainable therapies for the post-antibiotic era.},
}

@article {pmid42164989,
year = {2025},
author = {Taumar, D and Singh, AP and Sharma, H and Chaudhary, V},
title = {RNA Modifications as Drug Targets: Unlocking the Therapeutic Potential of the Epitranscriptome.},
journal = {Current genomics},
volume = {26},
number = {7},
pages = {802-818},
pmid = {42164989},
issn = {1389-2029},
abstract = {INTRODUCTION: The epitranscriptome covers reversible changes to RNA. These changes help control gene expression by making RNA more stable, easier to use, or more prone to degradation. They are increasingly implicated in disease development and offer a promising target for treatment. This review covers RNA alterations and their potential clinical applications.

METHODS: The systematic analysis of the peer-reviewed literature was performed, including experimental, clinical, and computational research. The biochemical properties and biological functions were used to classify RNA modifications. In addition, we evaluated current therapeutic options, including small molecules, CRISPR/Cas-based technologies, and RNA-targeted approaches.

RESULTS: RNA undergoes immense changes, including N6-methyladenosine (m6A), 5-methylcytosine (m5C), and pseudouridine. By these modifications, the genes are regulated by coordinated writers, erasers, and readers. Targeted therapeutics aim to regulate changes in cancer, neurodegenerative disease, and viral diseases. Several limitations remain, including overly general framing, suboptimal clarity in conveying the message, and inadequate response mechanisms.

DISCUSSION: The epitranscriptome provides an additional regulatory layer with significant therapeutic applications. New techniques appear to be successful. Researchers, in turn, should be more specific. These therapies require improved delivery vehicles and reduced side effects not associated with the treatment to be safe and effective for the patient.

CONCLUSION: Therapy targeting RNA-based modifications is a breakthrough in disease treatment. These chemical modifications affect gene function and expression patterns. The next step in the research requires overcoming current limitations to fully realize the therapeutic potential of epitranscriptomics-based interventions.},
}

@article {pmid42165133,
year = {2026},
author = {Liu, B and Klatt, D and Harris, C and McGuinness, M and Brendel, C and Williams, DA},
title = {Optimized lentivirus-derived virus-like particles for efficient delivery of Cas9-based genome editors.},
journal = {Nucleic acids research},
volume = {54},
number = {10},
pages = {},
doi = {10.1093/nar/gkag500},
pmid = {42165133},
issn = {1362-4962},
support = {INV-021791/GATES/Gates Foundation/United States ; },
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/genetics/metabolism ; Proprotein Convertase 9/genetics ; Animals ; *Lentivirus/genetics ; HEK293 Cells ; Mice ; Genetic Vectors/genetics ; Ribonucleoproteins/genetics ; Virion/genetics ; },
abstract = {Implementation of therapeutic genome editing requires a potent, versatile, and transient delivery system to enable safe and effective in vivo applications. Here, we report on an optimized virus-like particle (VLP) platform for protein-based delivery of Cas9 ribonucleoproteins and Cas9-derived base editors and prime editors, termed LV-VLP-MA, that enables flexible editor deployment. By systematically engineering a panel of truncated Gag-Cas9 fusion variants, we identify a minimal MA-Cas9 configuration that maximizes editor packaging while effectively preserving efficient particle production and functional delivery. Systematic refinement of VLP production parameters enhances particle yield, supporting robust editing activity across diverse genomic targets. Importantly, systemic administration of LV-VLP-MA mediates efficient in vivo editing of the Pcsk9 locus with functional target suppression, establishing proof-of-concept for therapeutic application. Together, these results define a programmable, modular VLP-based platform that combines potency, flexibility, and transient delivery to expand the scope of in vivo genome engineering for therapeutic development.},
}

*Gene Editing/methods
Humans
*CRISPR-Cas Systems
*CRISPR-Associated Protein 9/genetics/metabolism
Proprotein Convertase 9/genetics
Animals
*Lentivirus/genetics
HEK293 Cells
Mice
Genetic Vectors/genetics
Ribonucleoproteins/genetics
Virion/genetics

@article {pmid42165845,
year = {2026},
author = {Oxendine, J and Ibarra-Reyes, E and Ma, J and Li, C and Baron, S and Hwang, AE and Wang, R and Rodriguez-Leal, D},
title = {Optimization of Agrobacterium-mediated transformation of commercial heirloom tomato cultivars to develop novel traits via CRISPR/Cas9 genome editing.},
journal = {Planta},
volume = {264},
number = {1},
pages = {},
pmid = {42165845},
issn = {1432-2048},
support = {89365//California Tomato Research Institute/ ; MD-PSLA-243084//University of Maryland/ ; Startup funds//University of Maryland/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Solanum lycopersicum/genetics/growth & development ; Plants, Genetically Modified/genetics ; *Agrobacterium/genetics ; Transformation, Genetic ; Plant Breeding/methods ; Genome, Plant ; },
abstract = {Trait development for commercial heirloom tomatoes can be advanced by optimization of tissue culture and transformation via Agrobacterium and CRISPR/Cas9 mutagenesis. Genetic improvement using new genome editing approaches relies on the efficient delivery of the CRISPR/Cas system in the vegetable crop tomato. Previous protocols for tomato transformation have primarily focused on a handful of cultivars (M82, Ailsa Craig, Microtom, Sweet-100) with very little commercial relevance, and it is not clear if these protocols can be implemented directly in other commercially relevant varieties. Heirloom tomatoes are sought for their deep and diverse flavor but have not been subjected to systematic crop improvement via conventional breeding or biotechnology approaches such as transgenesis or genome editing. Therefore, we tested the transformation and regeneration capacity of six different heirloom cultivars known for their superior taste and market relevance in the US. Subsequently, we optimized rooting conditions and used the GRF4-GIF1 chimeric developmental regulator to successfully recover transgenic plants. Finally, we evaluated the efficiency of targeted genetic modification using the CRISPR/Cas9 genome editing system in several of these cultivars. We demonstrate that our optimizations led to successful transformation of several heirloom varieties, including the generation of edited plants for target genes modifying plant architecture and flowering time. Our results set the foundation for a biotechnology platform to deliver improved traits to local and regional heirloom varieties using genome editing.},
}

*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Solanum lycopersicum/genetics/growth & development
Plants, Genetically Modified/genetics
*Agrobacterium/genetics
Transformation, Genetic
Plant Breeding/methods
Genome, Plant

@article {pmid42165978,
year = {2026},
author = {Zhu, B},
title = {Nanomaterial-nucleic acid probe synergy: accelerating rapid pathogen detection and antimicrobial susceptibility testing in bloodstream infections.},
journal = {Folia microbiologica},
volume = {},
number = {},
pages = {},
pmid = {42165978},
issn = {1874-9356},
abstract = {Bloodstream infections (BSIs) remain among the most lethal clinical syndromes, driven in large part by diagnostic delays that compel empiric, broad-spectrum antimicrobial therapy and expose patients to avoidable toxicity and resistance selection. Conventional blood culture-based workflows, although diagnostically definitive, are intrinsically slow, often requiring 24-72 h, and are therefore poorly matched to the time-critical demands of sepsis management, where each hour of delayed appropriate therapy measurably increases mortality. In this context, nano-enabled nucleic acid diagnostics represent a promising but largely preclinical strategy for improving analytical sensitivity and turnaround time. This critical translational review examines how engineered nanomaterials spanning plasmonic and magnetic nanoparticles, fluorescent quantum dots, upconversion nanoparticles, and two-dimensional materials synergize with programmable nucleic acid recognizers, including aptamers, CRISPR/Cas effectors, DNAzymes, and conformational probes, to enable rapid, ultrasensitive detection of pathogens and resistance determinants directly from whole blood. Rather than reviewing nanomaterials and nucleic acid probes as separate toolkits, this article focuses on how their co-design at the nano-bio interface enables clinically actionable whole-blood diagnostics. We elucidate how convergence engineering at the nano-bio interface governs signal amplification, background suppression, and assay robustness in complex biological matrices. Particular emphasis is placed on front-end enrichment strategies, optical and magnetic transduction mechanisms, and multiplexed readout architectures that together enable species-level identification and early antimicrobial susceptibility profiling within clinically relevant timeframes, typically ~ 1-6 h in research settings. Beyond analytical performance, we critically assess interconnected translational barriers including batch-to-batch reproducibility, standardization of bioconjugation protocols, antifouling strategies, and evolving regulatory frameworks, which collectively govern the trajectory from laboratory innovation to clinical adoption. At present, direct-from-blood phenotypic antimicrobial susceptibility testing remains technically challenging, and clinical adoption is limited by reproducibility, matrix tolerance, and workflow integration. By integrating mechanistic insight with clinical positioning, this review frames nano-probe diagnostics as promising candidates for next-generation BSI management that may support more timely and precise therapy once analytical robustness, standardization, and clinical validation are achieved.},
}

@article {pmid42166245,
year = {2026},
author = {Lim, J and Van, AB and Wester, M and Koprowski, K and Valera, E and Bashir, R},
title = {Amplification-free dual-blocking autocatalytic CRISPR-Cascade for attomolar DNA detection with low nonspecific signal.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {21},
pages = {e2537414123},
doi = {10.1073/pnas.2537414123},
pmid = {42166245},
issn = {1091-6490},
support = {R01EB032725 A//HHS | NIH (NIH)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Methicillin-Resistant Staphylococcus aureus/genetics/isolation & purification ; RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; *DNA/analysis/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *DNA, Bacterial/genetics ; Ribonucleoproteins/genetics ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {Autocatalytic CRISPR architecture offers amplification-free nucleic acid detection by directly linking target recognition to self-reinforcing ribonucleoprotein (RNP) generation. However, spontaneous background activation remains a key barrier, because strand invasion or unwinding events can initiate unintended amplification and diminish assay specificity. Here, we introduce a dual-blocking CRISPR-Cascade design that independently cages both the guide RNA and trigger DNA, establishing an intrinsic AND gate to raise the effective kinetic barrier for unintended RNP formation. This strategy suppresses leakage by approximately 3- to 18-fold relative to single blocking configurations in full Cascade reactions, while preserving rapid detection (10 min), achieving single-copy sensitivity, and enabling quantitative detection. When paired with a competitive guide RNA decoy, the system further reduces background signals without affecting true target detection. Finally, we demonstrate robust Methicillin-resistant Staphylococcus aureus detection from whole blood in under 40 min including the sample purification and extraction. These results establish dual-blocking as a generalizable molecular gating framework for constructing leakage-resistant, amplification-free CRISPR systems suitable for rapid and decentralized diagnostics.},
}

*CRISPR-Cas Systems/genetics
Methicillin-Resistant Staphylococcus aureus/genetics/isolation & purification
RNA, Guide, CRISPR-Cas Systems/genetics
Humans
*DNA/analysis/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*DNA, Bacterial/genetics
Ribonucleoproteins/genetics
Nucleic Acid Amplification Techniques/methods

@article {pmid42166840,
year = {2026},
author = {Simonneau, B and Baghdoyan, S and Cailleret, M and Simon, S and Ruckebusch, O and Vrablikova, B and Giraud-Triboult, K and Kassar, LE and Fanen, P and Duriez, B},
title = {Generation of two iPSC lines carrying two cystic fibrosis rare intronic mutations c.1585-1G>A and c.1680-886A>G in the CFTR gene of the parental line PCIi033-A using CRISPR/Cas tools.},
journal = {Stem cell research},
volume = {94},
number = {},
pages = {104016},
doi = {10.1016/j.scr.2026.104016},
pmid = {42166840},
issn = {1876-7753},
abstract = {CFTR gene mutations are responsible for Cystic Fibrosis. For half a decade, a triple therapy has been available for patients carrying the most frequent mutation: p.F508del. Among classified mutations, intronic mutations are rare, and no therapeutic strategies have yet been developed for such patients. We genome edited the parental iPSC line PCIi033-A to introduce two intronic mutations: A.s.Cas12a for c.1585-1G>A (PCIi033-A-8) and SpCas9 for c.1680-886A>G (PCIi033-A-9). Both cell lines have normal morphology and karyotype, conserved pluripotency, and differentiate into the three germ layers. Obtaining iPSC-derived mutant lung epithelia would be an efficient tool for testing new therapeutic strategies.},
}

@article {pmid41937145,
year = {2026},
author = {Wu, X and Jin, S and Pan, Y and Zhen, W and Yu, S and Zhang, Y and Xu, F and Wang, R and Wu, M and Sun, W and Xu, J and Zang, X and Zhang, H},
title = {Immune-evasive stem cells: engineering tolerance and reprogramming microenvironments for regenerative therapy.},
journal = {Stem cell research & therapy},
volume = {17},
number = {1},
pages = {},
pmid = {41937145},
issn = {1757-6512},
support = {2023xkfyts01//the 2023 Disciplinary Construction Project in the School of Dentistry, Anhui Medical University/ ; YQYB2024013//Anhui Province Outstanding Young Teachers Development Program/ ; },
mesh = {Humans ; *Regenerative Medicine/methods ; Gene Editing ; *Immune Tolerance ; Animals ; *Cellular Reprogramming ; *Induced Pluripotent Stem Cells/immunology ; CRISPR-Cas Systems ; *Stem Cell Transplantation ; },
abstract = {Stem cell transplantation (SCT) holds significant promise for regenerative medicine, yet immune rejection remains a major obstacle. To address this, recent advances leverage CRISPR/Cas9 to engineer hypoimmunogenic induced pluripotent stem cells. These modified cells lack classical immune recognition markers (HLA class I/II) yet retain immune-tolerant molecules such as HLA-E, HLA-G, and CD47, enabling their universal use across different individuals. Additionally, mesenchymal stem cell-derived exosomes and immune checkpoint modulators (e.g., PD-L1) have shown clinical effectiveness by reducing graft-versus-host disease and autoimmune reactions. They achieve this through mechanisms such as suppressing inflammatory T-cell activation, promoting regulatory T-cell expansion, and modulating macrophage polarization. Despite these advances, several challenges remain. One key concern is the potential tumorigenic risk caused by genomic instability during genome editing and long-term cell expansion. Emerging precision editing platforms, including base editing and prime editing, provide strategies to reduce double-strand DNA break-induced chromosomal rearrangements and improve genomic safety. Future research priorities include integrating AI-based immune profiling, precision genome editing, and advanced 3D-bioprinting technologies. Together, these innovations represent a paradigm shift toward developing safer, more effective, universally compatible stem cell therapies for diseases previously deemed untreatable.},
}

Humans
*Regenerative Medicine/methods
Gene Editing
*Immune Tolerance
Animals
*Cellular Reprogramming
*Induced Pluripotent Stem Cells/immunology
CRISPR-Cas Systems
*Stem Cell Transplantation

@article {pmid41975095,
year = {2026},
author = {Guan, K and Ocampo, RF and Matheus Carnevali, PB and Castelle, CJ and Gonzalez-Osorio, L and Castanzo, DT and Thomas, NC and Brothers, M and Dangerfield, TL and Hooper, MM and West, MS and Appleby, NM and Krudop, I and Lamothe, RC and Aliaga Goltsman, DS and Alexander, LM and Butterfield, CN and Johnson, KA and Brown, CT and Taylor, DW},
title = {Comparative characterization of Cas12f orthologs reveals mechanistic features underlying enhanced genome editing efficiency.},
journal = {Nature structural & molecular biology},
volume = {33},
number = {5},
pages = {756-767},
pmid = {41975095},
issn = {1545-9985},
support = {R35 GM138348/GM/NIGMS NIH HHS/United States ; },
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems ; *CRISPR-Associated Proteins/genetics/chemistry/metabolism ; RNA, Guide, CRISPR-Cas Systems/metabolism/genetics ; *Eubacteriales/enzymology/genetics ; Models, Molecular ; HEK293 Cells ; },
abstract = {Miniature CRISPR-Cas12f nucleases are attractive candidates for therapeutic genome editing because of their compact size and compatibility with adeno-associated virus (AAV) delivery. However, editing efficiencies in mammalian cells are lower than those of larger systems. The extensive phylogenetic diversity of Cas12f suggests unexplored mechanistic variation with the potential for optimization. Here we identify and characterize a naturally occurring Cas12f ortholog discovered through metagenomics, Alistipes sp. Cas12f (Al3Cas12f), which supports robust genome editing in human cells. Through structural, biochemical and kinetic analyses, we compare Al3Cas12f to two recently described orthologs, Oscillibacter sp. Cas12f and Ruminiclostridium herbifermentans Cas12f. These orthologs present divergent architectures and regulatory features governing protospacer-adjacent motif recognition, guide RNA (gRNA) binding, dimerization and DNA cleavage. Notably, Al3Cas12f achieves efficient R-loop formation through a stable dimer interface and a naturally optimized gRNA. Leveraging these structural insights, we generate an engineered Al3Cas12f variant (RKK) that increases editing and improves activity across several tested genomic loci. By overcoming locus-dependent variability and an apparent potency threshold, this engineered compact editor seems to expand the feasibility of low-dose, AAV-compatible therapeutic genome editing. Our results elucidate mechanistic determinants of Cas12f activity and offer a framework for engineering compact genome editors that may bear therapeutic potential.},
}

*Gene Editing/methods
Humans
*CRISPR-Cas Systems
*CRISPR-Associated Proteins/genetics/chemistry/metabolism
RNA, Guide, CRISPR-Cas Systems/metabolism/genetics
*Eubacteriales/enzymology/genetics
Models, Molecular
HEK293 Cells

@article {pmid42156407,
year = {2026},
author = {Sa, Y and Liu, C and Yang, L and Yue, L and Zhu, L and Guo, Y and Wang, R and Wang, Y and Feng, Y and Wang, Y and Zhang, Y and Wang, W and Xie, Y},
title = {Structural basis for dual mechanism of Cas2/3 nuclease inhibition by anti-CRISPR protein AcrIF19.},
journal = {Nature communications},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41467-026-73156-3},
pmid = {42156407},
issn = {2041-1723},
support = {32301058//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32371329, 32301055，32400565，32560225//National Natural Science Foundation of China (National Science Foundation of China)/ ; ZR202211190090//Natural Science Foundation of Shandong Province (Shandong Provincial Natural Science Foundation)/ ; tsqn202306146//Taishan Scholar Project of Shandong Province/ ; },
abstract = {CRISPR-Cas systems are prokaryotic immune mechanisms often targeted by phage-encoded anti-CRISPR (Acr) proteins. This study characterizes AcrIF19, a potent inhibitor of the type I-F system in Pectobacterium atrosepticum. The cryo-EM structure of the apo Cas2/3 and Cas2/3-AcrIF19 complex reveals a dual inhibitory mechanism. AcrIF19 employs a negatively charged β2-β3 loop to sterically occlude the non-target DNA strand entry channel, acting as a competitive inhibitor to disrupt Cas2/3 recruitment. Concurrently, this steric occlusion impedes ssDNA-mediated allosteric activation, which locks the critical helix-like loop motif in an inhibitory conformation and thereby abrogates DNA cleavage activity. AcrIF19 represents an anti-CRISPR protein inhibiting Cas2/3 via two different mechanisms, integrating a competitive ssDNA inhibitor with an allosteric blockade to suppress both target recruitment and DNA cleavage.},
}

@article {pmid42157911,
year = {2025},
author = {Waqar, Z and Sethi, P and Jain, D and Singh, K and Alsaidan, OA and Alzarea, SI and Gupta, JK and Saxena, S and Sharma, MC},
title = {Precision Medicine in Neurodegenerative Diseases: Genomic Approaches to Target Amyloid-β, Tau, and Alpha-Synuclein Pathways.},
journal = {Current genomics},
volume = {26},
number = {6},
pages = {469-494},
pmid = {42157911},
issn = {1389-2029},
abstract = {Neurodegenerative diseases, including Alzheimer's and Parkinson's disease, are characterized by the pathological aggregation of proteins such as amyloid-β, tau, and alpha-synuclein. These hallmark proteins play central roles in disease progression and represent promising targets for therapeutic intervention. Advances in precision medicine, driven by genomic technologies such as CRISPR-Cas systems, RNA-based therapies, and high-throughput sequencing, have enabled the development of tailored strategies to modulate these pathological pathways. This review examines the integration of genomic approaches in targeting amyloid-β, tau, and alpha-synuclein, emphasizing their potential to mitigate disease progression and improve patient outcomes. We highlight current progress in preclinical and clinical studies, discuss challenges associated with translating these therapies into clinical practice, and explore future directions for achieving therapeutic precision in neurodegenerative disorders. By examining the interplay of genetic, molecular, and therapeutic innovations, this review underscores the transformative potential of genomic medicine in addressing the unmet needs of neurodegenerative disease treatment.},
}

@article {pmid42158919,
year = {2026},
author = {Zhou, C and Arimura, SI},
title = {TALENs and Related Technologies for Editing Nuclear and Organellar Genomes in a Model Plant, Arabidopsis thaliana.},
journal = {Bio-protocol},
volume = {16},
number = {9},
pages = {e5668},
pmid = {42158919},
issn = {2331-8325},
abstract = {Plant genome editing is a powerful approach for modifying plant DNA to investigate gene function and to engineer desirable traits. Several genome-editing technologies have been developed, among which CRISPR/Cas systems and transcription activator-like effector nucleases (TALENs) are widely used to introduce targeted double-stranded DNA breaks. While CRISPR/Cas systems are highly efficient for nuclear genome editing, their application to plant organellar genomes remains limited, largely due to difficulties in guide RNA delivery into mitochondria and chloroplasts. Here, we present a detailed and reproducible protocol for constructing TALEN-based binary vectors for targeted genome editing in Arabidopsis thaliana. This protocol describes the assembly of TALE repeat arrays, the generation of nuclear-, mitochondrial-, and plastid-targeted TALEN expression vectors using MultiSite Gateway cloning, and subsequent Agrobacterium-mediated plant transformation and genotyping. The workflow enables the production of nTALENs, mitoTALENs, and ptpTALENs using a unified vector design strategy. In addition, the protocol briefly outlines the construction principles of TALE-based cytidine deaminases (TALECDs) for targeted C-to-T base editing in plant organellar genomes. The protocol provides a flexible and robust framework for plant nuclear and organellar genome editing and can be readily adapted to different target genes and experimental purposes. Its modular design and compatibility with standard molecular cloning techniques make it accessible to laboratories aiming to perform precise genome manipulation in plants. Key features • Requires experience in basic molecular cloning and Arabidopsis transformation; suitable for laboratories performing plant nuclear and organellar genome editing. • Enables construction of nuclear-, mitochondrial-, and plastid-targeted TALENs using a unified MultiSite Gateway-based vector system. • Provides a modular workflow for assembling large TALEN binary vectors compatible with Agrobacterium-mediated transformation in Arabidopsis thaliana. • Includes optional extension to TALE-based cytidine deaminases for targeted C-to-T base editing in plant mitochondrial and plastid genomes.},
}

@article {pmid42159153,
year = {2026},
author = {Gonçalves, LDS and Domingues, WB and Nunes, LS and Blödorn, EB and Dellagostin, EN and Martins, AWDS and Acosta, IB and Corcini, CD and Varela, AS and Fróes, CN and Campos, VF},
title = {First Report of CRISPR-Cas9 Ribonucleoprotein Delivery Into Teleost Spermatozoa With Preserved Membrane and Genomic Integrity.},
journal = {Molecular reproduction and development},
volume = {93},
number = {5},
pages = {e70114},
doi = {10.1002/mrd.70114},
pmid = {42159153},
issn = {1098-2795},
support = {23/2551-0000181-0//Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul/ ; 22/2551-0001645-6//Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul/ ; 24/2551-0002158-2//Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul/ ; 440636/2022-1//Conselho Nacional de Desenvolvimento Científico e Tecnológico/ ; 407610/2024-3//Conselho Nacional de Desenvolvimento Científico e Tecnológico/ ; AUXPE #2537/2018//Coordenação de Aperfeiçoamento de Pessoal de Nível Superior/ ; },
mesh = {Animals ; Male ; *Spermatozoa/metabolism/cytology ; *Ribonucleoproteins/genetics/metabolism ; *CRISPR-Cas Systems ; *Cichlids/genetics ; *Cell Membrane/metabolism ; Electroporation/methods ; Sperm Motility ; DNA Fragmentation ; },
abstract = {Delivery of large molecular complexes into spermatozoa remains a challenge in reproductive biotechnology. Teleost sperm possess highly compacted chromatin and minimal cytoplasmic volume, limiting intracellular access of ribonucleoprotein assemblies. Here, we evaluated whether CRISPR-Cas9 ribonucleoprotein (RNP) complexes can be introduced into Nile tilapia spermatozoa using capillary electroporation while preserving cellular integrity. RNP uptake was quantified via EGFP fluorescence, and sperm performance was assessed through motility, membrane integrity, mitochondrial activity, and DNA fragmentation analyses. Approximately 10%-13% of spermatozoa exhibited detectable EGFP signal following electroporation. Increased pulse numbers reduced motility and mitochondrial activity, whereas membrane integrity was preserved and no increase in DNA fragmentation was detected. A single pulse (1250 V, 40 ms) provided the best balance between RNP uptake and functional preservation. These findings support capillary electroporation as a minimally disruptive strategy for CRISPR-RNP delivery in teleost reproductive biology.},
}

Animals
Male
*Spermatozoa/metabolism/cytology
*Ribonucleoproteins/genetics/metabolism
*CRISPR-Cas Systems
*Cichlids/genetics
*Cell Membrane/metabolism
Electroporation/methods
Sperm Motility
DNA Fragmentation

@article {pmid40601102,
year = {2026},
author = {Jong, HL and Yuen, KS and Jin, DY and Hoe, SLL and Ideris, A and Tan, CH and Lam, SK and Lim, YM and Cheong, SK},
title = {Generation of Leucine-Rich Repeat Kinase 2 (LRRK2) Knockout Neuroblastoma Cells SH-SY5Y by CRISPR/Cas9-Mediated Genome Editing.},
journal = {Biochemical genetics},
volume = {64},
number = {3},
pages = {4423-4455},
pmid = {40601102},
issn = {1573-4927},
mesh = {Humans ; *Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/genetics/metabolism ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Neuroblastoma/genetics/pathology ; Cell Line, Tumor ; *Gene Knockout Techniques/methods ; },
abstract = {Leucine-rich repeat kinase 2 (LRRK2) is associated with Parkinson's disease, despite its low expression in the brain. Pathogenic mutations in LRRK2 enhance kinase activity and contribute to the disease's pathogenesis. Neuroblastoma SH-SY5Y cells, which also exhibit low LRRK2 expression, are extensively used as a model for Parkinson's disease. While less prominent, low-expression genes can play crucial roles in cellular processes, development, and disease. Knocking out such genes poses specific challenges, including difficulties in detection, incomplete knockout, and compensatory mechanisms that can obscure phenotypic changes. This study develops a strategy to knockout low-expression LRRK2 in SH-SY5Y cells effectively. Our approach employs a double-cut and multiple guide RNAs strategy, optimized electroporation parameters to enhance CRISPR/Cas9 plasmid delivery, refined clonal expansion technique, and a sensitive protein detection protocol. We successfully generate LRRK2 knockout SH-SY5Y cells using CRISPR/Cas9, with the knockout efficiency validated by PCR analysis, sequencing, and Western blot analysis.},
}

Humans
*Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/genetics/metabolism
*CRISPR-Cas Systems
*Gene Editing/methods
*Neuroblastoma/genetics/pathology
Cell Line, Tumor
*Gene Knockout Techniques/methods

@article {pmid40853436,
year = {2026},
author = {Yıldız, Bİ and Karabağ, K},
title = {Targeted Gene Editing in Honey Bees Using Liposome-Based CRISPR-Cas9.},
journal = {Biochemical genetics},
volume = {64},
number = {3},
pages = {4477-4492},
pmid = {40853436},
issn = {1573-4927},
support = {FDK-2022-6006//Akdeniz Üniversitesi/ ; },
mesh = {Animals ; Bees/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Liposomes/chemistry ; Male ; },
abstract = {Genome editing technologies have revolutionized molecular biology, enabling precise manipulation of gene functions across diverse organisms. In this study, we introduce a novel liposome-mediated delivery system for CRISPR-Cas9 components targeting the Juvenile Hormone Acid Methyltransferase (JHAMT) gene in honey bees (Apis mellifera anatoliaca). This approach leverages drone sperm cells as vectors for CRISPR-Cas9 transfection, overcoming the technical challenges of embryo microinjection in honey bees, such as low survival rates and labor-intensive procedures. The study involved artificial insemination of queen bees with transfected sperm and subsequent evaluation of gene-editing efficiency across generations.Our findings demonstrate the successful generation of both heterozygous and homozygous mutants, with gene-editing efficiencies reaching approximately 43%. This innovative method highlights the potential of liposome-mediated delivery systems for non-invasive, efficient, and scalable genome editing in eusocial insects. The results pave the way for broader applications in honey bee genetic research, offering a viable alternative to traditional methods. Furthermore, this study underscores the importance of genetic tools in advancing apiculture and addressing ecological challenges linked to pollinator health.},
}

Animals
Bees/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
*Liposomes/chemistry
Male

@article {pmid41793179,
year = {2026},
author = {Dong, K and Hu, H and Wang, H and Zheng, Z and Cheng, S and Shu, W and Liu, R and Xin, X and Huang, S and Qian, D and Xiao, X and Fu, Q and Wang, H},
title = {A Single-Enzyme Activated CRISPR-Cas12a Nano System via Subtly Balanced dsDNA for Kinetic-Gated UDG Detection and Spatiotemporal Cellular Imaging.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {28},
pages = {e23400},
doi = {10.1002/advs.202523400},
pmid = {41793179},
issn = {2198-3844},
support = {2023YFC2705400//The National Key Research and Development Plan/ ; 82472965//National Natural Science Foundation project/ ; W2521097//National Natural Science Foundation project/ ; 2024EIA002//Hubei Provincial Central Government-Guided Local Science and Technology Fund Development Project/ ; 2025AFD286//Hubei Provincial National Natural Science Foundation/ ; 2025XHYN047//Free Innovation Pre-research Fund of Union Hospital/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *DNA/metabolism/genetics ; *Uracil-DNA Glycosidase/metabolism/genetics ; Kinetics ; Humans ; *Endodeoxyribonucleases/metabolism/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; DNA Repair ; Bacterial Proteins ; },
abstract = {Uracil-DNA glycosylase (UDG) is a key enzyme in base excision repair and an important biomarker for genomic stability and disease. In many reported sensing systems, uracil excision is coupled to signal generation through additional downstream BER processing steps, resulting in an indirect readout of UDG activity. Here, we report a single-enzyme activated CRISPR-Cas12a nanosystem driven by a subtly balanced double-stranded DNA (dsDNA) substrate. This dsDNA serves as a kinetic gatekeeper that maintains Cas12a in an inert state until UDG-mediated uracil excision disrupts the balance, lowering the energy barrier for crRNA invasion and initiating Cas12a trans-cleavage. This conformationally gated mechanism directly converts a uracil excision event into an amplified CRISPR response without requiring sequential enzymatic processing. The system achieves a 1840-fold discrimination ratio and an ultralow detection limit of 5 × 10[-7] U/mL. Furthermore, a genetically encoded variant enables nuclear localization of Cas12a and dsDNA sensors for in situ imaging of endogenous UDG. The platform visualizes UDG dynamics across distinct cell cycle phases, realizing spatiotemporal mapping of repair activity in living cells. This work introduces a new activation paradigm for CRISPR-Cas12a via subtly balanced dsDNA and establishes a generalizable strategy for precise molecular sensing in complex biological environments.},
}

*CRISPR-Cas Systems/genetics
*DNA/metabolism/genetics
*Uracil-DNA Glycosidase/metabolism/genetics
Kinetics
Humans
*Endodeoxyribonucleases/metabolism/genetics
*CRISPR-Associated Proteins/metabolism/genetics
DNA Repair
Bacterial Proteins

@article {pmid41806318,
year = {2026},
author = {Sun, J and Yang, X and Jiang, W and Ji, C and Wu, Y and Sun, H and Liu, X and Yamamoto, M and Tsukamoto, T and Nomura, S and Zhao, J and Ruan, Y and Li, H and Wang, X},
title = {In Vivo CRISPR Screening Identifies the Glutamate Receptor GRIA2 as Promoting Peritoneal Metastasis of Gastric Cancer via Calcium-Dependent β-Catenin Activation.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {28},
pages = {e21746},
doi = {10.1002/advs.202521746},
pmid = {41806318},
issn = {2198-3844},
support = {ZY2024-003//Scientific Research Foundation of Zhongshan Hospital/ ; },
mesh = {*Stomach Neoplasms/genetics/pathology/metabolism ; Animals ; Humans ; *Receptors, AMPA/genetics/metabolism ; Mice ; *Peritoneal Neoplasms/secondary/genetics/metabolism ; *beta Catenin/metabolism/genetics ; Cell Line, Tumor ; *Calcium/metabolism ; Cell Movement/genetics ; Wnt Signaling Pathway/genetics ; CRISPR-Cas Systems/genetics ; Glycogen Synthase Kinase 3 beta/metabolism ; },
abstract = {Peritoneal metastasis is the most lethal manifestation of gastric cancer, with a median survival of less than one year, highlighting the need for new therapeutic targets. Through an in vivo genome-wide CRISPR/Cas9 screen, we identified GRIA2, an AMPA-type glutamate receptor subunit, as a key driver of peritoneal metastasis. GRIA2 promotes gastric cancer cell migration, invasion, stemness, and adhesion to mesothelial cells in a glutamate-dependent manner. Mechanistically, glutamate activates GRIA2, enhancing its interaction with GSK-3β and inducing calcium influx, inhibiting GSK-3β kinase activity and stabilizing β-catenin, thereby activating the Wnt/β-catenin signaling pathway. Single-cell RNA sequencing revealed that cancer-associated fibroblasts are the primary source of glutamate in the peritoneal microenvironment, which establishes a paracrine axis that enhances GRIA2-driven metastasis. Pharmacological inhibition of AMPA receptors with NBQX and Selurampanel suppressed peritoneal metastasis in both cell line-derived and patient-derived organoid xenograft (PDOX) mouse models. In clinical analysis, GRIA2 expression in peritoneal metastases correlated with the levels of β-catenin and phosphorylated GSK-3β (serine 9), with high GRIA2 expression predicting poor prognosis. These findings suggest that GRIA2 is a novel therapeutic target, and AMPA receptor antagonists are promising agents for treating gastric cancer peritoneal metastasis.},
}

*Stomach Neoplasms/genetics/pathology/metabolism
Animals
Humans
*Receptors, AMPA/genetics/metabolism
Mice
*Peritoneal Neoplasms/secondary/genetics/metabolism
*beta Catenin/metabolism/genetics
Cell Line, Tumor
*Calcium/metabolism
Cell Movement/genetics
Wnt Signaling Pathway/genetics
CRISPR-Cas Systems/genetics
Glycogen Synthase Kinase 3 beta/metabolism

@article {pmid41957628,
year = {2026},
author = {Yang, W and Wu, J and Zhou, Y and Yu, Z and Shang, X and Huang, J},
title = {PfVPS4, an ESCRT AAA-ATPase, is essential for asexual proliferation and gametocyte sexual conversion in Plasmodium falciparum.},
journal = {Parasites & vectors},
volume = {19},
number = {1},
pages = {},
pmid = {41957628},
issn = {1756-3305},
support = {32170071//National Natural Science Foundation of China/ ; 32200450//National Natural Science Foundation of China/ ; 32300051//National Natural Science Foundation of China/ ; 2023JJ40798//Natural Science Foundation of Hunan Province/ ; 2023JJ30651//Natural Science Foundation of Hunan Province/ ; },
mesh = {*Plasmodium falciparum/genetics/growth & development/enzymology/physiology ; *Endosomal Sorting Complexes Required for Transport/genetics/metabolism ; *Protozoan Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *Adenosine Triphosphatases/genetics/metabolism ; Gene Knockdown Techniques ; Erythrocytes/parasitology ; Reproduction, Asexual ; Humans ; *ATPases Associated with Diverse Cellular Activities/genetics/metabolism ; Life Cycle Stages ; },
abstract = {BACKGROUND: Malaria, caused by Plasmodium spp., remains a major global health threat. Among them, Plasmodium falciparum is the most pathogenic, and its asexual intraerythrocytic proliferation is the pathological basis. This process has enormous biosynthetic demands and highly relies on the coordinated function of the endomembrane and vesicular transport systems. The transition from asexual proliferation to sexual differentiation similarly involves remodeling of internal membrane complexes, membrane reshaping, and precise protein sorting. In eukaryotic cells, the Endosomal Sorting Complexes Required for Transport (ESCRT) complex is a core machinery for membrane remodeling and endosomal development. However, how the ESCRT system regulates the complex life cycle of Plasmodium, particularly during intraerythrocytic proliferation and sexual conversion, remains an important unresolved question.

METHODS: In this study, using Plasmodium falciparum as a model system, we applied CRISPR-Cas9-mediated homologous recombination to achieve conditional knockdown of PfVPS4, the core ATPase of the ESCRT complex - vacuolar protein sorting-associated protein 4 (PfVPS4). Western blotting and immunofluorescence assays were used to assess PfVPS4 abundance and subcellular localization. Tightly synchronized cultures were used to evaluate its effects on parasite growth, merozoite numbers, and gametocyte conversion rate. In vitro protein purification, enzyme kinetics, and site-directed mutagenesis were performed to identify the impact of key residues on PfVPS4 ATPase activity and to validate the synergistic activation by its cofactor PfVta1. In addition, multiple sequence alignment and AlphaFold3 modeling were used to predict and display structural features before and after mutation of key sites.

RESULTS: We successfully generated conditional knockdown lines in both Pf3D7 and PfNF54 parasite strains, enabling effective knockdown at different stages of the intraerythrocytic cycle and during gametocytogenesis. Knockdown of PfVPS4 led to an 84% reduction in asexual progeny parasite numbers, decreased merozoite numbers, and a 46% reduction in gametocyte conversion rate, without affecting subsequent gametocyte maturation. Biochemical assays showed that PfVPS4 ATPase activity is optimal at pH 7.5 and 37°C, and is dependent on Mg²⁺, with a Vmax of 2.23 ± 0.053 U/mg and a Km of 0.086 mM. Site-directed mutagenesis validated the essential role of the canonical catalytic residues (D213, E214) and the species-specific key residues (T161, I288) in maintaining enzymatic activity, and confirmed that the cofactor PfVta1 significantly enhances PfVPS4 activity.

CONCLUSION: PfVPS4 is essential for normal asexual blood-stage replication and efficient sexual conversion in Plasmodium falciparum. Its knockdown severely disrupts intraerythrocytic proliferative homeostasis and reduces gametocyte conversion, indicating that this protein has a broader role in coordinating parasite proliferation and transmission. Given its essentiality, species‑specific residues, and regulation by PfVta1, PfVPS4 and its complex are attractive antimalarial drug targets.},
}

*Plasmodium falciparum/genetics/growth & development/enzymology/physiology
*Endosomal Sorting Complexes Required for Transport/genetics/metabolism
*Protozoan Proteins/genetics/metabolism
CRISPR-Cas Systems
*Adenosine Triphosphatases/genetics/metabolism
Gene Knockdown Techniques
Erythrocytes/parasitology
Reproduction, Asexual
Humans
*ATPases Associated with Diverse Cellular Activities/genetics/metabolism
Life Cycle Stages

@article {pmid41975467,
year = {2026},
author = {Song, HY and Cao, H and Huang, SB and Elsheikha, HM and Zheng, Z and Lu, XS and Tian, X and Zheng, XN and Zhu, XQ},
title = {Functional characterization of 11 novel rhoptry proteins in the type I RH strain of Toxoplasma gondii using the CRISPR-Cas9 system.},
journal = {Parasites & vectors},
volume = {19},
number = {1},
pages = {},
pmid = {41975467},
issn = {1756-3305},
support = {2022Y309//the Graduate Innovation Project of Shanxi Province/ ; U2202201//the NSFC-Yunnan Joint Fund/ ; 2021YFC2300800 and 2021YFC2300802//National Key Research and Development Program of China/ ; 2021XG001//the Special Research Fund of Shanxi Agricultural University for High-level Talents/ ; },
mesh = {*Toxoplasma/genetics/pathogenicity/metabolism ; *Protozoan Proteins/genetics/metabolism ; Animals ; *CRISPR-Cas Systems ; Mice ; Virulence ; Gene Editing ; Female ; Virulence Factors/genetics ; Mice, Inbred BALB C ; Gene Knockout Techniques ; Toxoplasmosis/parasitology ; },
abstract = {BACKGROUND: Rhoptry proteins (ROPs) are secreted effectors that play important roles in the virulence of Toxoplasma gondii by facilitating host cell invasion and immune modulation. Although many ROPs have been predicted, their specific functions remain largely unexplored. This study investigates the roles of 11 previously uncharacterized ROPs in T. gondii biology, with a focus on their contributions to virulence.

METHODS: Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated genome editing was employed to generate epitope-tagged and knockout mutants for each candidate ROP in the T. gondii RHΔku80 strain. Subcellular localization was determined via immunofluorescence microscopy in both tachyzoite and bradyzoite stages. In vitro assays assessed parasite invasion, replication, egress, and plaque formation. In vivo virulence was evaluated in mouse infection models. To explore molecular mechanisms underlying virulence attenuation, we performed transcriptomic profiling of RHΔrop64 and RHΔrop65 knockout strains.

RESULTS: All 11 candidate ROPs exhibited rhoptry localization in both tachyzoite and bradyzoite stages. Despite no apparent in vitro growth defects, deletion of ROP64 and ROP65 led to significant attenuation of virulence in mice, with ROP64 showing the most pronounced effect. Transcriptome analysis revealed downregulation of key immune-modulatory genes, including ROP5, ROP39, TgIST, and PLP1. In addition, RHΔrop64 exhibited broader suppression of ROPs than RHΔrop65, suggesting it has a more pronounced role in immune modulation.

CONCLUSIONS: ROP64 and ROP65 are critical to T. gondii virulence, likely through modulation of the parasite's immune-evasive machinery. Their regulatory influence on effector expression underscores their importance in host adaptation. Importantly, the RHΔrop64 mutant displays characteristics of an attenuated strain with potential for vaccine development against toxoplasmosis.},
}

*Toxoplasma/genetics/pathogenicity/metabolism
*Protozoan Proteins/genetics/metabolism
Animals
*CRISPR-Cas Systems
Mice
Virulence
Gene Editing
Female
Virulence Factors/genetics
Mice, Inbred BALB C
Gene Knockout Techniques
Toxoplasmosis/parasitology

@article {pmid41999750,
year = {2026},
author = {Thege, FI and Hoskins, A and Kramer, A and Salim, I and Seetharaman, A and Fowlkes, N and Rajapakshe, KI and Maitra, A and Wörmann, SM},
title = {An autochthonous CRISPR activation screening platform for characterizing tissue-specific oncogene selection.},
journal = {Cell reports. Medicine},
volume = {7},
number = {5},
pages = {102759},
doi = {10.1016/j.xcrm.2026.102759},
pmid = {41999750},
issn = {2666-3791},
mesh = {Animals ; *Oncogenes/genetics ; Humans ; Mice ; *Lung Neoplasms/genetics/pathology ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Cas Systems/genetics ; *Pancreatic Neoplasms/genetics/pathology ; Organ Specificity/genetics ; },
abstract = {Human adenocarcinomas exhibit tissue-specific mutation and copy-number patterns that suggest diverse selective pressures and distinct oncogene dependencies. Here, we use our FiCASCan platform to test whether in vivo CRISPR activation screening can recapitulate oncogene selection during tumor initiation. Using CRISPRa-competent PPKS mice and intranasal or intraductal delivery of pooled lentivirus, we screen frequently amplified and mutated genes in autochthonous lung and pancreas cancer models. We observe strong selection for Egfr, Myc, Sox2, and Pik3cb activation in lung tumors and near-complete dominance of Myc in pancreatic tumors, revealing striking tissue-specific differences. In our model, Sox2 activation suppresses Nkx2-1 signaling and drives aggressive mucinous lung adenocarcinoma. MYC activation in the pancreas mirrors MYC amplification in human PDAC, including the emergence of an immune-cold microenvironment. Overall, our findings show that in vivo CRISPR activation screening faithfully captures oncogene selection and provides a powerful approach for studying tumor initiation and progression.},
}

Animals
*Oncogenes/genetics
Humans
Mice
*Lung Neoplasms/genetics/pathology
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*CRISPR-Cas Systems/genetics
*Pancreatic Neoplasms/genetics/pathology
Organ Specificity/genetics

@article {pmid42083769,
year = {2026},
author = {Chen, Z and Mao, K and Meng, H and Gao, C and Lv, H and Li, X and Zheng, Q and Yang, Z and Hamza, IA and Tu, C and Zhang, H},
title = {String-Powered Microfluidic Chip Integrating Heparin-Mediated One-Pot RT-RPA/CRISPR-Cas12a for Multiplex Detection of HFMD Viruses.},
journal = {Analytical chemistry},
volume = {98},
number = {19},
pages = {14315-14329},
doi = {10.1021/acs.analchem.6c00663},
pmid = {42083769},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems ; *Hand, Foot and Mouth Disease/virology/diagnosis ; *Heparin/chemistry ; Humans ; *Lab-On-A-Chip Devices ; Wastewater/virology ; Nucleic Acid Amplification Techniques ; },
abstract = {Hand, foot, and mouth disease (HFMD) is a common childhood infection caused by enteroviruses, which exhibit distinct regional and seasonal epidemiological patterns. Wastewater-based epidemiology is a crucial tool for monitoring population infection dynamics and viral subtype distribution. However, the lack of effective on-site viral detection methods limits timely early warning and effective surveillance of infectious disease outbreaks. This study developed a one-pot RT-RPA/CRISPR-Cas12a assay-based, string-powered flywheel microfluidic chip for the multiplex detection of HFMD viruses in wastewater. First, by leveraging the regulatory effect of heparin sodium on CRISPR/Cas12a activity, a one-pot RT-RPA/CRISPR-Cas12a system was constructed to detect four major subtypes of HFMD virus (EV-A71, CV-A16, CV-A6, and CV-A10). Subsequently, this method was integrated into a pull-wire, flywheel-type, dual-axis centrifugal microfluidic chip, named the Heparin-Inhibited CRISPR-Associated System Chip (HICAS-Chip), enabling integrated enrichment, purification, elution, and multiplexed detection. The HICAS-Chip allowed visual detection of nucleic acids at 10 aM sensitivity within 1 h, corresponding to the sensitivity of the one-pot RT-RPA/CRISPR-Cas12a assay. During a year-long wastewater monitoring program in Guiyang City, China, the HICAS-Chip identified EV-A71 and CV-A10 as the predominant circulating subtypes, with incidence peaks observed in June, November, and December. The wastewater detection results obtained using HICAS-Chip showed high concordance (95.83%) with RT-qPCR assays. This platform provides an efficient portable device for the early detection and continuous monitoring of HFMD epidemic trends by wastewater-based epidemiology.},
}

*CRISPR-Cas Systems
*Hand, Foot and Mouth Disease/virology/diagnosis
*Heparin/chemistry
Humans
*Lab-On-A-Chip Devices
Wastewater/virology
Nucleic Acid Amplification Techniques

@article {pmid42087437,
year = {2026},
author = {Yang, Y and Liu, Y and Xu, H and Zhou, F and Ji, X and He, Z},
title = {CRISPR/Cas13a-Driven Catalytic Hairpin Assembly of a Quantum Dot Nanobeacon for Viral RNA Detection.},
journal = {Analytical chemistry},
volume = {98},
number = {19},
pages = {14163-14172},
doi = {10.1021/acs.analchem.5c08146},
pmid = {42087437},
issn = {1520-6882},
mesh = {*Quantum Dots/chemistry ; *RNA, Viral/analysis/genetics ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; *Influenza A Virus, H1N1 Subtype/genetics/isolation & purification ; SARS-CoV-2/genetics/isolation & purification ; Limit of Detection ; Humans ; Nucleic Acid Amplification Techniques ; COVID-19/diagnosis ; },
abstract = {Influenza A (H1N1), which is a highly contagious respiratory pathogen, poses a serious public health threat. Its effective control necessitates rapid and accurate detection methods. Here, we developed a novel quantum dot (QDs) nanobeacon-based biosensor that synergistically integrates the precise target recognition and trans-cleavage activity of CRISPR/Cas13a with the isothermal, enzyme-free signal amplification power of catalytic hairpin assembly (CHA) for sensitive detection of H1N1 RNA. This system achieves dual signal amplification, enabling detection with a remarkably low limit of detection (LOD) of 95.2 aM within 40 min. Excellent specificity distinguishes H1N1 from other common influenza viruses. Crucially, the programmability of crRNA endows the method with broad versatility. Beyond influenza virus detection, it was successfully applied to SARS-CoV-2 RNA detection, achieving an LOD of 87.0 aM, demonstrating its potential for diverse pathogen diagnostics. These results collectively demonstrate the high performance and adaptability of this diagnostic platform, highlighting its significant promise for future applications in the rapid and sensitive detection of diverse viral pathogens.},
}

*Quantum Dots/chemistry
*RNA, Viral/analysis/genetics
*CRISPR-Cas Systems
*Biosensing Techniques/methods
*Influenza A Virus, H1N1 Subtype/genetics/isolation & purification
SARS-CoV-2/genetics/isolation & purification
Limit of Detection
Humans
Nucleic Acid Amplification Techniques
COVID-19/diagnosis

@article {pmid42153625,
year = {2026},
author = {Djouad, ME and Bendaha, MEA and Belaouni, HA and Bensalah, F},
title = {Comparative genomics of Lacticaseibacillus paracasei strain 69Bis reveals encoded probiotic traits and genomic specificities.},
journal = {FEMS microbiology letters},
volume = {},
number = {},
pages = {},
doi = {10.1093/femsle/fnag060},
pmid = {42153625},
issn = {1574-6968},
abstract = {This study provides a comprehensive characterization of Lacticaseibacillus paracasei strain 69Bis, isolated from traditional fermented goat milk collected in Biskra (southeast Algeria). Phenotypic investigations revealed robust probiotic traits across all isolates, including NaCl tolerance at 10%, acid resistance at pH 2.0 for 3 h, bile tolerance at 0.3%, and survival in simulated gastrointestinal digestion. Adhesion properties included high auto-aggregation and hydrophobicity. The strain exhibited potent antimicrobial activity, particularly against Pseudomonas aeruginosa. Whole-genome sequencing yielded a genome of 2.9 Mbp, with 46.38% GC and 2,969 protein-coding sequences, enriched in carbohydrate and amino acid metabolism. Functional annotation highlighted genomic features associated with probiotic functions, including adhesion factors (acm, fimA, lapA), stress response pathways, and exopolysaccharide synthesis (eps gene family). Three biosynthetic gene clusters were detected, encoding bacteriocins and terpene synthesis. The absence of known acquired antimicrobial resistance genes and the susceptibility to all tested antibiotics indicate a favourable safety profile. The presence of CRISPR-Cas systems was confirmed, which may contribute to genomic stability and protection against phages. Comparative genomics, through genome-based taxonomy, confirmed the assignment to Lacticaseibacillus paracasei. Pan-genome analysis of 122 genomes from the same species (including 69Bis) revealed an open pan-genome (6,878 genes, 25.76% core), along with several unique and rare determinants linked to probiotic traits in 69Bis. Overall, these results position strain 69Bis as a promising candidate for use in functional foods, particularly in the context of nutrition in arid regions.},
}

@article {pmid42153634,
year = {2026},
author = {Shang, W and Lyu, Z and Chen, G},
title = {Harnessing Nature's Algorithm: From Test Tubes to Autonomous In Vivo Evolution.},
journal = {Biotechnology journal},
volume = {21},
number = {5},
pages = {e70237},
doi = {10.1002/biot.70237},
pmid = {42153634},
issn = {1860-7314},
support = {2024JJ4053//Hunan Provincial Natural Science Foundation/ ; 2025PT5021//Scientific Research Program of FuRong Laboratory/ ; },
mesh = {*Directed Molecular Evolution/methods ; *Algorithms ; Machine Learning ; Mutagenesis ; CRISPR-Cas Systems ; },
abstract = {Directed evolution (DE) enables the engineering of biomolecules without prior structural knowledge. However, traditional step-wise DE is constrained by limited screening throughput. To more efficiently navigate epistatic fitness landscapes, the field is increasingly adopting autonomous, continuous in vivo evolution systems. This review critically examines the molecular architectures and engineering principles driving this transition. We evaluate strategies for continuous genetic diversification-ranging from orthogonal replication systems (e.g., OrthoRep, T7-ORACLE) to CRISPR-guided mutagenesis (e.g., EvolvR)-with a focus on the fundamental trade-off between mutational load and host viability. Furthermore, we analyze the biophysical constraints of screening and the kinetic demands of coupling real-time selection with ultra-fast mutagenesis, as exemplified by phage-assisted continuous evolution (PACE). Crucially, we explore the functional integration of machine learning (ML), highlighting how active learning models and zero-shot predictions via protein language models (PLMs) can resolve epistatic complexities and mitigate the latency of next-generation sequencing. Finally, we discuss the multidimensional hardware and algorithmic bottlenecks currently impeding the realization of fully closed-loop biofoundries, and assess the strategic implications of these technologies for accelerating the engineering of complex therapeutics.},
}

*Directed Molecular Evolution/methods
*Algorithms
Machine Learning
Mutagenesis
CRISPR-Cas Systems

@article {pmid42153784,
year = {2026},
author = {Estay-Ahumada, CE and Roux, M and Ciocca, D and El-Kholti, N and Birling, MC and Rossolillo, P and Felder-Schmittbuhl, MP and Hicks, D},
title = {Abca4 Knockdown in the Cone-Rich Rodent Psammomys Obesus Leads to Stargardt's Disease-Like Progressive Retinal Degeneration.},
journal = {Investigative ophthalmology & visual science},
volume = {67},
number = {5},
pages = {47},
doi = {10.1167/iovs.67.5.47},
pmid = {42153784},
issn = {1552-5783},
mesh = {Animals ; Gerbillinae ; *ATP-Binding Cassette Transporters/genetics ; Electroretinography ; Stargardt Disease/genetics ; Disease Models, Animal ; *Retinal Cone Photoreceptor Cells/metabolism/pathology ; Tomography, Optical Coherence ; *Macular Degeneration/genetics/congenital/metabolism ; *Retinal Degeneration/genetics/metabolism ; CRISPR-Cas Systems ; Gene Knockdown Techniques ; Genetic Vectors ; Dependovirus/genetics ; },
abstract = {PURPOSE: Mutations in the gene ABCA4 coding for photoreceptor-specific ATP-binding cassette subfamily A member 4, are responsible for Stargardts disease type 1 (STGD1), the most common form of inherited macular degeneration. We recently showed that injection of viral vectors expressing CRISPR/Cas9 tools directed against Abca4 into young Sand Rat (Psammomys obesus) eyes led to extensive structural and functional retinal degeneration resembling STGD1. Here we provide further evidence that this is highly likely due to specific knockdown of Abca4 and not off-target errors.

METHODS: We performed subretinal injections of Adeno-Associated Virus-CRISPR/Cas9-Abca4 constructs into postnatal (∼P15) Psammomys obesus. Eyes were examined by noninvasive exploration (ocular coherence tomography, fundus and electroretinography) at 15-60 days after injection. Additionally, subgroups were euthanized over the same time period, and ocular tissue was used for immunochemical analyses.

RESULTS: RNAscope analysis of injected eyes showed knockdown of Abca4, rhodopsin and cone transducin mRNA in transduced regions; neighbouring tissue that was not transduced showed robust expression of all three. Injection of control AAV, expressing CAS9 alone, induced only mild glial activation. Statistically significant decreases in visual responses to light flashes were only seen in eyes injected with the fully active CRISPR/Cas9-Abca4 probes.

CONCLUSIONS: Taken together, these data rule out off-target effects as responsible for the observed degeneration, and indicate that Psammomys obesus faithfully recapitulates many of the features seen in human STGD1, thus positioning it as an important research opportunity to further explore genotype-phenotype relationships and test putative therapeutic approaches.},
}

Animals
Gerbillinae
*ATP-Binding Cassette Transporters/genetics
Electroretinography
Stargardt Disease/genetics
Disease Models, Animal
*Retinal Cone Photoreceptor Cells/metabolism/pathology
Tomography, Optical Coherence
*Macular Degeneration/genetics/congenital/metabolism
*Retinal Degeneration/genetics/metabolism
CRISPR-Cas Systems
Gene Knockdown Techniques
Genetic Vectors
Dependovirus/genetics

@article {pmid42154426,
year = {2026},
author = {Mayuri, K and Saravanan, KM and Somala, CS and Selvaraj, C and Anand, T and Vickram, S},
title = {CRISPR-Cas and Infectious Diseases: A Decade of Translational Advances in Molecular Biotechnology.},
journal = {Biochemical genetics},
volume = {},
number = {},
pages = {},
pmid = {42154426},
issn = {1573-4927},
abstract = {CRISPR-Cas systems have emerged as a versatile tool for diagnosing, treating, and preventing infectious diseases. This review highlights translational advancements in CRISPR-Cas-based applications, concentrating on the past decades in diagnostics, therapeutic genome editing, and vaccine development. The article highlights key platforms like DETECTR and SHERLOCK, which enable rapid, sensitive pathogen detection, and explores CRISPR-Cas9 systems in therapeutic strategies for directly targeting viral genomes and combating antimicrobial resistance. It also examines the role of CRISPR-Cas9 in engineering live-attenuated and personalized neoantigen vaccines. Principal findings demonstrate a clear progression from experimental proof-of-concept to preclinical applications primarily in CRISPR-based diagnostics and the engineering of live-attenuated vaccine candidates, whereas translation in CRISPR-based therapeutics and personalized neoantigen vaccines for infectious diseases remains at earlier, more exploratory stages. CRISPR-based diagnostics have progressed further toward clinical evaluation than therapeutics due to delivery and safety constraints, while personalized neoantigen vaccines are included mainly as an emerging, comparative concept for infectious diseases rather than a mature application. This review uniquely integrates CRISPR-based diagnostics, therapeutics, and vaccine development within a single infectious disease framework, critically assesses their current maturity, and systematically highlights technical, regulatory, and ethical barriers alongside realistic future priorities. The review concludes that while CRISPR-Cas holds transformative potential for infectious disease management, significant challenges in delivery efficiency, off-target effects, and ethical regulation must be addressed to ensure safe and equitable clinical translation.},
}

@article {pmid41884948,
year = {2026},
author = {Ekstrand, F and Ruhrmann, S and Bacos, K and Bartel, S and Jellema, P and Rots, MG and Ling, C and Prinz, CN},
title = {Nanopore Electroporation: A New Delivery Method Within the Field of Epigenetic Editing.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {22},
number = {28},
pages = {e13858},
doi = {10.1002/smll.202513858},
pmid = {41884948},
issn = {1613-6829},
support = {682206//ERC CoG NanoPokers/ ; //Swedish Research Council (VR)/ ; //Novo Nordisk foundation/ ; //Region Skåne/ ; Dnr 2009-1039//Strategic Research Area Exodiab/ ; //The Crafoord Foundation/ ; //NanoLund/ ; //The Swedish Diabetes Foundation/ ; ITM-17//The Swedish foundation for Strategic Research/ ; IRC15-0067//The Swedish foundation for Strategic Research/ ; },
mesh = {*Electroporation/methods ; *Nanopores ; *Gene Editing/methods ; *Epigenesis, Genetic/genetics ; CRISPR-Cas Systems/genetics ; Animals ; Humans ; Insulin-Secreting Cells/metabolism ; Insulin/genetics/metabolism ; Epigenome Editing ; },
abstract = {Epigenetic modifications influence gene expression and contribute to type 2 diabetes (T2D), but establishing causality requires targeted modulation of specific genes. CRISPR-dCas9-based tools offer this potential, yet β-cells are notoriously difficult to transfect, and efficient, non-viral delivery methods are lacking. Here, we developed nanopore-mediated electroporation to deliver a CRISPR interference (CRISPRi) system to clonal INS1 β-cells, achieving targeted downregulation of insulin expression. Cells were seeded atop a nanopore substrate with CRISPRi plasmids in solution below. Mild electric pulses generated transient nanoscale pores in the membrane, enabling electrophoretic delivery of plasmids into the cytosol while preserving high cell viability. The CRISPRi system comprised the transcriptional repressor Krueppel-associated Box Domain (KRAB) fused to an inactive Cas9 (dCas9), guided to the transcription start site of the insulin-1 gene (Ins1) by a single guide RNA (sgRNA). After transfection, Ins1 expression was significantly reduced, demonstrating effective modulation of gene expression in this difficult-to-transfect cell type. This nanopore electroporation approach provides a robust, safe, and efficient platform for delivering CRISPR-dCas9-based epigenetic editors in pancreatic β-cells. By enabling precise gene regulation, it opens avenues for mechanistic studies of epigenetic contributions to T2D and potentially other challenging cell systems.},
}

*Electroporation/methods
*Nanopores
*Gene Editing/methods
*Epigenesis, Genetic/genetics
CRISPR-Cas Systems/genetics
Animals
Humans
Insulin-Secreting Cells/metabolism
Insulin/genetics/metabolism
Epigenome Editing

@article {pmid41950922,
year = {2026},
author = {Kang, T and Bleris, L},
title = {Cellular-state control using ribozyme-scaffolded miRNA-sensing and CRISPR-mediated actuation.},
journal = {Cell reports methods},
volume = {6},
number = {5},
pages = {101379},
doi = {10.1016/j.crmeth.2026.101379},
pmid = {41950922},
issn = {2667-2375},
mesh = {*MicroRNAs/genetics/metabolism ; Humans ; *RNA, Catalytic/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; Epithelial-Mesenchymal Transition/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cell Line, Tumor ; },
abstract = {Cellular transitions between states are fundamental to development, adaptation, and pathological processes, but monitoring and guiding these transitions using endogenous signals remain challenging. MicroRNAs (miRNAs) represent a powerful modality, as distinct cell states are characterized by unique miRNA expression signatures. Here, we introduce a state-specific miRNA-directed CRISPR system for detecting and responding to epithelial-to-mesenchymal transition (EMT), a critical process in development, wound healing, and cancer metastasis. This system leverages EMT-specific miRNAs to regulate activation of type II polymerase-driven ribozyme-single-guide RNA (sgRNA) constructs, which direct CRISPR-based effectors to modulate gene expression. Using this approach, we demonstrate selective elimination of cells that have undergone mesenchymal transition and dynamic filtering of cell populations. This system provides a versatile platform for precise activation of CRISPR-Cas9 effectors using endogenous, state-specific cues. Integrating miRNA signatures with CRISPR technology to monitor, modulate, and reprogram cell-state transitions paves the way for applications in regenerative medicine, cancer therapy, and beyond.},
}

*MicroRNAs/genetics/metabolism
Humans
*RNA, Catalytic/metabolism/genetics
*CRISPR-Cas Systems/genetics
Epithelial-Mesenchymal Transition/genetics
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Cell Line, Tumor

@article {pmid42000058,
year = {2026},
author = {Carvalho, C and Hérivaux, A and Mello, T and Bastide, F and Thomas, O and Guillemette, T and Saulnier, P and Souza Dos Santos, AL and Papon, N and Bouchara, JP},
title = {TRXR2, a thioredoxin reductase-encoding gene, contributes to protection against the oxidative stress and virulence in Scedosporium apiospermum.},
journal = {Microbial pathogenesis},
volume = {216},
number = {},
pages = {108498},
doi = {10.1016/j.micpath.2026.108498},
pmid = {42000058},
issn = {1096-1208},
mesh = {*Oxidative Stress ; *Scedosporium/pathogenicity/genetics/enzymology/drug effects ; Virulence/genetics ; Animals ; Hyphae ; *Thioredoxin-Disulfide Reductase/genetics/metabolism ; *Thioredoxin Reductase 2/genetics/metabolism ; Reactive Oxygen Species/metabolism ; Fungal Proteins/genetics/metabolism ; Spores, Fungal ; Cell Wall/metabolism/ultrastructure ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Antifungal Agents/pharmacology ; Larva/microbiology ; Gene Expression Regulation, Fungal ; Benzene Derivatives ; },
abstract = {Scedosporium apiospermum has received an increased attention over the past decades, especially because of its low susceptibility to current antifungals and its clinical relevance in cystic fibrosis. Nevertheless, little is still known about its pathogenic mechanisms. During the inflammatory reaction, macrophages and neutrophils release antimicrobial compounds, especially reactive oxygen species (ROS). To cope with ROS, pathogens have developed various strategies, including synthesis of some immunoprotective secondary metabolites, and enzymatic mechanisms relying on antioxidant enzymes. A recent transcriptomic study showed that the TRXR2 gene, encoding one of its two thioredoxin reductases (TrxRs), is overexpressed upon exposure to oxidative stress. To investigate the role of TrxR2, the encoding gene was disrupted using the CRISPR-Cas9 technology. An ultrastructural study revealed that TRXR2 gene knock-out induced the loss of the outer cell wall layer of hyphae, thereby increasing their surface hydrophobicity. The effects on the tolerance of the fungus to chemically-induced oxidative stresses were also investigated, revealing a higher susceptibility to cumene hydroperoxide. Additionally, an overexpression of BoyT gene (encoding the other TrxR) was seen as a compensatory mechanism, but bioinformatic analysis also suggested distinct roles for the two TrxRs in relation with distinct subcellular localizations. Finally, conidia exhibited a reduced capacity to adhere to epithelial cells and a diminished virulence in the Galleria mellonella model. In conclusion, these results suggest that TrxR2 plays a contributory role in the pathogenicity of S. apiospermum, but is not a major determinant of its virulence, which justifies future studies on the other components of the thioredoxin system.},
}

*Oxidative Stress
*Scedosporium/pathogenicity/genetics/enzymology/drug effects
Virulence/genetics
Animals
Hyphae
*Thioredoxin-Disulfide Reductase/genetics/metabolism
*Thioredoxin Reductase 2/genetics/metabolism
Reactive Oxygen Species/metabolism
Fungal Proteins/genetics/metabolism
Spores, Fungal
Cell Wall/metabolism/ultrastructure
CRISPR-Cas Systems
Gene Knockout Techniques
Antifungal Agents/pharmacology
Larva/microbiology
Gene Expression Regulation, Fungal
Benzene Derivatives

@article {pmid42019502,
year = {2026},
author = {Vereecke, N and Behrmann, M and Khare, A and Dekker, JP},
title = {Improved Cas9-targeted nanopore sequencing facilitates ultra-deep analysis of genomic variation.},
journal = {Cell reports methods},
volume = {6},
number = {5},
pages = {101410},
doi = {10.1016/j.crmeth.2026.101410},
pmid = {42019502},
issn = {2667-2375},
mesh = {*High-Throughput Nucleotide Sequencing/methods ; *Nanopore Sequencing/methods ; Staphylococcus aureus/genetics ; *CRISPR-Cas Systems/genetics ; Bacteroides fragilis/genetics ; *Nanopores ; *Genomics/methods ; *CRISPR-Associated Protein 9/metabolism ; Genome, Bacterial ; *Genetic Variation ; },
abstract = {We present nanopore adapter-enriched Cas9-targeted sequencing (nAECATS), a method permitting inexpensive, ultra-deep, selective long-read sequencing of targeted regions in native, unamplified DNA. This method modifies previous Cas9-targeted sequencing approaches through the inclusion of a bead-based capture step that exploits the poly(T)8 stretch within the R10.4.1 ligation adapter for additional purification. Testing on a 10 kb Bacteroides fragilis genomic region achieved 90% on-target yield with 51,000× coverage from a single Flongle flow cell (353-fold increase versus whole-genome sequencing). Applied to a variable-length (>20 kb) Staphylococcus aureus genomic target containing dynamic gene amplifications conferring antibiotic resistance, nAECATS achieved 46,000× coverage and 74% on-target yield, revealing 2-4 tandem amplifications at single-cell resolution. While efficiency decreased with longer fragments (up to 41 kb), substantial enrichment improvements were demonstrated. We anticipate that nAECATS ultra-deep sequencing will find broad application for a wide range of biological questions in pro- and eukaryotic (epi)genomics and microbiology.},
}

*High-Throughput Nucleotide Sequencing/methods
*Nanopore Sequencing/methods
Staphylococcus aureus/genetics
*CRISPR-Cas Systems/genetics
Bacteroides fragilis/genetics
*Nanopores
*Genomics/methods
*CRISPR-Associated Protein 9/metabolism
Genome, Bacterial
*Genetic Variation

@article {pmid42053710,
year = {2026},
author = {Panchuk, IO and Grigorieva, OV and Kurshakova, EV and Nagieva, SE and Shchagina, OA and Levchenko, OA and Pozhitnova, VO and Voronina, ES and Tabakov, VY and Smirnikhina, SA and Lavrov, AV},
title = {Generation of an Induced Pluripotent Stem Cell Line from a Duchenne Muscular Dystrophy Patient Carrying the DMD p.Ser429Ter (c.1286C>G) Nonsense Mutation.},
journal = {Bulletin of experimental biology and medicine},
volume = {180},
number = {4},
pages = {548-554},
pmid = {42053710},
issn = {1573-8221},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology/pathology ; *Muscular Dystrophy, Duchenne/genetics/pathology/metabolism ; Kruppel-Like Factor 4 ; *Codon, Nonsense/genetics ; Cell Differentiation ; *Dystrophin/genetics/metabolism ; Cell Line ; Male ; Fibroblasts/metabolism/cytology ; Octamer Transcription Factor-3/genetics/metabolism ; CRISPR-Cas Systems ; Cellular Reprogramming ; Sendai virus/genetics ; },
abstract = {An induced pluripotent stem cell (iPSC) line was successfully generated from dermal fibroblasts of a patient with Duchenne muscular dystrophy (DMD) harboring the pathogenic nonsense variant c.1286C>G (p.Ser429Ter) in the DMD gene using non-integrating Sendai virus reprogramming. The iPSC clone exhibited typical pluripotent stem cell morphology, expressed key pluripotency markers (OCT4, SSEA4, NANOG, and TRA-1-60), and retained trilineage differentiation potential. The cell line had a normal karyotype, and elimination of reprogramming vectors (OCT3/4, SOX2, KLF4, and c-MYC) was confirmed. This isogenic cell model provides a valuable platform for investigating DMD pathogenesis associated with this specific mutation and for developing targeted therapeutic approaches, including CRISPR/Cas9-mediated gene correction.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology/pathology
*Muscular Dystrophy, Duchenne/genetics/pathology/metabolism
Kruppel-Like Factor 4
*Codon, Nonsense/genetics
Cell Differentiation
*Dystrophin/genetics/metabolism
Cell Line
Male
Fibroblasts/metabolism/cytology
Octamer Transcription Factor-3/genetics/metabolism
CRISPR-Cas Systems
Cellular Reprogramming
Sendai virus/genetics

@article {pmid42080223,
year = {2026},
author = {Zhao, S and Huang, S and Li, N and Wang, N and Yu, Z and Liang, X and Yang, Z and Guo, H},
title = {Piperazine-Derived Diamine Lipid Nanoparticles Targeting to the Liver for Delivering Clustered Regularly Interspaced Short Palindromic Repeat Editing of PCSK9 to Durably Maintain Plasmatic Low-Density Lipoprotein Cholesterol in Low Levels.},
journal = {ACS applied bio materials},
volume = {9},
number = {10},
pages = {4465-4476},
doi = {10.1021/acsabm.6c00032},
pmid = {42080223},
issn = {2576-6422},
mesh = {*Proprotein Convertase 9/genetics/metabolism ; *Nanoparticles/chemistry ; Humans ; Animals ; Mice ; *Gene Editing ; *Liver/metabolism/drug effects ; *Cholesterol, LDL/blood ; *Piperazine/chemistry ; *Lipids/chemistry ; *Biocompatible Materials/chemistry/pharmacology/chemical synthesis ; Particle Size ; Materials Testing ; Mice, Inbred C57BL ; Hep G2 Cells ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Cardiovascular diseases remain a leading cause of morbidity and mortality worldwide, and durable suppression of low-density lipoprotein cholesterol (LDL-C) via genome editing represents a promising therapeutic strategy. Here, we report the rational design of a piperazine-derived bis-tertiary amine ionizable lipid (M10) and its optimized lipid nanoparticle formulation (M10-F4) for efficient and liver-targeted CRISPR/Cas9 delivery. Compared with benchmark lipids such as SM-102, M10 enables a reduced molar ratio of ionizable lipid while maintaining high nucleic acid encapsulation efficiency (>80%) and forming stable, spherical nanoparticles. The piperazine-based multi-cationic core confers an optimized apparent pKa of 6.56, facilitating endosomal escape through enhanced protonation under acidic conditions. Confocal microscopy in HepG2 and Huh-7 cells reveals efficient cellular uptake and enhanced cytosolic release of RNA cargo with minimal lysosomal entrapment. In vivo, M10-F4 exhibits strong liver tropism following systemic administration. A single intravenous dose mediates robust PCSK9 gene editing in C57BL/6 mice, resulting in sustained reductions of circulating PCSK9 and LDL-C levels under both normal and high-fat diet conditions for up to 48 days, accompanied by decreased hepatic PCSK9 expression. Importantly, acute safety evaluation in BALB/c mice showed no obvious signs of short-term systemic toxicity, including stable body weight, minimal induction of inflammatory cytokines (IL-6, TNF-α, and CXCL-10), no significant elevation of liver enzymes, and normal gross organ morphology. Collectively, this work establishes M10-F4 as a molecularly engineered, liver-targeted LNP platform in which ionizable lipid architecture and formulation composition enable effective in vivo genome editing with favorable tolerability, highlighting the importance of rational materials design at the materials-bio interface for cardiometabolic gene-editing applications.},
}

*Proprotein Convertase 9/genetics/metabolism
*Nanoparticles/chemistry
Humans
Animals
Mice
*Gene Editing
*Liver/metabolism/drug effects
*Cholesterol, LDL/blood
*Piperazine/chemistry
*Lipids/chemistry
*Biocompatible Materials/chemistry/pharmacology/chemical synthesis
Particle Size
Materials Testing
Mice, Inbred C57BL
Hep G2 Cells
CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42119627,
year = {2026},
author = {Guo, K and Yu, S and Yang, N and Wang, X and Liu, Q and Fu, Y and Liu, J},
title = {Serial passage is associated with virulence attenuation in Neospora caninum and transcriptomic remodeling of invasion- and chronic-stage-associated pathways.},
journal = {Microbial pathogenesis},
volume = {216},
number = {},
pages = {108549},
doi = {10.1016/j.micpath.2026.108549},
pmid = {42119627},
issn = {1096-1208},
mesh = {*Neospora/pathogenicity/genetics/growth & development ; Virulence/genetics ; Animals ; Gene Expression Profiling ; *Transcriptome ; Serial Passage ; Cattle ; Protozoan Proteins/genetics ; Host-Parasite Interactions/genetics ; Coccidiosis/parasitology/veterinary ; CRISPR-Cas Systems ; },
abstract = {N. caninum is an obligate intracellular apicomplexan parasite and a major cause of abortion in cattle worldwide. However, the molecular basis underlying virulence attenuation during long-term in vitro passage remains poorly understood. Here, we performed comparative transcriptomic profiling between the virulent Nc1-09 strain and its attenuated derivative Nc1 to investigate transcriptional changes associated with virulence reduction. A total of 487 differentially expressed genes (DEGs) were identified. Functional enrichment analyses revealed prominent changes in pathways related to protein modification, host-parasite interaction, motility, and metabolism. Notably, multiple members of the surface antigen glycoprotein (SRS) family displayed marked transcriptional shifts, consistent with altered host cell interaction and immune-related functions. CRISPR/Cas9-mediated disruption of three representative DEGs (NCLIV_016320, NCLIV_024950, and NCLIV_022830) did not reproduce the attenuated phenotype, suggesting that disruption of these individual genes is insufficient to account for the full attenuated phenotype under the conditions tested. Gene set enrichment analysis further demonstrated systematic downregulation of protein modification-associated pathways in the attenuated strain. Overall, the data indicate that virulence attenuation in Neospora caninum （N. caninum） is associated with coordinated transcriptional remodeling across multiple functional pathways rather than discrete gene loss.},
}

*Neospora/pathogenicity/genetics/growth & development
Virulence/genetics
Animals
Gene Expression Profiling
*Transcriptome
Serial Passage
Cattle
Protozoan Proteins/genetics
Host-Parasite Interactions/genetics
Coccidiosis/parasitology/veterinary
CRISPR-Cas Systems

@article {pmid42144912,
year = {2026},
author = {Guo, X and Tian, T and Zhou, X},
title = {Split crRNAs Enhance Cas12a Diagnostic Performance.},
journal = {Chembiochem : a European journal of chemical biology},
volume = {27},
number = {10},
pages = {e70383},
doi = {10.1002/cbic.70383},
pmid = {42144912},
issn = {1439-7633},
mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; },
abstract = {CRISPR RNA (crRNA) is the key guide molecule in the CRISPR-Cas12a system, directing the Cas protein to recognize target sequences. It consists of a repeat-derived stem loop that binds Cas12a and helps stabilize the ribonucleoprotein complex, and a spacer region that base-pairs with the target and determines recognition specificity. Recently, multiple studies have shown that crRNAs can be split and reassembled in vitro in diverse ways. These split-and-reconfigured strategies have enabled detection schemes that are more flexible than full-length crRNAs, cover a broader range of targets, and achieve higher signal-to-background ratios. Here, we focus on split crRNA strategies for CRISPR-Cas12a and systematically summarize existing split crRNA-based detection platforms. We outline their design principles, reaction mechanisms, and performance features, and we synthesize how these approaches improve key metrics-including target scope, sensitivity, specificity, and controllability. Finally, we discuss the major advantages and current limitations of split crRNA strategies and highlight directions for further design optimization and translational applications. Schematic overview of split crRNA strategies for enhancing CRISPR-Dx performance. These advances are mainly reflected in four aspects: broadening the target range, thereby enabling Cas12a to be applied to the detection of short RNAs, structured RNAs, and certain non-nucleic acid targets; improving sensitivity by enhancing detection signals through reassembly-dependent activation, cascade amplification, or auxiliary activation strategies; increasing specificity by strengthening the discrimination of single-nucleotide differences through stepwise recognition and conditional assembly; and enhancing controllability by achieving on-demand activation of Cas12a activity via light, enzymes, small molecules, or proximity effects.},
}

*CRISPR-Cas Systems
*CRISPR-Associated Proteins/metabolism/genetics
*Endodeoxyribonucleases/metabolism/genetics
*Bacterial Proteins/metabolism/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism

@article {pmid42145154,
year = {2026},
author = {Dong, R and Zhang, Y and Yuan, G and Yue, X and Ding, Y and Zeng, X and Xiao, H},
title = {A Novel Strategy to Produce CAR-γδ T Cells via Site-Directed Gene Integration by a Combination of CRISPR/Cas9 and AAV.},
journal = {Cancer medicine},
volume = {15},
number = {5},
pages = {e71918},
doi = {10.1002/cam4.71918},
pmid = {42145154},
issn = {2045-7634},
support = {82170141//National Natural Science Foundation of China/ ; 82470209//National Natural Science Foundation of China/ ; },
mesh = {*Dependovirus/genetics ; Animals ; *CRISPR-Cas Systems ; Humans ; Mice ; *Immunotherapy, Adoptive/methods ; *Receptors, Chimeric Antigen/genetics/immunology/metabolism ; *Receptors, Antigen, T-Cell, gamma-delta/genetics/immunology/metabolism ; *T-Lymphocytes/immunology/metabolism ; Genetic Vectors/genetics ; Cell Line, Tumor ; },
abstract = {Chimeric antigen receptor (CAR)-αβ T cells are commonly employed in tumor therapy but hindered by some limitations. γδ T cells are promising substrates for CAR therapy for their major histocompatibility complex (MHC)-unrestricted recognition manner and innate immune function. Here, we established a novel method for generating CAR-γδ T cells. We utilized the clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) method to interrupt the TCR delta chain constant region (TRDC) sequence, followed by the site-directed insertion of the CAR sequence into the TRDC locus via homologous complementation mediated by adeno-associated virus (AAV) gene delivery. We optimized electroporation parameters for Cas9/ribonucleoproteins (RNP) delivery and infection conditions for CAR-gene carrying AAV in γδ T cells. These optimizations facilitated efficient TCR knockout and site-directed CAR insertion, ultimately yielding functional CAR-γδ T cells. In vitro experiments demonstrated that these newly prepared CAR-γδ T cells could stimulate cytokine production, kill tumor cells as well as exhibit robust proliferative potential and memory-like phenotype. These state-of-the-art CAR-γδ T cells could reduce tumor burden and extend the survival period of tumor-bearing mice.},
}

*Dependovirus/genetics
Animals
*CRISPR-Cas Systems
Humans
Mice
*Immunotherapy, Adoptive/methods
*Receptors, Chimeric Antigen/genetics/immunology/metabolism
*Receptors, Antigen, T-Cell, gamma-delta/genetics/immunology/metabolism
*T-Lymphocytes/immunology/metabolism
Genetic Vectors/genetics
Cell Line, Tumor

@article {pmid42145193,
year = {2026},
author = {Yin, XY and Wang, SZ},
title = {High-throughput identification methods of genomic functional variation in post-GWAS era and their application in agricultural animals.},
journal = {Yi chuan = Hereditas},
volume = {48},
number = {5},
pages = {451-470},
doi = {10.16288/j.yczz.25-319},
pmid = {42145193},
issn = {0253-9772},
mesh = {Animals ; *Genome-Wide Association Study/methods ; *Genetic Variation ; Gene Editing ; *Genomics/methods ; CRISPR-Cas Systems ; },
abstract = {Genome-wide association study (GWAS) has identified a large number of genetic variations that are significantly associated with human diseases and animal and plant economic traits. However, the majority of these variants are located in non-coding regions of the genome, which makes it challenging to accurately pinpoint functional variants of biological significance from a vast number of candidate loci. In the post-GWAS era, high-throughput analytical approaches, such as high-throughput reporter gene analysis, CRISPR/Cas9-based gene editing technologies, and epigenetic analyses, have become powerful tools for systematically uncovering functional variants in the genome. These methods not only enable efficient identification of functional variants but also help elucidate the mechanisms through which they regulate gene expression, thereby clarifying the molecular basis underlying trait formation or disease pathogenesis. In this review, we systematically summarize current high-throughput strategies for identifying functional genomic variants, highlight their applications and recent advances in major agricultural animal species, and outline future research directions, with the aim of providing a reference for subsequent studies in related fields.},
}

Animals
*Genome-Wide Association Study/methods
*Genetic Variation
Gene Editing
*Genomics/methods
CRISPR-Cas Systems

@article {pmid42148306,
year = {2026},
author = {Jezierski, A and Huang, J and Desbiens, L and Benabdallah, B and McComb, S and Beausejour, C},
title = {Induced pluripotent stem cells as platforms for engineering NK cell immunotherapies.},
journal = {Frontiers in cell and developmental biology},
volume = {14},
number = {},
pages = {1810206},
pmid = {42148306},
issn = {2296-634X},
abstract = {Human induced pluripotent stem cells (iPSCs) are transforming adoptive cell therapy by combining unlimited self-renewal, broad differentiation potential, and high amenability to genome engineering. These attributes make iPSCs a versatile source for the development of standardized immune effector cells at industrial scale, enabling a shift from patient- or donor-restricted cell products toward true off-the-shelf immunotherapies that can be improved through iterative genome engineering. iPSC-derived natural killer (iNK) cells are the most clinically advanced and exemplify the platform's advantages over conventional autologous or donor-sourced approaches. Unlike autologous therapies, which require labor-intensive and expensive personalized clinical-grade manufacturing, and are constrained by variable quality and genetic intractability of donor products, iPSC technology supports the creation of renewable, clonally defined master cell banks as uniform starting material for NK-cell therapy products. Advances in CRISPR/Cas-based editing now permit multiplex introduction of functional traits, enhanced cytokine signaling, antibody-dependent cytotoxicity, checkpoint resistance, optimized trafficking, safety switches, and increasing signal complexity, directly at the pluripotent or progenitor stages; ultimately allowing for fully-programmable iNK cells with customizable potency and persistence. Early clinical studies of iNK products validate the feasibility, safety, and therapeutic potential of this approach, but also underscore the need for continued refinement of differentiation protocols, manufacturing pipelines, and regulatory standards to ensure efficacy, genomic stability, phenotypic maturity, and long-term safety. This review outlines current breakthroughs and future directions of iNK cell therapies, emphasizing how programmable iPSC chassis platforms are enabling modular and off-the-shelf targeted immunotherapies.},
}

@article {pmid42148710,
year = {2026},
author = {Du, T and He, J and Wang, Y and Xie, X and Chen, J and Xu, Z and Zhou, C and Sun, W},
title = {An Evolutionarily Conserved Function of Grainyhead in Orchestrating Insect Wing Development.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {40},
number = {10},
pages = {e71927},
doi = {10.1096/fj.202600640R},
pmid = {42148710},
issn = {1530-6860},
support = {CSTB2025NSCQ-GPX0734//CSTC | Natural Science Foundation of Chongqing Municipality/ ; 32070499//MOST | National Natural Science Foundation of China (NSFC)/ ; 2025CDJ-IAISYB-022//MOE | Fundamental Research Funds for the Central Universities (Fundamental Research Fund for the Central Universities)/ ; },
mesh = {Animals ; *Wings, Animal/growth & development/metabolism ; *Insect Proteins/genetics/metabolism ; *Drosophila melanogaster/genetics/growth & development/metabolism ; *Bombyx/genetics/growth & development/metabolism ; Gene Expression Regulation, Developmental ; *Transcription Factors/genetics/metabolism ; Female ; CRISPR-Cas Systems ; Male ; },
abstract = {The development of insect appendages is governed by deeply conserved genetic programs, even as developmental strategies diverge widely across taxa. In this study, we identify the conserved transcription factor Grainyhead (Grh) as a crucial regulator of insect wing development. In the silkworm Bombyx mori, CRISPR/Cas9-mediated knockout of BmGrh did not compromise larval viability but resulted in severe wing defects in adults, including crumpled and non-expandable wings. Similarly, tissue-specific knockdown of DmGrh in Drosophila melanogaster wing imaginal discs led to pupal lethality, a sex-biased eclosion rate, and adults exhibiting crumpled wings with disrupted wing hair polarity. Comparative transcriptomics revealed that loss of DmGrh function predominantly downregulates genes associated with cuticle structure and extracellular matrix organization in both species. In Drosophila, chromatin immunoprecipitation further confirmed that DmGrh directly binds to regulatory regions of these downregulated cuticle-related genes. Through functional screening, we identified the cuticle protein gene cpr65Ea as a key downstream effector, whose knockdown recapitulated the wing morphogenesis and eclosion defects observed in DmGrh RNA interference individuals. Moreover, extending our investigation to an agricultural pest, RNAi-mediated silencing of SfGrh in the white-backed planthopper (Sogatella furcifera) impaired wing expansion and completely abolished flight ability. Our findings establish Grh as an evolutionarily conserved regulator of insect wing development and highlight its potential as a novel genetic target for pest management by disrupting flight capacity and dispersal.},
}

Animals
*Wings, Animal/growth & development/metabolism
*Insect Proteins/genetics/metabolism
*Drosophila melanogaster/genetics/growth & development/metabolism
*Bombyx/genetics/growth & development/metabolism
Gene Expression Regulation, Developmental
*Transcription Factors/genetics/metabolism
Female
CRISPR-Cas Systems
Male

@article {pmid42149441,
year = {2026},
author = {Klijnhout, JA and Senders, EAW and van den Bogaard, EH and Smits, JPH},
title = {Genetic Engineering of Human Keratinocytes Using CRISPR/Cas9 Ribonucleoprotein Complexes or Modified Cas9-Encoding mRNAs.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3031},
number = {},
pages = {87-101},
pmid = {42149441},
issn = {1940-6029},
mesh = {Humans ; *Keratinocytes/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *RNA, Messenger/genetics ; *Ribonucleoproteins/genetics/metabolism ; *Genetic Engineering/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics ; Cells, Cultured ; },
abstract = {CRISPR/Cas9 is a straightforward genome-editing technique that is implemented across disciplines and research areas. However, in keratinocytes, CRISPR/Cas9 can be particularly difficult due to variable genome-editing efficiency, reduced cell viability, and difficulties during (sub)cloning of gene-edited keratinocyte populations. Here, we provide a step-by-step detailed protocol for the genetic manipulation of human (primary) keratinocytes, including widely accepted procedures for the analysis of CRISPR/Cas9 efficiency, (sub)cloning procedures to select heterozygous or homozygous keratinocytes, and off-target genome-editing analysis.},
}

Humans
*Keratinocytes/metabolism/cytology
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*RNA, Messenger/genetics
*Ribonucleoproteins/genetics/metabolism
*Genetic Engineering/methods
RNA, Guide, CRISPR-Cas Systems/genetics
CRISPR-Associated Protein 9/genetics
Cells, Cultured

@article {pmid42150000,
year = {2026},
author = {Hu, Q and Lin, J and Cui, H and Zhao, X and Wu, M and Wei, S and Deng, M and Wang, B and Liu, T and Gao, X and Huang, Q and Liang, Y and Liu, S and Yang, H and Lin, HB and Li, G},
title = {Cas13-Mediated RNA Base Editing for the Treatment of Hereditary Hypertrophic Cardiomyopathy.},
journal = {Circulation},
volume = {153},
number = {20},
pages = {1598-1602},
doi = {10.1161/CIRCULATIONAHA.125.076905},
pmid = {42150000},
issn = {1524-4539},
}

@article {pmid40335752,
year = {2026},
author = {Kannan, S and Altae-Tran, H and Zhu, S and Xu, P and Strebinger, D and Oshiro, R and Faure, G and Moeller, L and Pham, J and Mears, KS and Ni, HM and Macrae, RK and Zhang, F},
title = {Evolution-guided protein design of IscB for persistent epigenome editing in vivo.},
journal = {Nature biotechnology},
volume = {44},
number = {5},
pages = {759-770},
pmid = {40335752},
issn = {1546-1696},
mesh = {*Gene Editing/methods ; Humans ; *Protein Engineering/methods ; *Epigenome/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genome, Human ; CRISPR-Cas Systems ; Epigenome Editing ; },
abstract = {Naturally existing enzymes have been adapted for a variety of molecular technologies, with enhancements or modifications to the enzymes introduced to improve the desired function; however, it is difficult to engineer variants with enhanced activity while maintaining specificity. Here we engineer the compact Obligate Mobile Element Guided Activity (OMEGA) RNA-guided endonuclease IscB and its guiding RNA (ωRNA) by combining ortholog screening, structure-guided protein domain design and RNA engineering, and deep learning-based structure prediction to generate an improved variant, NovaIscB. We show that the compact NovaIscB achieves up to 40% indel activity (~100-fold improvement over wild-type OgeuIscB) on the human genome with improved specificity relative to existing IscBs. We further show that NovaIscB can be fused with a methyltransferase to create a programmable transcriptional repressor, OMEGAoff, that is compact enough to be packaged in a single adeno-associated virus vector for persistent in vivo gene repression. This study highlights the power of combining natural diversity with protein engineering to design enhanced enzymes for molecular biology applications.},
}

*Gene Editing/methods
Humans
*Protein Engineering/methods
*Epigenome/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Genome, Human
CRISPR-Cas Systems
Epigenome Editing

@article {pmid40555761,
year = {2026},
author = {Niu, X and Tang, W and Liu, Y and Mo, B and Yu, Y and Liu, Y and Wei, W},
title = {Prime editor-based high-throughput screening reveals functional synonymous mutations in human cells.},
journal = {Nature biotechnology},
volume = {44},
number = {5},
pages = {832-844},
pmid = {40555761},
issn = {1546-1696},
support = {NSFC 31930016//National Natural Science Foundation of China (National Science Foundation of China)/ ; tsqn202312362//Taishan Scholar Foundation of Shandong Province/ ; },
mesh = {Humans ; *High-Throughput Screening Assays/methods ; *Silent Mutation/genetics ; Machine Learning ; Mutation/genetics ; RNA Splicing/genetics ; Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Synonymous mutations are generally considered neutral, while their roles in the human genome remain largely unexplored. Here we use the PEmax system to create a library of 297,900 engineered prime-editing guide RNAs and perform extensive screening to identify synonymous mutations affecting cell fitness. Unlike recent findings in yeast, group-level analyses show that synonymous mutations diverge from nonsynonymous mutations in fitness effects yet exhibit similar phenotypic distributions relative to negative controls. Following rigorous quality control, only a small subset demonstrated measurable effects. For these functional mutations, we develop a specialized machine learning tool and uncover their impact on various biological processes such as messenger RNA splicing and transcription, supported by multifaceted experimental evidence. We find that synonymous mutations can alter RNA folding and affect translation, as demonstrated by PLK1_S2. By integrating screening data with our model, we predict clinically deleterious synonymous mutations. This research deepens our understanding of synonymous mutations, providing insights for clinical disease studies.},
}

Humans
*High-Throughput Screening Assays/methods
*Silent Mutation/genetics
Machine Learning
Mutation/genetics
RNA Splicing/genetics
Gene Editing/methods
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid41692169,
year = {2026},
author = {Hu, Y and Wang, Y and Wang, S and Jiao, Y and Tao, R and Yao, S},
title = {Reporter-based screening identifies small-molecule CBL0137 as an enhancer of CRISPR cytosine base editor and prime editor via p53 activation and NF-κB inhibition.},
journal = {New biotechnology},
volume = {93},
number = {},
pages = {54-74},
doi = {10.1016/j.nbt.2026.02.002},
pmid = {41692169},
issn = {1876-4347},
mesh = {Humans ; *Gene Editing/methods ; *Tumor Suppressor Protein p53/metabolism ; *NF-kappa B/metabolism/antagonists & inhibitors ; *CRISPR-Cas Systems/genetics ; *Cytosine/metabolism ; *Genes, Reporter ; HEK293 Cells ; },
abstract = {Recently developed CRISPR base editors (BEs) and prime editors (PEs) enable precise genome editing without inducing double-strand breaks, making them highly promising tools for therapeutic applications. However, their efficiency remains a major barrier to clinical translation, particularly at difficult-to-target sites. To address this limitation, we used a high-throughput GFP reporter system responsive to cytosine base editor (CBE) activity to screen small molecules involved in DNA damage response, cell cycle, and apoptosis pathways. This screen identified CBL0137 as a candidate that significantly enhanced editing efficiency at both the reporter and endogenous target sites, with up to an 80 % improvement. Mechanistic studies revealed that CBL0137 acts through activation of the p53 pathway and inhibition of NF-κB. Interestingly, the enhancement was largely specific to CBEs, while in PEs, CBL0137 selectively improved multi-site mutations and fragment insertions without affecting single-point edits or deletions. Collectively, these results identify CBL0137 as a selective enhancer of specific genome editing activities, providing a strategy to increase editing efficiency and advancing the translational potential of next-generation genome editing tools for therapeutic applications.},
}

Humans
*Gene Editing/methods
*Tumor Suppressor Protein p53/metabolism
*NF-kappa B/metabolism/antagonists & inhibitors
*CRISPR-Cas Systems/genetics
*Cytosine/metabolism
*Genes, Reporter
HEK293 Cells

@article {pmid41881989,
year = {2026},
author = {Ren, X and Zheng, L and Liu, Y and Maliskova, L and Tam, TW and Sun, Y and Liu, H and Cui, X and Lee, J and Takagi, MA and Li, B and Ren, B and Wang, W and Shen, Y},
title = {CRISPR tiling deletion screens reveal functional enhancers and allelic compensation effects (ACE) on SIN3A transcription.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41881989},
issn = {2041-1723},
support = {UM1HG009402//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; P30DK063720//U.S. Department of Health & Human Services | NIH | National Institute of Diabetes and Digestive and Kidney Diseases (National Institute of Diabetes & Digestive & Kidney Diseases)/ ; S101S10OD021822-01//U.S. Department of Health & Human Services | NIH | NIH Office of the Director (OD)/ ; 1S10OD028511-01//U.S. Department of Health & Human Services | NIH | NIH Office of the Director (OD)/ ; },
mesh = {Humans ; *Enhancer Elements, Genetic/genetics ; *Repressor Proteins/genetics/metabolism ; Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Transcription, Genetic ; *Sin3 Histone Deacetylase and Corepressor Complex/genetics/metabolism ; Cell Differentiation/genetics ; Promoter Regions, Genetic ; Alleles ; *Dosage Compensation, Genetic ; Neurons/metabolism ; Gene Expression Regulation ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Precise transcriptional regulation is critical for cellular function and development, yet the mechanism of this process remains poorly understood for many genes. To gain a deeper understanding of the regulation of neuropsychiatric disease risk genes, we identify a total of 39 functional enhancers for four dosage-sensitive genes, APP, FMR1, MECP2, and SIN3A, using CRISPR tiling deletion screening in human induced pluripotent stem cell (iPSC)-induced excitatory neurons. More importantly, we discover that allelic enhancer deletions at SIN3A could be compensated by increased transcriptional activities from the other intact allele. Such allelic compensation effects (ACE) on transcription are stably maintained during differentiation and, once established, cannot be reversed by ectopic SIN3A expression. Further, ACE at SIN3A occurs through dosage sensing by the promoter. Together, our findings unravel a regulatory compensation mechanism that ensures stable and precise transcriptional output for SIN3A, and potentially other dosage-sensitive genes.},
}

Humans
*Enhancer Elements, Genetic/genetics
*Repressor Proteins/genetics/metabolism
Induced Pluripotent Stem Cells/metabolism/cytology
*CRISPR-Cas Systems/genetics
*Transcription, Genetic
*Sin3 Histone Deacetylase and Corepressor Complex/genetics/metabolism
Cell Differentiation/genetics
Promoter Regions, Genetic
Alleles
*Dosage Compensation, Genetic
Neurons/metabolism
Gene Expression Regulation
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid41882347,
year = {2026},
author = {Akdoğan, E and Lundgren, SM and Kamber, RA and Bassik, MC and Collins, SR},
title = {Parallel CRISPR screens reveal pathways controlling the cell surface levels of the attractant receptor FPR1.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {},
pmid = {41882347},
issn = {2399-3642},
support = {P30CA093373//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; DP2HD094656//U.S. Department of Health & Human Services | NIH | Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)/ ; R01 GM148769/GM/NIGMS NIH HHS/United States ; S10OD018223//U.S. Department of Health & Human Services | NIH | NIH Office of the Director (OD)/ ; DP2 HD094656/HD/NICHD NIH HHS/United States ; R01GM148769//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; CA259218//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; },
mesh = {Humans ; *Receptors, Formyl Peptide/metabolism/genetics ; Endocytosis ; *CRISPR-Cas Systems ; Signal Transduction ; ADP-Ribosylation Factor 6 ; HEK293 Cells ; *Cell Membrane/metabolism ; },
abstract = {Chemoattractants generate strong chemotactic and cytotoxic responses in immune cells by activating cognate receptors. Cell surface receptor levels control sensitivity, which is critical for achieving effective responses without excessive inflammation. The surface levels of the attractant receptor FPR1 are controlled through a balance of delivery and removal, which responds to receptor activation and other stimuli. While this regulation is critical for appropriate responses, the underlying mechanisms remain unclear, including the roles of classic endocytosis regulators. We address these questions using both focused and genome-scale approaches. We find that the receptor kinase GRK6 acts in parallel with GRK2 and GRK3 to trigger internalization, and that internalization uses a β-arrestin-independent pathway, as well as pathways involving β-arrestin1 and 2. Moreover, we use an integrated analysis of two parallel CRISPR/Cas9 screens to classify regulators of FPR1 biogenesis, surface expression, recycling, and endocytosis. We identify the formin mDia1 and the small GTPase ARF6 as specific regulators of FPR1 internalization, which we confirm using chemical inhibitors in primary human neutrophils. Finally, we find that ARF6 contributes to the β-arrestin-independent pathway. Together, our results provide a systems overview of the control of FPR1 surface levels and offer insights into alternative endocytosis mechanisms used by chemoattractant receptors.},
}

Humans
*Receptors, Formyl Peptide/metabolism/genetics
Endocytosis
*CRISPR-Cas Systems
Signal Transduction
ADP-Ribosylation Factor 6
HEK293 Cells
*Cell Membrane/metabolism

@article {pmid41882878,
year = {2026},
author = {Yang, Y and Han, Z and Li, G and Li, Z and Shao, C and Li, W and Wang, J and Luan, J and Cui, Y and Han, J},
title = {Biallelic inactivation of EXT1 in patient-derived iPSCs confirms the "Two-hit" hypothesis in hereditary multiple osteochondromas.},
journal = {Bioscience trends},
volume = {20},
number = {2},
pages = {245-253},
doi = {10.5582/bst.2026.01046},
pmid = {41882878},
issn = {1881-7823},
mesh = {Humans ; *N-Acetylglucosaminyltransferases/genetics/metabolism ; Exostosin 1 ; *Exostoses, Multiple Hereditary/genetics/pathology ; *Induced Pluripotent Stem Cells/metabolism ; Cell Differentiation/genetics ; Mutation ; CRISPR-Cas Systems ; Chondrocytes/metabolism/cytology ; Chondrogenesis/genetics ; Alleles ; Organoids ; },
abstract = {Hereditary Multiple Osteochondromas (HMO) is a rare autosomal dominant skeletal disorder caused by heterozygous loss-of-function mutations in EXT1 or EXT2, which encode glycosyltransferases essential for heparan sulfate (HS) biosynthesis. Whether haploinsufficiency alone suffices or biallelic inactivation is required for osteochondroma formation remains a central unresolved question. In this study, we employed CRISPR/Cas9 combined with PiggyBac transposon technology to introduce a second pathogenic mutation (c.1883+1G>T) into patient-derived induced pluripotent stem cells (iPSCs) carrying a heterozygous EXT1 c.1126C>T mutation. This approach enabled the generation of isogenic iPSC lines: wild-type (WT), single-mutant (SM), and double-mutant (DM). These iPSCs were differentiated through induced mesenchymal stem cells (iMSCs) into chondrocytes. Biallelic EXT1 mutation in DM cells led to significant upregulation of SOX9, COL2A1, and ACAN, elevated glycosaminoglycan (GAG) levels, and markedly reduced HS, whereas SM cells remained indistinguishable from WT. Three-dimensional (3D) chondrogenic organoid cultures revealed that DM organoids were enlarged and structurally disorganized, partially recapitulating key histopathological features of osteochondromas. Transcriptomic analysis identified the Wnt signaling pathway as the most significantly enriched pathway among differentially expressed genes following EXT1 loss. Collectively, these findings provide direct human cellular evidence that complete EXT1 inactivation-not haploinsufficiency-drives aberrant chondrogenesis, likely through impaired sequestration of morphogen ligands, thereby supporting the Two-hit pathogenic model.},
}

Humans
*N-Acetylglucosaminyltransferases/genetics/metabolism
Exostosin 1
*Exostoses, Multiple Hereditary/genetics/pathology
*Induced Pluripotent Stem Cells/metabolism
Cell Differentiation/genetics
Mutation
CRISPR-Cas Systems
Chondrocytes/metabolism/cytology
Chondrogenesis/genetics
Alleles
Organoids

@article {pmid42001795,
year = {2026},
author = {Radszuweit, P and Fitzel, R and Bruestl, S and Hentrich, T and Korkmaz, F and Mankel, B and González-Menéndez, I and Rudat, S and Marschalek, R and Erkner, E and Keppeler, H and Schairer, R and Luib, L and Mezger, M and Quintanilla-Martinez, L and Schulze-Hentrich, J and Lengerke, C and Schneidawind, D and Schneidawind, C},
title = {Characterizing the impact of MLL fusion variants and fusion partners on leukemia plasticity using a human CRISPR-engineered MLL-rearranged leukemia model.},
journal = {Neoplasia (New York, N.Y.)},
volume = {77},
number = {},
pages = {101308},
pmid = {42001795},
issn = {1476-5586},
mesh = {Humans ; *Myeloid-Lymphoid Leukemia Protein/genetics ; *Oncogene Proteins, Fusion/genetics ; Animals ; Mice ; *Histone-Lysine N-Methyltransferase/genetics ; *CRISPR-Cas Systems ; *Gene Rearrangement ; *Leukemia/genetics/pathology/metabolism ; Disease Models, Animal ; Gene Expression Profiling ; Cell Line, Tumor ; },
abstract = {Acute leukemias involving KMT2A (MLL) rearrangements are aggressive hematologic malignancies associated with a poor prognosis, especially in infants. The majority of MLL breakpoints are located within the breakpoint cluster region spanning exons 8-14, with AFF1 (AF4) and MLLT3 (AF9) being the most frequent fusion partners. To study the contribution of different fusion partners and breakpoint locations to leukemogenesis, we created a human CRISPR/Cas9-based model. We introduced MLL::AF4 or MLL::AF9 fusions with MLL breakpoints in intron 9 or 11, respectively, into human cord blood-derived CD34[+] cells from the same donor. Compared to healthy control cells, all MLL-rearranged cells showed increased proliferation and stemness, as well as an altered immunophenotype characterized by the upregulation of leukemic markers. Transcriptomic profiling revealed breakpoint- and partner-specific gene expression patterns that influence the characteristics of the disease. Notably, even after prolonged in vitro culture MLL(intron 9)::AF9 cells displayed robust colony formation in semisolid media and engrafted robustly in NOD scid gamma mice. The cells still exhibited high lineage plasticity, switching from a myeloid to a B-lymphoid identity in vivo. In conclusion, this model enables the mechanistic dissection of MLL fusion variants in vitro and in vivo, providing a foundation for developing targeted therapies for MLL-rearranged leukemias.},
}

Humans
*Myeloid-Lymphoid Leukemia Protein/genetics
*Oncogene Proteins, Fusion/genetics
Animals
Mice
*Histone-Lysine N-Methyltransferase/genetics
*CRISPR-Cas Systems
*Gene Rearrangement
*Leukemia/genetics/pathology/metabolism
Disease Models, Animal
Gene Expression Profiling
Cell Line, Tumor

@article {pmid42102651,
year = {2026},
author = {Walus, M and Kida, E and Golabek, AA},
title = {Lysosomal storage, mitochondrial pathology, and autophagy in knockout of tripeptidyl peptidase 1 in human neuroblastoma cells in vitro.},
journal = {Molecular genetics and metabolism},
volume = {148},
number = {2},
pages = {110130},
doi = {10.1016/j.ymgme.2026.110130},
pmid = {42102651},
issn = {1096-7206},
mesh = {Humans ; Tripeptidyl-Peptidase 1 ; *Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/genetics/deficiency/metabolism ; *Autophagy/genetics ; *Mitochondria/pathology/metabolism/genetics ; *Lysosomes/metabolism/pathology/genetics ; *Neuronal Ceroid-Lipofuscinoses/genetics/pathology/metabolism ; *Aminopeptidases/genetics/metabolism/deficiency ; *Serine Proteases/genetics/metabolism/deficiency ; Cell Line, Tumor ; Gene Knockout Techniques ; *Neuroblastoma/genetics/pathology ; CRISPR-Cas Systems ; },
abstract = {Deficiency of tripeptidyl-peptidase 1 (TPP1; EC 3.4.14.9), a lysosomal enzyme encoded by the CLN2 gene, is associated with the lysosomal storage disorder - classic late infantile neuronal ceroid lipofuscinosis (CLN2 disease). The classic form of CLN2 disease leads to the accumulation of autofluorescent lysosomal storage and a massive loss of neurons with gliosis in the brain. The major component of the storage is subunit c of mitochondrial ATP synthase, a highly hydrophobic, 75-aminoacid polypeptide. We developed an in vitro model of CLN2 disease by knocking out CLN2 in the human neuroblastoma cells SH-SY5Y by using CRISPR/cas9 technology. We focused on defining the pattern of deposition of subunit c, factors contributing to subunit c accumulation, and subcellular morphometry to identify differences between the model cells knockout (KO) and controls. Implementation of acetone for cell fixation allowed us to: i. identify higher levels of subunit c in the mitochondria of KO cells than controls; ii. characterize in detail subunit c inclusions, also present in controls; iii. identify other mitochondrial proteins colocalizing with subunit c in inclusions; and iv. detect mitochondrial pathology in degenerating cells often accompanied by deposition of subunit c. Differentiation of cells with retinoic acid and brain-derived neurotrophic factor led to a substantial increase in the levels of subunit c and to significant differences in the levels of autophagy-related proteins between KO and control cells. Inhibition of induced autophagy by bafilomycin A1 (Baf.A1) decreased subunit c levels in controls but not in KO cells, whereas the levels of subunit c were unaffected by Baf.A1 treatment upon basal autophagy. Finally, subcellular morphometry showed differences in the number and size of vesicular structures immunostained for autophagy-related proteins between KO cells and controls upon both induced and basal autophagy, further supporting the association of TPP1 deficiency with autophagy.},
}

Humans
Tripeptidyl-Peptidase 1
*Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/genetics/deficiency/metabolism
*Autophagy/genetics
*Mitochondria/pathology/metabolism/genetics
*Lysosomes/metabolism/pathology/genetics
*Neuronal Ceroid-Lipofuscinoses/genetics/pathology/metabolism
*Aminopeptidases/genetics/metabolism/deficiency
*Serine Proteases/genetics/metabolism/deficiency
Cell Line, Tumor
Gene Knockout Techniques
*Neuroblastoma/genetics/pathology
CRISPR-Cas Systems

@article {pmid42138076,
year = {2026},
author = {Hale, AT and Kundishora, AJ and Kalailingam, P and Barak, T and Duy, PQ and Ramundo, CM and Fan, B and Li, Q and Brastianos, PK and Shankar, GM and Alper, SL and Kleinstiver, BP and Musolino, PL and Kahle, KT},
title = {Towards precision medicine for brain arteriovenous malformations.},
journal = {The Journal of clinical investigation},
volume = {136},
number = {10},
pages = {},
pmid = {42138076},
issn = {1558-8238},
mesh = {Humans ; *Precision Medicine ; *Intracranial Arteriovenous Malformations/genetics/therapy/metabolism/pathology ; Animals ; Mutation ; Proto-Oncogene Proteins B-raf/genetics/metabolism ; },
abstract = {Recent advances in cerebrovascular genomics, single-cell biology, pharmacology, and gene editing technology are transforming our understanding of brain arteriovenous malformations (bAVMs) - a leading cause of pediatric hemorrhagic stroke. Once considered static anatomical defects, bAVMs are now recognized as dynamic, genetically driven lesions resulting from somatic mutations in KRAS, BRAF, and pathways involved in arteriovenous specification, angiogenesis, and vascular remodeling. By integrating human genetics, animal models, and endovascular innovations, researchers have uncovered convergent mechanisms that link endothelial Ras/MAPK hyperactivation to abnormal vessel growth and higher rupture risk. These insights provide a foundation for precision medicine approaches that combine molecular diagnostics - such as liquid or endoluminal biopsies - with mutation-specific pharmacotherapies and emerging CRISPR-based gene editing strategies. We suggest that genotype-guided interventions, tailored by spatial and developmental cerebrovascular context, could ultimately reclassify bAVMs from surgically incurable malformations to treatable molecular conditions.},
}

Humans
*Precision Medicine
*Intracranial Arteriovenous Malformations/genetics/therapy/metabolism/pathology
Animals
Mutation
Proto-Oncogene Proteins B-raf/genetics/metabolism

@article {pmid42143446,
year = {2026},
author = {Martin, L and Rostami, S and Schuster, I and Qin, PZ and Rajan, R},
title = {Helicity of the bridge helix of Cas12a regulates on-target DNA cleavage efficiency and off-target cleavage propensity.},
journal = {Biochemical and biophysical research communications},
volume = {823},
number = {},
pages = {153760},
doi = {10.1016/j.bbrc.2026.153760},
pmid = {42143446},
issn = {1090-2104},
abstract = {CRISPR-Cas systems comprise a CRISPR RNA (crRNA)-guided CRISPR-associated (Cas) nuclease for providing immune protection. The complementary base pairing between crRNA and the invader genome leads to the formation of an "R-loop", which triggers the nuclease activity of the Cas protein, effectively neutralizing the invasion. This molecular mechanism has been repurposed for genome applications using Cas9 and Cas12a. Cas12a has several favorable features for applications including its smaller size, crRNA processing ability, and creation of staggered double-stranded DNA (dsDNA) cleavage. Gene editing with these Cas proteins, however, has some setbacks due to off-target and non-specific DNA cleavages. To increase the specificity in DNA cleavage, we introduced proline/alanine substitutions at different positions along a conserved arginine/lysine-rich "bridge helix" (BH) of Cas12a that plays an integral role in mediating conformational changes needed for DNA cleavage. Cleavage kinetics analyses reveal that enhanced helical integrity of the BH of Francisella novicida Cas12a provided by alanine substitutions increases DNA cleavage efficiency, while reducing the ability of the variants to discriminate DNA mismatches. Proline substitutions demonstrate an opposite effect by reducing the efficiency of cleaving on-target DNA, but almost completely abolishing linearization of a target with a mismatch in the middle of the R-loop. These results parallel those reported for Cas9 and show that balancing the helicity of BH through rational amino acid substitutions can finetune Cas12a's off-target profiles. This may provide a strategy for enhancing specificity of Cas12a in genome manipulation.},
}

@article {pmid42143634,
year = {2026},
author = {Mahendrarajan, V and Sankaranarayanan, GN and Muthukaliannan, GK and Easwaran, N},
title = {Triphala Modulates the Membrane Vesicle Transcriptome of Enterococcus durans VIT3 to Influence Antibiotic Response and Probiotic Functions.},
journal = {Molecular biotechnology},
volume = {},
number = {},
pages = {},
pmid = {42143634},
issn = {1559-0305},
support = {SG20210254//VIT University/ ; },
abstract = {Membrane vesicles (MVs) are critical mediators of bacterial communication, physiology, and host interactions. This study explores how triphala, a polyherbal formulation, modulates the RNA cargo of MVs in Enterococcus durans VIT3 isolate under varied stress conditions. The isolated MVs from all treatment conditions (triphala, antibiotics, or sequential combinations) were spherical, intact, with negative zeta potential and sizes ranging from 56.4 to 462.3 nm. Cytotoxicity assays indicated no toxicity toward CaCo2 cells under the tested conditions. Transcriptomic profiling suggested that triphala pretreatment enhanced relative expression of genes linked to oxidative stress defense (ohrB, sodA), envelope integrity (dlt operon, pbp, mprF), and CRISPR-Cas-associated genes (cas1). In contrast, antibiotic first regimens suppressed stress and defense-associated genes, underscoring treatment order effects. Notably, triphala antibiotic sequence condition showed coordinated expression patterns within ATP synthase, arginine deiminase, and CRISPR loci, suggesting operon-level transcriptional organization of metabolic and stress-response pathways. STRING network analysis further supported clustering of genes showing relatively higher expression into functionally related molecules. Collectively, these findings suggest a potential role for triphala in shaping MV RNA cargo, offering a conceptual framework for synbiotic strategies aimed at supporting microbial stress resilience. All transcriptomic observations are based on pooled MV samples, which represent exploratory expression trends rather than statistically inferred differential expression or functional outcomes.},
}

@article {pmid42144412,
year = {2026},
author = {Kasapoglu, MZ and Acar, I and Gumustop, I and Erol, I and Kurt, IC and Ortakci, F},
title = {Pangenomics of Limosilactobacillus fermentum reveals genomic diversity and bacteriocin activity against Staphylococcus aureus and Escherichia coli.},
journal = {Scientific reports},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41598-026-51219-1},
pmid = {42144412},
issn = {2045-2322},
support = {MGA-2024-45355//Bilimsel Araştırma Projeleri Birimi, İstanbul Teknik Üniversitesi/ ; },
abstract = {Limosilactobacillus fermentum is a versatile lactic acid bacterium with significant probiotic and biotechnological potential, yet the genomic determinants underlying its ecological adaptation and therapeutic applications remain underexplored. This study performed a comparative genomic analysis of 52 L. fermentum strains, with a focus on the novel strain ATT-06 isolated from traditional Turkish shalgam. We uncovered substantial genetic diversity, evidenced by an open pan-genome (18,647 genes) and a small core genome (718 genes). CRISPR-Cas systems were prevalent (46 strains), with Type-IE and Type-IIA being most common and mutually exclusive from Type-IC. Strain ATT-06, which uniquely produced gamma-aminobutyric acid at 17.74 µg/mL, harbored a Type-IIA CRISPR system and a single prophage. In silico molecular docking and dynamics simulations revealed that the bacteriocin Lactococcin, encoded by strain ATA-LTC-Lf170503, exhibited strong binding affinities (ΔG: -8.1 to -13.0 kcal/mol) against Rho proteins of Staphylococcus aureus and Escherichia coli, outperforming Acidocin A. These findings highlight the genomic plasticity and adaptive mechanisms of L. fermentum, and position strain ATT-06 as a promising probiotic candidate with potential neuroactive and antimicrobial applications.},
}

@article {pmid41104514,
year = {2026},
author = {Hu, Y and Zeng, Z and Ming, X and Yin, S and Guan, Y and Gao, L and Li, D},
title = {Construction of pathogenic Sec16a mutation mouse model using CRISPR/Cas9.},
journal = {Animal models and experimental medicine},
volume = {9},
number = {3},
pages = {537-545},
doi = {10.1002/ame2.70082},
pmid = {41104514},
issn = {2576-2095},
support = {HZKY20220076//The Chunhui Program of the ministry of Education/ ; 21JC1402200//the Shanghai Municipal Commission of Science and Technology/ ; 22YF1437700//the Shanghai Municipal Commission of Science and Technology/ ; 23HC1400700//the Shanghai Municipal Commission of Science and Technology/ ; 32200732//National Natural Science Foundation of China/ ; 2022YFC3400200//National Key Research and Development Program of China/ ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Disease Models, Animal ; *Mutation ; Mice ; *Vesicular Transport Proteins/genetics ; Humans ; Mice, Inbred C57BL ; Male ; },
abstract = {BACKGROUND: SEC16A is a pivotal protein that facilitates the transport of proteins from the endoplasmic reticulum to the Golgi apparatus. Utilizing the protein structure function database, a potentially pathogenic mutation site (NM_014866.1: c.4606C>G(p.L1536V)) was pinpointed within the conserved central core region of the human SEC16A protein, a component integral to the COPII complex assembly.

METHODS: Leveraging information on human gene mutations and aligning human and mouse protein amino acid sequences, the Sec16aL1551V/L1551V mouse model was successfully developed using CRISPR/Cas9 technology.

RESULTS: Two behavioral experiments, namely novel object recognition and cued fear conditioning, revealed that Sec16aL1551V/L1551V mice demonstrated a phenotype of neurological impairment, evidenced by diminished abilities in learning and memory. Furthermore, while undergoing tail suspension, the Sec16aL1551V/L1551V mice displayed a distinctive limb clasping behavior, a characteristic typically associated with mouse models of chronic neurodegenerative diseases.

CONCLUSION: The Sec16aL1551V/L1551V mouse model developed in this study providing a powerful tool for better understanding of the pathogenic mechanisms of Sec16a gene mutations in brain dysfunction diseases.},
}

Animals
*CRISPR-Cas Systems/genetics
*Disease Models, Animal
*Mutation
Mice
*Vesicular Transport Proteins/genetics
Humans
Mice, Inbred C57BL
Male

@article {pmid41700779,
year = {2026},
author = {Alberio, V and Savy, V and Felipe, MY and Fernandez-Martín, R and Salamone, DF},
title = {Simultaneous CRISPR-on activation of TFAP2C and SMARCA4 promotes development of trophoblast-fate cells in bovine embryos†.},
journal = {Biology of reproduction},
volume = {114},
number = {5},
pages = {1598-1609},
doi = {10.1093/biolre/ioag041},
pmid = {41700779},
issn = {1529-7268},
support = {PICT 2018-04632//ANPCyT from the Ministry of Science and Technology, Argentina/ ; },
mesh = {Animals ; *Trophoblasts/physiology/metabolism/cytology ; Cattle/embryology ; *Transcription Factor AP-2/genetics/metabolism ; Gene Expression Regulation, Developmental ; Female ; *Embryonic Development/genetics ; *Transcription Factors/genetics/metabolism ; *DNA Helicases/genetics/metabolism ; Cell Differentiation/genetics ; *Nuclear Proteins/genetics/metabolism ; Embryo, Mammalian ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Pregnancy ; },
abstract = {Assisted reproductive techniques are widely used to produce domestic animal embryos for commercial or research purposes. In cattle, abnormal trophoblast cell differentiation during embryo development causes pregnancy and placentation failures. The CRISPR-on system has been successfully used in bovine embryos to individually activate early trophoblast lineage genes TFAP2C and SMARCA4. This study evaluates the effect of CRISPR-on activation of early and advanced trophoblast-related genes in bovine zygotes to promote trophectoderm differentiation. In one experiment, RNA was microinjected to simultaneously activate TFAP2C and SMARCA4; in another, circular DNA was microinjected to induce CDX2 or GATA3 expression separately. Controls included groups without small guide RNAs (sgRNAs, SHAM) and non-injected embryos (IVF). RNA microinjection transiently and simultaneously increased TFAP2C, SMARCA4, and downstream gene expression, producing a prolonged effect beyond individual gene activation. This led to a significant increase in trophectoderm cells at the blastocyst stage. Individual activation of CDX2 and GATA3 was effective, also inducing upstream genes without altering trophectoderm cell percentages. Neither RNA nor DNA microinjection affected blastocyst production compared to IVF. This work demonstrates the successful increase of trophoblast cells in bovine embryos using CRISPR-on, offering a useful strategy for IVF or SCNT embryos. The CRISPR-dCas9VP160 system may also aid understanding of trophoblast lineage signaling during development.},
}

Animals
*Trophoblasts/physiology/metabolism/cytology
Cattle/embryology
*Transcription Factor AP-2/genetics/metabolism
Gene Expression Regulation, Developmental
Female
*Embryonic Development/genetics
*Transcription Factors/genetics/metabolism
*DNA Helicases/genetics/metabolism
Cell Differentiation/genetics
*Nuclear Proteins/genetics/metabolism
Embryo, Mammalian
*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
Pregnancy

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

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

@article {pmid41874563,
year = {2026},
author = {Fang, J and Simon, JM and Wang, T and Gao, Y and Bi, X and Hu, L and Liao, C and Zhang, C and Adachi, Y and Zhou, J and Liu, H and Liang, Q and Nathan, JA and Mani, R and Brugarolas, J and Zhang, Q},
title = {Genome-wide CRISPR screen identifies a cytokine-enhancer circuit driving HIF-2α activation in renal cancer.},
journal = {The Journal of clinical investigation},
volume = {136},
number = {10},
pages = {},
doi = {10.1172/JCI201639},
pmid = {41874563},
issn = {1558-8238},
support = {P50 CA196516/CA/NCI NIH HHS/United States ; },
mesh = {*Basic Helix-Loop-Helix Proteins/genetics/metabolism/biosynthesis ; Humans ; *Kidney Neoplasms/genetics/metabolism/pathology ; *Carcinoma, Renal Cell/genetics/metabolism/pathology ; STAT3 Transcription Factor/metabolism/genetics ; Mice ; Animals ; Suppressor of Cytokine Signaling 3 Protein/genetics/metabolism ; Cell Line, Tumor ; *Gene Expression Regulation, Neoplastic ; Janus Kinase 1/metabolism/genetics ; *CRISPR-Cas Systems ; *Enhancer Elements, Genetic ; *Cytokines/metabolism/genetics ; Signal Transduction ; *Neoplasm Proteins/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Von Hippel-Lindau Tumor Suppressor Protein/genetics ; },
abstract = {Resistance to HIF-2α inhibitors such as belzutifan underscores the need to better understand how HIF-2α is transcriptionally regulated in clear cell renal cell carcinoma (ccRCC). Here, we uncover a cytokine-driven enhancer mechanism that sustains HIF-2α expression through the JAK1/STAT3 signaling pathway. Using a genome-wide CRISPR screen in von Hippel-Lindau-deficient (VHL-deficient) ccRCC cells, we identified SOCS3 as a key negative regulator of HIF-2α. Mechanistically, loss of SOCS3 activates JAK1/STAT3 signaling, leading to the recruitment of STAT3 to distal enhancers upstream of endothelial PAS domain-containing protein (EPAS1) that physically loop to its promoter to drive HIF-2α transcription. This cytokine-enhancer circuit was recapitulated in samples from patients with ccRCC and functionally validated using CRISPR interference (CRISPRi), which disrupted enhancer-promoter looping and reduced tumor growth in HIF-2α-dependent models. SOCS3 overexpression or pharmacologic inhibition of JAK1/STAT3 markedly suppressed HIF-2α expression and tumor progression both in vitro and in vivo. Unlike prior studies focusing on VHL/HIF occupancy-driven enhancer activation, this work defines a trans-acting cytokine-JAK1/STAT3 pathway that transcriptionally controls EPAS1. Together, these findings reveal a targetable enhancer mechanism that sustains HIF-2α expression and suggest that combined inhibition of JAK1/STAT3 and HIF-2α may overcome therapeutic resistance in kidney cancer.},
}

*Basic Helix-Loop-Helix Proteins/genetics/metabolism/biosynthesis
Humans
*Kidney Neoplasms/genetics/metabolism/pathology
*Carcinoma, Renal Cell/genetics/metabolism/pathology
STAT3 Transcription Factor/metabolism/genetics
Mice
Animals
Suppressor of Cytokine Signaling 3 Protein/genetics/metabolism
Cell Line, Tumor
*Gene Expression Regulation, Neoplastic
Janus Kinase 1/metabolism/genetics
*CRISPR-Cas Systems
*Enhancer Elements, Genetic
*Cytokines/metabolism/genetics
Signal Transduction
*Neoplasm Proteins/genetics/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats
Von Hippel-Lindau Tumor Suppressor Protein/genetics

@article {pmid41915438,
year = {2026},
author = {Ma, Z and Jia, W and Zhou, X and Liu, J and Li, Q and Chang, R and Shiqi, G and Yuan, N and Chen, Z and Lan, P},
title = {In vivo CRISPR screens identify CBX4 as an epigenetic regulator for cancer immunotherapy.},
journal = {The Journal of clinical investigation},
volume = {136},
number = {10},
pages = {},
doi = {10.1172/JCI200564},
pmid = {41915438},
issn = {1558-8238},
mesh = {Animals ; Mice ; Humans ; *Epigenesis, Genetic/immunology ; *Immunotherapy ; *Tumor Microenvironment/immunology/genetics ; *CRISPR-Cas Systems ; *Polycomb-Group Proteins/genetics/immunology ; *Ligases/genetics/immunology ; Cell Line, Tumor ; *Neoplasm Proteins/genetics/immunology ; *Neoplasms/genetics/immunology/therapy ; *Liver Neoplasms/immunology/genetics/therapy ; },
abstract = {Epigenetic dysregulation is associated with immune evasion and immune checkpoint blockade (ICB) resistance. Here, using in vivo CRISPR/Cas9 screens targeting epigenetics-related factors in mouse tumor models treated with ICB, we identified chromobox 4 (CBX4) as a key negative regulator of the immune tumor microenvironment (TME). Single-cell RNA-seq and spatial transcriptomics analyses of patients receiving neoadjuvant anti-programmed cell death protein 1 (anti-PD-1) therapy revealed high CBX4 expression in both tumor cells and immunosuppressive tumor-associated macrophage subpopulations, with preferential accumulation in nonresponders. Deficiency of CBX4 in macrophages or tumor cells induced robust antitumor immunity and increased infiltration and the cytotoxic activity of CD8+ T cells and NK cells, thereby heightening the sensitivity of ICB treatment. Mechanistically, CBX4 targeted H3K9me3- and H3K27me3-marked endogenous retroelements such as RLTR4-Mm-int. Loss of CBX4 derepressed retrotransposons, activating cytosolic RNA-sensing pathways and triggering the type I IFN response, ultimately leading to a robustly inflamed TME. Moreover, we uncovered a negative correlation between CBX4 expression, immune responses, and retrotransposon levels, and were able to determine the prognosis of patients with hepatocellular carcinoma (HCC) undergoing ICB therapy. Our study establishes CBX4 as an epigenetic immune checkpoint through the epigenetic silencing of retrotransposons, remodeling the immune TME and thus providing a promising therapeutic target to enhance tumor immunogenicity and overcome immunotherapy resistance.},
}

Animals
Mice
Humans
*Epigenesis, Genetic/immunology
*Immunotherapy
*Tumor Microenvironment/immunology/genetics
*CRISPR-Cas Systems
*Polycomb-Group Proteins/genetics/immunology
*Ligases/genetics/immunology
Cell Line, Tumor
*Neoplasm Proteins/genetics/immunology
*Neoplasms/genetics/immunology/therapy
*Liver Neoplasms/immunology/genetics/therapy

@article {pmid41926741,
year = {2026},
author = {Driscoll, CS and Kim, J and Knott, JG},
title = {CRISPR-mediated editing of cis-regulatory elements in early mouse embryos: a tool for studying pluripotency gene regulation.},
journal = {Reproduction (Cambridge, England)},
volume = {171},
number = {5},
pages = {},
doi = {10.1093/reprod/xaag042},
pmid = {41926741},
issn = {1741-7899},
support = {HD095371//National Institutes of Child Health and Development/ ; //Michigan State University AgBioResearch/ ; HD087166//T32 doctoral fellowship from the NICHD/ ; },
mesh = {Animals ; Mice ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Gene Expression Regulation, Developmental ; Female ; *Transcription Factor AP-2/genetics/metabolism ; *Blastocyst/metabolism ; *Embryonic Development/genetics ; Octamer Transcription Factor-3/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Pluripotent Stem Cells/metabolism ; },
abstract = {In brief Cis-regulatory elements and transcription factor binding motifs play crucial roles in regulating the spatial and temporal patterns of gene expression during development. This study tested the utility of CRISPR/Cas9 as a tool to interrogate the function of transcription factor AP2 gamma motifs in pluripotency gene expression during mouse preimplantation embryo development. Abstract Clustered regularly interspaced short palindromic repeat/CRISPR-associated nuclease 9 (CRISPR/Cas9) is a highly efficient tool that enables the generation of gene knockouts, knock-ins, and single base substitutions in a variety of organisms. Recently, we used CRISPR to examine the activity of cis-regulatory elements (CREs) in mouse preimplantation embryos. However, there is limited information on the feasibility of using CRISPR in preimplantation embryos to interrogate the function of select transcription factor (TF) binding motifs located within critical CREs in pluripotency genes. In the current study we employed CRISPR to disrupt TF AP2 gamma (TFAP2C) binding motifs located within key CREs involved in the regulation of Pou5f1 and Sox2 expression in early embryos. Microinjection of ribonucleoprotein complexes containing Cas9 and single guide RNAs (sgRNAs) targeting TFAP2C motifs located within a distal enhancer and proximal promoter substantially impaired Pou5f1 and Sox2 expression, respectively. Quantification of the editing efficiencies at each targeted CRE revealed that the targeting sgRNA sequences and the number of sgRNAs injected influenced the overall editing rates. Lastly, we investigated whether TFAP2C-induced activation of Sox2 expression in 2-cell embryos required TFAP2C motifs located within the Sox2 proximal promoter. CRISPR-mediated editing of these motifs diminished the activation of Sox2 expression. In summary, these findings indicate that CRISPR/Cas9 is a feasible approach for editing TF motifs in preimplantation embryos and provide evidence that TFAP2C directly contributes to Pou5f1 and Sox2 expression in preimplantation embryos.},
}

Animals
Mice
*Gene Editing/methods
*CRISPR-Cas Systems
*Gene Expression Regulation, Developmental
Female
*Transcription Factor AP-2/genetics/metabolism
*Blastocyst/metabolism
*Embryonic Development/genetics
Octamer Transcription Factor-3/genetics/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats
*Pluripotent Stem Cells/metabolism

@article {pmid41957993,
year = {2026},
author = {Duhalde, MA and Martino, RA and Smania, A and Alvarez, HM and Hernández, MA},
title = {CRISPR/Cas9 and Cytidine Base-Editing Systems for Efficient Genome Engineering in Oleaginous Rhodococcus.},
journal = {ACS synthetic biology},
volume = {15},
number = {5},
pages = {1926-1942},
doi = {10.1021/acssynbio.6c00019},
pmid = {41957993},
issn = {2161-5063},
mesh = {*Rhodococcus/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Cytidine/genetics/metabolism ; Plasmids/genetics ; Genome, Bacterial/genetics ; DNA End-Joining Repair/genetics ; },
abstract = {Oleaginous Rhodococcus strains can degrade diverse compounds and synthesize and accumulate large amounts of intracellular lipids, making them attractive platforms for biotechnological applications. However, efficient genome editing in Rhodococcus remains challenging, and new molecular tools are needed to advance the understanding of its metabolism, stress responses, and cellular physiology. Here, we describe the nonhomologous end joining (NHEJ) system in rhodococci and implement an efficient genome-editing system based on a CRISPR/Cas9 nuclease approach that utilizes this repair mechanism, eliminating the need for donor DNA templates. In addition, we report the first implementation of a cytidine base-editing (CBE) system, enabling precise single-nucleotide substitutions (C•G → T•A) in oleaginous Rhodococcus strains. Both strategies rely on a dual-plasmid CRISPR platform, resulting in two plasmid sets: pTipCas9/pCA71sgRNA and pTipBE/pCA71sgRNA. These systems enabled high rates of INDEL formation and C•G → T•A base conversions, with efficiencies of 70-80% and 75-85% at native genomic targets, respectively. Finally, for the CBE system, we implemented a Csy4-mediated sgRNA-processing module to support multiplex genome editing, enabling the simultaneous modification of multiple loci. Together, these tools outperform recombination-based approaches and will facilitate the study of complex metabolic pathways and the development of genetic strategies for biotechnological applications in Rhodococcus, while also being transferable to other actinobacteria.},
}

*Rhodococcus/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Cytidine/genetics/metabolism
Plasmids/genetics
Genome, Bacterial/genetics
DNA End-Joining Repair/genetics

@article {pmid41974167,
year = {2026},
author = {Peng, Z and Liu, Z and Wei, S and Zhu, Y and Jia, X and Xiao, W and Dong, Z and Hu, H and Lu, C and Pan, M and Chen, P},
title = {Genome-Wide CRISPR/Cas9 Screening Reveals BmM-ALP Orchestrates the Antioxidant Response and Metabolic Adaptations for Heat Resistance in Bombyx mori.},
journal = {ACS synthetic biology},
volume = {15},
number = {5},
pages = {1968-1979},
doi = {10.1021/acssynbio.6c00053},
pmid = {41974167},
issn = {2161-5063},
mesh = {Animals ; *Bombyx/genetics/metabolism/physiology ; *CRISPR-Cas Systems/genetics ; Reactive Oxygen Species/metabolism ; *Antioxidants/metabolism ; *Thermotolerance/genetics ; Adaptation, Physiological/genetics ; *Insect Proteins/genetics/metabolism ; Heat-Shock Response/genetics ; Animals, Genetically Modified ; },
abstract = {Climate change-induced heat stress represents an increasing threat to biodiversity, engendering the need to understand evolutionary adaptations to thermal extremes. Using Bombyx mori as an economically important insect and research model, we employed genome-wide CRISPR/Cas9 screening integrated with high-throughput sequencing to systematically identify adaptations for heat resistance. The analysis identified the Bombyx mori membrane-bound alkaline phosphatase-like gene (BmM-ALP) as a vital thermoregulator. BmM-ALP triggered a pleiotropic protective cascade by significantly decreasing reactive oxygen species (ROS), suppressing apoptosis, and reprogramming mitochondrial metabolism via the phosphorylation of Vitamin B1 (VB1), suggesting a potential role in sustaining oxidative phosphorylation and enhancing energy metabolism under stress. Transgenic validation experiments confirmed the consistency of this strategy, and the BmM-ALP-OE strain displayed significantly enhanced thermal tolerance and prolonged survival under high-temperature stress. Beyond establishing BmM-ALP as a key gene of heat resistance in silkworms, this study uncovered a regulatory axis linking BmM-ALP to Vitamin B1 metabolism, illustrating a novel connection between energy homeostasis and thermal adaptation. The findings provide new insights that can be applied to breeding resistant strains in agriculture and biodiversity conservation in the context of global warming.},
}

Animals
*Bombyx/genetics/metabolism/physiology
*CRISPR-Cas Systems/genetics
Reactive Oxygen Species/metabolism
*Antioxidants/metabolism
*Thermotolerance/genetics
Adaptation, Physiological/genetics
*Insect Proteins/genetics/metabolism
Heat-Shock Response/genetics
Animals, Genetically Modified

@article {pmid41979903,
year = {2026},
author = {Stuckless, EE and Gai, LS and Slattery, SS and Dempsey, KH and Browne, TS and Gloor, GB and Edgell, DR},
title = {PHYCUT: Scalable Multiplex CRISPR/Cas9 Editing for Genome Engineering in the Diatom Phaeodactylum tricornutum.},
journal = {ACS synthetic biology},
volume = {15},
number = {5},
pages = {1850-1865},
doi = {10.1021/acssynbio.5c00843},
pmid = {41979903},
issn = {2161-5063},
mesh = {*Diatoms/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Glycosylation ; Plasmids/genetics ; Fucose/metabolism ; },
abstract = {Diatoms are globally significant microalgae that contribute ∼20% of oxygen production and exhibit remarkable metabolic diversity. The marine diatom Phaeodactylum tricornutum has emerged as a promising synthetic biology platform for the bioproduction of recombinant proteins, supported by a human-like N-linked glycosylation pathway. However, its α(1,3)-linked core fucose is potentially immunogenic in humans and thus limits its biopharmaceutical applications. One hurdle to efficient genome engineering in P. tricornutum is the lack of a robust system for simultaneous CRISPR/Cas9 editing at multiple sites. To overcome this limitation, we develop PHYCUT (Phaeodactylum tricornutum Csy4-Cas9 multiplex tool), a versatile plasmid-based CRISPR/Cas9 system that uses the Csy4 endoribonuclease to process multiguide RNA arrays. To highlight PHYCUT applications, we demonstrate multiplex editing of all three FucT genes responsible for α(1,3) fucosylation in P. tricornutum, yielding strains with reduced fucosylation of secreted proteins. PHYCUT enables facile, multiplexed genome engineering in diatoms and provides a foundation for humanizing the P. tricornutum glycosylation pathway to support next-generation algal biotechnology.},
}

*Diatoms/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Glycosylation
Plasmids/genetics
Fucose/metabolism

@article {pmid42014015,
year = {2026},
author = {Li, R and Xie, L and Hu, J and Liu, B and Zhang, H and Qian, H},
title = {Genome-wide CRISPR knockout screening identifies novel disease-associated genes in retinal pigment epithelium cells.},
journal = {Experimental eye research},
volume = {268},
number = {},
pages = {111032},
doi = {10.1016/j.exer.2026.111032},
pmid = {42014015},
issn = {1096-0007},
mesh = {*Retinal Pigment Epithelium/metabolism/pathology ; Humans ; Gene Knockout Techniques ; *Retinal Degeneration/genetics/metabolism/pathology ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Genome-Wide Association Study ; Cell Survival ; *CRISPR-Cas Systems ; *Gene Expression Regulation/physiology ; Cells, Cultured ; *Eye Proteins/genetics ; },
abstract = {Dysfunction and degeneration of retinal pigment epithelium (RPE) cells are common pathological features observed in various retinal degenerative diseases. It has been proposed to treat these diseases by either protecting RPE cells or replacing them with new RPE cells derived from stem cells. However, the development of effective therapeutic strategies is still limited due to the insufficient understanding of the pathogenic factors involved in retinal degeneration and their impact on the function and survival of RPE cells. In this study, we employed genome-scale CRISPR knockout (KO) screening in human RPE cells to identify genes critical for RPE cell survival. Over 300 genes were identified, including well-established housekeeping genes as well as several candidate genes previously linked to retinal degeneration, many of which still lack comprehensive investigation. Among these, we further validated PRPF38B, which was both enriched in our screening and highlighted in a prior family-based linkage study, as essential for RPE cell survival, thus confirming the effectiveness of our approach. As a component of the spliceosome, we found that PRPF38B is crucial for functions specific to RPE cells, offering new insights into its role in retinal degeneration. Our study presents a novel approach for investigating risk genes associated with retinal diseases and may inspire future research on RPE cells and vision disorders.},
}

*Retinal Pigment Epithelium/metabolism/pathology
Humans
Gene Knockout Techniques
*Retinal Degeneration/genetics/metabolism/pathology
*Clustered Regularly Interspaced Short Palindromic Repeats
Genome-Wide Association Study
Cell Survival
*CRISPR-Cas Systems
*Gene Expression Regulation/physiology
Cells, Cultured
*Eye Proteins/genetics

@article {pmid42044361,
year = {2026},
author = {Huang, J and Zhang, C and Li, J and Ren, H and Yang, K and Zhang, Y},
title = {Comparative Evaluation of Engineered Bacteria and Yeast for Oral Delivery of CRISPR/Cas9 Systems in Colon Cancer Therapy.},
journal = {ACS synthetic biology},
volume = {15},
number = {5},
pages = {1915-1925},
doi = {10.1021/acssynbio.6c00006},
pmid = {42044361},
issn = {2161-5063},
mesh = {*CRISPR-Cas Systems/genetics ; Animals ; *Colonic Neoplasms/therapy/genetics ; Mice ; *Escherichia coli/genetics/metabolism ; Humans ; Administration, Oral ; Gene Editing/methods ; Cell Line, Tumor ; Female ; Genetic Therapy/methods ; Mice, Inbred BALB C ; },
abstract = {Colorectal cancer (CRC) poses a serious threat to human health. CRISPR/Cas9 technology offers new therapeutic strategies for the management of this disease, but its oral application is severely hindered by the limitations of suitable delivery systems. Herein, we develop and compare two separate orally delivered, genetically and chemically modified CRISPR/Cas9 delivery platforms based on E. coli BL21 and P. pastoris X33, which upon colonization in the intestine, secreted extracellular vesicles carrying the Cas9 protein and ART1-targeting sgRNA for tumor-specific gene disruption. Arginine ADP-ribosyltransferase 1 (ART1) plays a crucial role in the biological regulation of colon cancer, which was for the first time to the best of our knowledge, employed in vivo as a target gene in this study. Furthermore, we employed polydopamine (PDA) coating and gastrointestinal synthetic epithelial lining systems to facilitate microbial viability and intestinal retention, establishing on site cell factories for sustained CRISPR secretion. In subcutaneous tumor-bearing murine models, both delivery systems demonstrated comparable antitumor efficacy with significant tumor suppression. Taken together, the genetically modified microbial platform using bacterial and yeast strategies shows great potential and broad therapeutic versatility, offering a promising CRISPR-based solution for CRC treatment.},
}

*CRISPR-Cas Systems/genetics
Animals
*Colonic Neoplasms/therapy/genetics
Mice
*Escherichia coli/genetics/metabolism
Humans
Administration, Oral
Gene Editing/methods
Cell Line, Tumor
Female
Genetic Therapy/methods
Mice, Inbred BALB C

@article {pmid42052944,
year = {2026},
author = {Chen, Z and Hong, W and Wei, X and Li, Y and Feng, T and Ke, X and Li, X and Wang, Y and Hang, H and Tian, X and Chu, J},
title = {A Versatile tRNA-gRNA Array-Based CRISPR/Cas9 Platform Enabling Multiplex Genome Editing and Large-Fragment Engineering in Acremonium chrysogenum.},
journal = {ACS synthetic biology},
volume = {15},
number = {5},
pages = {1955-1967},
doi = {10.1021/acssynbio.6c00052},
pmid = {42052944},
issn = {2161-5063},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Acremonium/genetics/metabolism ; *RNA, Transfer/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Promoter Regions, Genetic/genetics ; Cephalosporins/biosynthesis ; Metabolic Engineering/methods ; Gene Knockout Techniques ; },
abstract = {Cephalosporin C (CPC)-derived antibiotics have played a vital role in improving human health and extending life expectancy. Acremonium chrysogenum remains the only microorganism capable of industrial-scale CPC production to date. However, the lack of efficient multiplex genome-editing tools has limited studies on its gene function, high-yield mechanisms as well as metabolic engineering. To overcome this limitation, a rapid and efficient CRISPR/Cas9-based multiplex genome-editing system was developed, driven by endogenous tRNA promoters, enabling one-step multilocus knockout, large-fragment DNA deletion, and gene overexpression in A. chrysogenum. Given that many strains lack visible phenotypes associated with specific genes, we introduced a visually distinguishable red phenotype by expressing the heterologous protein mCherry under a strong promoter. In the wild-type strain, 20 endogenous tRNA promoters were evaluated and compared to the heterologous Aspergillus nidulans PgpdA and Aspergillus fumigatus U6 promoters. The endogenous tRNA[Val] promoter showed the highest knockout efficiency (95.5%). The tRNA-gRNA array-based CRISPR/Cas9 system enabled double- and triple-site knockouts without donor DNA in industrial strain, with efficiencies of 50.0-83.3% and 14.3%, respectively. This is the first demonstration of simultaneous triple-site knockout in A. chrysogenum, especially in industrial strain. Using this system, we successfully deleted a 50.7-kb DNA fragment containing the sorbicillinoids biosynthetic gene cluster with nearly 100% efficiency and achieved overexpression of the key gene pcbAB involved in CPC biosynthesis in high-yield strain, increasing CPC titer from 5.59 g/L to 6.92 g/L with an improvement of 23.8%. Overall, this tRNA-gRNA array-based CRISPR/Cas9 multiplex gene-editing system provides an efficient and versatile platform for functional genomics and industrial strain engineering in A. chrysogenum.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Acremonium/genetics/metabolism
*RNA, Transfer/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics
Promoter Regions, Genetic/genetics
Cephalosporins/biosynthesis
Metabolic Engineering/methods
Gene Knockout Techniques

@article {pmid42068556,
year = {2026},
author = {Ban, H and Rondthaler, SN and Lebovich, M and Lora, MA and Ugbesia, B and Andrews, LB},
title = {Cross-Strain Transferability of CRISPRi Systems and Design Rules from Laboratory to Clinical Escherichia coli Strains.},
journal = {ACS synthetic biology},
volume = {15},
number = {5},
pages = {1993-2010},
doi = {10.1021/acssynbio.6c00075},
pmid = {42068556},
issn = {2161-5063},
support = {NSF DMR-1904901//NSF National Science Foundation/ ; NSF EES-1824090//NSF UMass ADVANCE program National Science Foundation/ ; NSF CBET-1943695//NSF National Science Foundaion/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Escherichia coli/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing/methods ; },
abstract = {CRISPR interference (CRISPRi) has emerged as a versatile approach for targeted gene repression in many organisms, including microbes and bacteria, due to the simple design of sequence-specific transcriptional silencing of gene expression. However, the strain-specific effects on repression efficiency and the host when translating a CRISPRi system from a laboratory strain to nonmodel strains are not well understood, yet they can present important limitations to its use. Here, we investigated the repression efficiency and toxicity of three CRISPRi systems (one dCas9 and two dCas12a variants) across four different Escherichia coli strains, including a laboratory K-12 strain (MG1655) and three nonmodel strains that are clinical isolates (probiotic Nissle 1917, uropathogenic CFT073, and uropathogenic UMN026). We evaluated the repression in each strain using sets of guide RNAs (gRNAs) targeting along the gene sequence and assayed cytotoxicity of expressing each dCas protein. Growth toxicity from expression of the different dCas proteins notably differed and showed high variation between some host strains. We also observed variable repression among the strains and notably poorer repression in multiple clinical strains. Therefore, we developed a dual gRNA CRISPRi system for enhanced gene silencing among the strains, which achieved up to 824-fold repression in CFT073. The results demonstrate that strain-specific design considerations can arise when a CRISPRi genetic system is transferred to a closely related bacterial strain. These findings provide insight into the relationships between criteria used for CRISPRi genetic design and in vivo activity across nonmodel E. coli strains, providing guidelines for diverse applications of these tools.},
}

*CRISPR-Cas Systems/genetics
*Escherichia coli/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Gene Editing/methods

@article {pmid42084552,
year = {2026},
author = {Huang, X and Liu, M and Chen, Y and Fang, L and Cao, Y},
title = {Genome-Scale CRISPRi Screening Identifies Gene Targets for Enhanced Octanoic Acid Tolerance and Production in Escherichia coli.},
journal = {ACS synthetic biology},
volume = {15},
number = {5},
pages = {1771-1778},
doi = {10.1021/acssynbio.6c00105},
pmid = {42084552},
issn = {2161-5063},
mesh = {*Escherichia coli/genetics/metabolism/drug effects ; *Caprylates/metabolism/pharmacology ; Metabolic Engineering/methods ; *CRISPR-Cas Systems/genetics ; Escherichia coli Proteins/genetics/metabolism ; Reactive Oxygen Species/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; NADP/metabolism ; Genome, Bacterial ; },
abstract = {Medium-chain fatty acids (MCFAs) are valuable precursors for biofuels and other commodity chemicals; however, the microbial biosynthesis of these compounds is severely constrained by cytotoxic effects. Here, we employed a genome-scale CRISPR interference (CRISPRi) library to systematically identify gene targets whose repression enhanced the tolerance to octanoic acid (C8) in Escherichia coli. Among the identified targets, repression of ygaM, gluQ, gatY, and talA enabled a 1.1- to 1.7-fold increase in C8 production relative to the parental strain MS-1. Mechanistic analyses revealed that the enhanced tolerance was associated with improved membrane properties, reduced reactive oxygen species (ROS) levels, and a shorter cell morphology. Further metabolic engineering to optimize NADPH availability increased C8 production to 1083 mg/L, representing a 2.3-fold increase over that of the MS-1 strain. This study provides new insights into engineering robust E. coli strains for MCFAs production and highlights the utility of genome-scale CRISPRi screening for identifying genetic determinants of microbial stress tolerance.},
}

*Escherichia coli/genetics/metabolism/drug effects
*Caprylates/metabolism/pharmacology
Metabolic Engineering/methods
*CRISPR-Cas Systems/genetics
Escherichia coli Proteins/genetics/metabolism
Reactive Oxygen Species/metabolism
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
NADP/metabolism
Genome, Bacterial

@article {pmid42136289,
year = {2026},
author = {Sorourian, S and Behbahani, AB},
title = {CRISPR-Cas Systems and CHO in Biopharmaceuticals: Unlocking New Possibilities in Gene Editing.},
journal = {Current pharmaceutical biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.2174/0113892010408149251016112221},
pmid = {42136289},
issn = {1873-4316},
abstract = {The CRISPR-Cas system has significantly advanced genome editing, offering superior efficiency, precision, and ease of use compared to traditional technologies such as Zinc Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs). CHO cells are a widely used mammalian cell line for large-scale therapeutic protein manufacturing due to their ability to produce human-like glycosylation patterns and grow in serum-free media. Recent CRISPR-based CHO cell engineering enables precise genetic modifications, improving productivity, stability, scalability, and reducing costs. This article highlights the transformative role of CRISPR technologies in addressing genetic disorders and expanding the frontiers of multiple scientific fields. It offers a comprehensive analysis of several CRISPR-Cas systems, including Cas9, Cas12, Cas13, and Cas14, emphasizing their unique structural features and functional capabilities. While Cas9 has dominated many genomeediting applications, the use of Cas13 in Chinese Hamster Ovary (CHO) cells has opened up promising RNA-targeting strategies. Moreover, the compact Cas14 system presents notable potential for applications requiring ultra-precise genome manipulation. With their critical role in therapeutic protein production, CHO cells have greatly benefited from CRISPR-enabled engineering, leading to measurable improvements in productivity, stability, and cost-efficiency. Key advancements in CRISPR delivery platforms, including both viral and nonviral vectors, are discussed alongside ongoing challenges such as off-target effects and regulatory considerations. Emerging trends such as base editing, prime editing, and the integration of artificial intelligence for system optimization are also explored. Altogether, the discussion underscores the pivotal contribution of CRISPR technologies to CHO cell engineering and their broader impact on the future of biopharmaceutical manufacturing.},
}

@article {pmid42137038,
year = {2026},
author = {Koshi, N and Kobayashi, M and Ezura, H and Miura, K},
title = {Enhancement of parthenocarpy and fruit set through genome editing in tomato variety for processing use.},
journal = {Plant biotechnology (Tokyo, Japan)},
volume = {43},
number = {1},
pages = {127-131},
pmid = {42137038},
issn = {1342-4580},
abstract = {Tomatoes are extremely important plants that are cultivated worldwide, with various varieties grown in different regions. The traits required can vary depending on the region and intended use. Parthenocarpy, a trait that confers numerous advantages, reduces the labor required for pollination and minimizes the incidence of poor fruit set owing to temperature fluctuations. Mutations in SlIAA9 induce parthenocarpy in tomatoes, and the introduction of this trait into processed varieties via genome editing suggests its potential to markedly shorten the breeding timeline. Genome editing has gained considerable attention as a breeding technique because it enables precise mutations in specific genes. However, only a few recent studies have reported examples of genome editing in Japanese tomato varieties for processing. In this study, we employed a genome-editing technique targeting SlIAA9 to induce parthenocarpy in the Japanese tomato variety Natsunokoma for processing purposes, thereby reducing the labor required for pollination. The null-segregant Sliaa9 mutant exhibited enhanced parthenocarpy and fruit set. These results suggest that improvements in fruit-bearing and parthenocarpic traits enhance the quality of tomato varieties that are mainly used for processing.},
}

@article {pmid42137600,
year = {2026},
author = {Schönberg, PY and Muñoz-Ovalle, Á and Saleh, HA and Crespo, E and Kuhnert, R and Michen, S and Loureiro, L and Temme, A and Feldmann, A and Buchholz, F},
title = {Epigenetic editing balances TCR suppression and persistence in CAR T cells.},
journal = {Molecular therapy. Advances},
volume = {34},
number = {2},
pages = {201712},
pmid = {42137600},
issn = {3117-387X},
abstract = {Allogeneic chimeric antigen receptor (CAR) T cell therapies offer a scalable, off-the-shelf option for cancer treatment, but their clinical use is limited by the risk of graft-versus-host disease (GvHD), mediated by the endogenous T cell receptor (TCR). Conventional strategies to eliminate TCR expression rely on genome editing tools such as CRISPR-Cas9 or base editing, which introduce permanent DNA changes and pose safety concerns. Here, we present an epigenetic editing approach that enables efficient, specific, and reversible silencing of the CD3ε gene, a critical component of the TCR complex, without altering the genome. We systematically optimized the epigenetic editor and guide RNA in a cell line and achieved robust TCR silencing in primary T and CAR T cells while preserving CAR expression, activation, and effector function. Transcriptome analysis confirmed minimal off-target effects. In vivo observations suggest the epigenetically silenced T cells to prevent GvHD while persisting longer than TCR-knockout cells, supporting the notion that transient TCR suppression may help balance safety and long-term efficacy. Our findings establish epigenetic editing as a non-genotoxic alternative to genome editing, offering a flexible and safer route to generate next-generation allogeneic CAR T cells.},
}

@article {pmid42137606,
year = {2026},
author = {Campbell, JM and Korpela, DM and Han, H and Zhao, S and Webster, DA and Nguyen, YAH and Koes, N and Aune, RO and Dagan, H and Milliken, R and Watts, JK and Murthy, N and Carlson, DF},
title = {Swine reporter model for preclinical evaluation and characterization of gene delivery vectors.},
journal = {Molecular therapy. Advances},
volume = {34},
number = {2},
pages = {201729},
pmid = {42137606},
issn = {3117-387X},
abstract = {Delivery of gene therapy vectors efficiently targeted to any somatic cell remains a key barrier for the development of genetic medicines. While rodent models provide insights into vector biodistribution and cellular tropism, their anatomical and physiological differences from humans limit their translational potential and studies in large animal models are often required. In this study, we developed a swine reporter model (SRM-1) to evaluate both viral and non-viral vector delivery in a large animal system. The SRM-1 model harbors a tdTomato reporter at the ROSA26 locus that can be activated by Cre recombinase or CRISPR-Cas reagents and allows for tracking of gene delivery vectors in vivo. To evaluate this model, we administered adeno-associated virus serotype 9 (AAV9) and lipid nanoparticles (LNPs) carrying messenger RNA (mRNA) systemically and found successful in vivo reporter activation across a variety of tissues. Intracerebroventricular (i.c.v.) administration of LNP-mRNA was also performed and demonstrated localized activation in cortical brain cells. In addition to systemic biodistribution studies, this model has utility for testing clinically relevant local administration methods, surgical and non-surgical, of delivery vectors. Our findings support the SRM-1 model as a valuable tool for advancing gene therapies from preclinical testing to clinical application.},
}

@article {pmid42137980,
year = {2026},
author = {Xiong, W and Zhou, E and Qi, Q and Liu, X and Zhang, K and Huang, S and Li, M and Wang, C and Zhou, X and Tian, T},
title = {Harnessing a single molecule for dual bioorthogonal regulation of RNA function and m6A methylation.},
journal = {Nucleic acids research},
volume = {54},
number = {9},
pages = {},
doi = {10.1093/nar/gkag438},
pmid = {42137980},
issn = {1362-4962},
support = {22377094//National Natural Science Foundation of China/ ; 22177089//National Natural Science Foundation of China/ ; 22037004//National Natural Science Foundation of China/ ; 91853119//National Natural Science Foundation of China/ ; 91753201//National Natural Science Foundation of China/ ; 22177088//National Natural Science Foundation of China/ ; 22377095//National Natural Science Foundation of China/ ; 22407104//National Natural Science Foundation of China/ ; 22407106//National Natural Science Foundation of China/ ; 22407107//National Natural Science Foundation of China/ ; 22377094//National Natural Science Foundation of China/ ; 2022YFA1502902//National Key Research and Development Program of China/ ; },
mesh = {Methylation ; Click Chemistry/methods ; *Adenosine/analogs & derivatives/metabolism/chemistry ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism/chemistry ; Humans ; *RNA/metabolism/chemistry/genetics ; CRISPR-Cas Systems ; Azides/chemistry ; RNA Processing, Post-Transcriptional ; },
abstract = {This study presents a multifunctional RNA regulation strategy that enables RNA molecules to undergo both bioorthogonal ligation and cleavage reactions within the same system. Using guide RNA (gRNA) as an example, we demonstrate on-demand inactivation via click chemistry and subsequent reactivation by light exposure. Applied to CRISPR-mediated, site-specific RNA methylation, this technology enables continuous, multistep programmable control, overcoming the one-way limitation of traditional methods. Results show that gRNA can stably tolerate both azide and photodegradable groups, facilitating efficient targeting of M3M14-dCas9 and dCas13b-M3M14 methylation systems to introduce m6A at specific RNA sites. Small molecules can turn off methylation through in situ click chemistry, while 365-nm light exposure rapidly restores gRNA function, allowing precise control over RNA methylation. This strategy highlights the efficiency and flexibility of progressive bioorthogonal RNA modulation and paves the way for multisite, dynamic regulation in complex biological systems.},
}

Methylation
Click Chemistry/methods
*Adenosine/analogs & derivatives/metabolism/chemistry
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism/chemistry
Humans
*RNA/metabolism/chemistry/genetics
CRISPR-Cas Systems
Azides/chemistry
RNA Processing, Post-Transcriptional

@article {pmid42138081,
year = {2026},
author = {Chang, LC and Eyler, CE and Lee, CL},
title = {Chromosomal instability induced by CRISPR/Cas9: implications for pancreatic cancer therapy.},
journal = {The Journal of clinical investigation},
volume = {136},
number = {10},
pages = {},
doi = {10.1172/JCI206028},
pmid = {42138081},
issn = {1558-8238},
mesh = {Humans ; *Pancreatic Neoplasms/genetics/therapy/pathology/metabolism ; *Chromosomal Instability ; *CRISPR-Cas Systems ; *DNA Breaks, Double-Stranded ; },
abstract = {Clinical management of pancreatic cancer (PC) remains severely limited, primarily due to the complex tumor microenvironment. Emerging DNA damage-targeted strategies have demonstrated considerable therapeutic potential in PC. In this issue of the JCI, Teh et al. employed cancer-specific multitarget sgRNAs to induce DNA double-strand breaks (DSBs), resulting in lethal effects in PC cells. Integrative bioinformatic and cytogenetic analyses revealed that CRISPR/Cas9-mediated DSBs provoked persistent chromosomal instability, ultimately leading to chromosome catastrophe and cell death. Compared with equivalent radiation-induced DSBs, these sgRNAs exhibited superior cytotoxicity and were able to eliminate cells resistant to a specific sgRNA via subsequent targeting at distinct genomic sites, highlighting a promising and innovative precision therapeutic approach for clinical treatment of PC.},
}

Humans
*Pancreatic Neoplasms/genetics/therapy/pathology/metabolism
*Chromosomal Instability
*CRISPR-Cas Systems
*DNA Breaks, Double-Stranded

@article {pmid42138085,
year = {2026},
author = {Teh, SSK and Kotwal, A and Bennett, A and Halper-Stromberg, E and Morsberger, L and Zamani, S and Shi, Y and Skaist, A and Zhu, Q and Bowland, K and Liang, H and Hruban, RH and Hung, CF and Anders, RA and Roberts, NJ and Scharpf, RB and Goldstein, M and Zou, YS and Eshleman, JR},
title = {Simultaneous CRISPR/Cas9-induced double-strand breaks are lethal in models of pancreatic cancer.},
journal = {The Journal of clinical investigation},
volume = {136},
number = {10},
pages = {},
doi = {10.1172/JCI190121},
pmid = {42138085},
issn = {1558-8238},
mesh = {*Pancreatic Neoplasms/genetics/pathology/therapy/metabolism ; *CRISPR-Cas Systems ; Humans ; *DNA Breaks, Double-Stranded ; Cell Line, Tumor ; Animals ; Mice ; Gene Editing ; },
abstract = {While radiation is an effective oncologic therapy, killing cancer by inducing DNA double-strand breaks (DSBs), it lacks specificity for neoplastic cells. We have previously adapted the CRISPR/Cas9 gene-editing technology as a cancer-specific treatment modality targeting somatic mutations in pancreatic cancer (PC). However, its tumoricidal potential remains unclear, especially in comparison with therapeutic doses of radiation. Here, we demonstrate that CRISPR/Cas9-induced DSBs are more cytotoxic in PCs than a comparable number of radiation-induced DSBs. We observed more than 90% tumor growth inhibition by targeting 9 sites with cancer-specific sgRNAs. Through both bioinformatics and cytogenetics analyses, we found that CRISPR/Cas9-induced DSBs triggered ongoing chromosomal rearrangements, with 87% of structural variants not directly produced from the initial CRISPR/Cas9-induced DSBs, and chromosomal instability peaking before cell death. By comparing the cytotoxicity of CRISPR/Cas9- and radiation-induced DSBs, we demonstrated that the number of DSBs required to achieve equitoxic effects was approximately 3 times higher for radiation than CRISPR/Cas9. Finally, we showed that PC cells that had survived CRISPR/Cas9 targeting retained susceptibility to subsequent CRISPR/Cas9-induced DSBs at different genomic sites with more than 87% growth inhibition. Together, our data support the therapeutic potential of CRISPR/Cas9 as an anticancer strategy.},
}

*Pancreatic Neoplasms/genetics/pathology/therapy/metabolism
*CRISPR-Cas Systems
Humans
*DNA Breaks, Double-Stranded
Cell Line, Tumor
Animals
Mice
Gene Editing

@article {pmid42138264,
year = {2026},
author = {Gao, M and Jiang, T and Si, W and Rong, Y and Hu, Y and He, X and Liu, BF and Chen, P and Yang, Y and Deng, Y and Wang, FB},
title = {Harnessing CRISPR-Cas12 and Microfluidics Chips for Multiplex Respiratory Pathogens Diagnosis.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.5c03226},
pmid = {42138264},
issn = {2379-3694},
abstract = {Respiratory pathogens jeopardize population health, particularly high-risk groups. CRISPR-Cas systems, as novel nucleic acid detection platforms, offer timely identification and have become a major research focus. This study presents a novel diagnostic workflow that combines recombinase polymerase amplification (RPA) for pre-amplification of pathogen nucleic acids with CRISPR-based detection. By combining microfluidic technology and portable imaging devices, this study developed a multiplex assay capable of simultaneously detecting seven clinically relevant pathogens in a single sample, including influenza A virus (FluA), influenza B virus (FluB), respiratory syncytial virus (HRSV) A and B, mycoplasma pneumoniae (MP), adenovirus (HAdv), and parainfluenza virus (HPIVs). Utilizing the POCT-CRISPR platform, simultaneous detection of seven respiratory pathogens can be achieved within approximately 30 min, achieving detection limits of 0.1-1 fM. This method streamlines the detection process, significantly reducing both the complexity of operations and the overall detection time. Clinical cohort validation demonstrated a detection efficiency of 99.63% sensitivity and 100% specificity. These results confirm the effectiveness and reliability of the detection method. Additionally, the 7-virus panel is estimated at approximately $32 per sample, a cost competitive with commercial multiplex qPCR detection kits ($15-$110 per sample) and substantially more economical than integrated cartridge-based syndromic platforms. The platform features simple operation, cost-effectiveness, short turnaround time, and reliable detection performance, making it highly suitable for point-of-care testing (POCT) at the grassroots level.},
}

@article {pmid42138716,
year = {2026},
author = {Lefrançois, G and Lavallée, E and Rowell, MC and Bourdeau, V and Mohebali, F and Bertomeu, T and Duman, AM and Nikolova, M and Tyers, M and Gravel, SP and Schmitzer, AR and Ferbeyre, G},
title = {The role of ATP synthase subunit e (ATP5I) in mediating the metabolic and antiproliferative effects of metformin in cancer cells.},
journal = {eLife},
volume = {13},
number = {},
pages = {},
doi = {10.7554/eLife.102680},
pmid = {42138716},
issn = {2050-084X},
support = {TFRI Project #1123//Terry Fox Research Institute/ ; Operating Grant 2016//Cancer Research Society/ ; 935858//Cancer Research Society/ ; 840633//Cancer Research Society/ ; RGPIN-2021-03128//Natural Sciences and Engineering Research Council of Canada/ ; Ganotec/Marc-André Pigeon Fund//Cancer Research Society/ ; 1054571//Cancer Research Society/ ; },
mesh = {*Metformin/pharmacology/metabolism ; Humans ; Cell Line, Tumor ; *Mitochondrial Proton-Translocating ATPases/metabolism/genetics ; *Cell Proliferation/drug effects ; Oxidative Phosphorylation/drug effects ; *Antineoplastic Agents/pharmacology ; Mitochondria/drug effects/metabolism ; Gene Knockout Techniques ; CRISPR-Cas Systems ; *Hypoglycemic Agents/pharmacology ; },
abstract = {Here, we identify the subunit e of F1F0-ATP synthase (ATP5I) as a target of metformin, a first-in-class antidiabetic biguanide. ATP5I maintains the stability of F1F0-ATP synthase dimers, which is crucial for shaping cristae morphology. We demonstrate that ATP5I interacts with a biguanide analogue in vitro, and disabling its expression by CRISPR-Cas9 in pancreatic cancer cells leads to the same phenotype as biguanide-treated cells, including mitochondrial morphology alterations, reduction of the NAD[+]/NADH ratio, inhibition of oxidative phosphorylation (OXPHOS), rescue of respiration by uncouplers, and a compensatory increase in glycolysis. Notably, metformin disrupts F1F0-ATP synthase oligomerization, leading to the accumulation of vestigial assembly intermediates in pancreatic and osteosarcoma cancer cells, a phenotype also observed upon ATP5I inactivation in pancreatic cancer cells. Moreover, ATP5I knockout (KO) cells exhibit resistance to the antiproliferative effects of biguanides, but reintroduction of ATP5I rescues the metabolic and antiproliferative effects of metformin and phenformin. Finally, a genome-wide CRISPR screening in NALM-6 lymphoma cells revealed that metformin-treated cells exhibit genetic interaction profiles similar to those observed with the F1F0-ATP synthase inhibitor oligomycin, but not with the complex I inhibitor rotenone. This provides unbiased support for the relevance of the newly proposed target.},
}

*Metformin/pharmacology/metabolism
Humans
Cell Line, Tumor
*Mitochondrial Proton-Translocating ATPases/metabolism/genetics
*Cell Proliferation/drug effects
Oxidative Phosphorylation/drug effects
*Antineoplastic Agents/pharmacology
Mitochondria/drug effects/metabolism
Gene Knockout Techniques
CRISPR-Cas Systems
*Hypoglycemic Agents/pharmacology

@article {pmid42139236,
year = {2026},
author = {Quiroz-Huanca, A and Vargas-Reyes, M and López, JD and Flores-Jimenez, K and Saldarriaga-Morán, S and Cifuentes, K and Alcántara, R},
title = {Thermal optimized PCR coupled to CRISPR-Cas12a for rapid detection of blaOXA-1 resistance gene.},
journal = {PloS one},
volume = {21},
number = {5},
pages = {e0337675},
doi = {10.1371/journal.pone.0337675},
pmid = {42139236},
issn = {1932-6203},
mesh = {*beta-Lactamases/genetics ; *CRISPR-Cas Systems/genetics ; *Escherichia coli/genetics/drug effects/isolation & purification/enzymology ; *Polymerase Chain Reaction/methods ; Animals ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {The β-lactams are critically important broad-spectrum antibiotics, widely used as first-line treatments; however, their effectiveness is increasingly compromised by β-lactamase enzymes. Among these, OXA-type enzymes have expanded to over 400 variants and are highly prevalent in Enterobacteriaceae. Current phenotypic and molecular detection tests have long turnaround times or require specialized equipment, respectively. In this study, we optimize a rapid molecular assay combining a PCR with modified thermal ramp rate (TRR) along with CRISPR-Cas12a fluorescence detection for blaOXA-1-harboring E. coli isolates. Using a commercial DNA Taq polymerase (TRR: 2.2 °C/s, annealing and extension hold time: 1 s), amplification time was reduced from 80 to 30 min, enabling detection within 50 min (PCR: 30 min; CRISPR: 20 min). With a locally produced enzyme (hold: 10 s), amplification time was 44 min. To demonstrate the practical application of the assay, we evaluated spiked poultry fecal samples achieving an analytical sensitivity of 8 CFU/reaction using commercial DNA Taq polymerase. The accelerated PCR:CRISPR workflow delivers results in less than one hour without compromising technical sensitivity (attomoles range), not requiring high technical expertise, and can be implemented in laboratories with basic molecular biology equipment.},
}

*beta-Lactamases/genetics
*CRISPR-Cas Systems/genetics
*Escherichia coli/genetics/drug effects/isolation & purification/enzymology
*Polymerase Chain Reaction/methods
Animals
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid42141309,
year = {2026},
author = {Qin, L and Liu, D and Wang, Z and Sun, X and Zhao, X},
title = {Molecular biology of Pleurotus mushrooms: genomic resources, genetic manipulation, and regulation mechanisms.},
journal = {Antonie van Leeuwenhoek},
volume = {119},
number = {6},
pages = {},
pmid = {42141309},
issn = {1572-9699},
support = {32402651//National Natural Science Foundation of China/ ; 2024AFB274//Hubei　Provincial　Natural　Science　Foundation　of　China/ ; Q20231511//Youth Talent Project of Scientific Research Plan of Hubei Provincial Education Department of China/ ; K2023058//Scientific Research Fund of Wuhan Institute of Technology/ ; },
mesh = {*Pleurotus/genetics ; *Genomics/methods ; *Genome, Fungal ; Gene Editing ; CRISPR-Cas Systems ; Gene Expression Regulation, Fungal ; RNA Interference ; },
abstract = {As one of the most widely cultivated edible mushrooms in the world, Pleurotus mushrooms are popular among people for their delicious taste and rich nutritional value. Because of their great economic value, the research on the molecular biology of Pleurotus spp. has been deepening in recent years. The study first summarized the current situation of genomic resources available for this genus. The whole genome sequencing of 14 species, including Pleurotus tuoliensis and Pleurotus ostreatus, provides reference data for mining functional genes. Although the genomic data for Pleurotus mushrooms are continuously increasing, actual instances of successful genetic transformation remain restricted. Research on the regulatory mechanisms of key genes at different developmental stages and under various environmental stresses is insufficient. Then, the application of gene editing methods (CRISPR/Cas9, RNAi, and gene overexpression) in Pleurotus mushrooms was systematically described. RNAi and gene overexpression technologies have become well-established and are routinely used in most Pleurotus mushrooms. However, the application of CRISPR/Cas9 technology is still limited to P. eryngii and P. ostreatus. This limitation is attributed to the difficulties in establishing genetic transformation systems and the low efficiency of homologous recombination. Furthermore, this review explored the value of multi-omics technologies in elucidating the molecular mechanisms of morphogenesis and stress responses. To address the lack of specific antibodies for non-model organisms, we evaluated the application potential of DAP-seq technology in Pleurotus mushrooms and discussed its limitations, including the risk of false positives arising from the absence of a genuine environment in vivo. The purpose of this review is to evaluate the current molecular biology research on Pleurotus spp., and to provide systematic technical support and insights for functional genomics research and the analysis of molecular mechanisms of complex traits in Pleurotus mushrooms.},
}

*Pleurotus/genetics
*Genomics/methods
*Genome, Fungal
Gene Editing
CRISPR-Cas Systems
Gene Expression Regulation, Fungal
RNA Interference

@article {pmid41397899,
year = {2026},
author = {Chowdhury, A and Garcia, BG and Zahoor, MA and ElMawla, NF and Davidson, AR and Wyatt, HDM and Maxwell, KL and Mahassine, A and Gehring, A and Feld, JJ},
title = {A Rapid Assay for Hepatitis C Virus RNA Detection Using Reverse-Transcription Loop-Mediated Isothermal Amplification-Coupled CRISPR-Cas12b-Based Strategy.},
journal = {The Journal of infectious diseases},
volume = {233},
number = {5},
pages = {840-847},
doi = {10.1093/infdis/jiaf609},
pmid = {41397899},
issn = {1537-6613},
mesh = {Humans ; *Hepacivirus/genetics/isolation & purification ; *Hepatitis C/diagnosis/virology ; *RNA, Viral/genetics/blood ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; Genotype ; Limit of Detection ; },
abstract = {BACKGROUND: Hepatitis C virus (HCV) diagnosis usually requires detection of antibody followed by HCV RNA. The requirement for 2 tests leads to major drop-offs in the cascade of care. Existing near-care HCV RNA tests have slow turnaround time and are expensive with limited availability. We aim to develop a cost-effective, rapid, and sensitive test for detection of HCV RNA to enhance screening, particularly in marginalized and remote populations.

METHODS: After RNA extraction from plasma, HCV RNA is reverse-transcribed and amplified using loop-mediated isothermal amplification with HCV-specific primers. The amplified HCV DNA is then detected via CRISPR-Cas12b with a fluorescence readout.

RESULTS: HCV RNA from patient samples with genotypes 1a, 1b, 2, 3a, and 4 was detected with high sensitivity and specificity. The lower limit of detection (LLOD) with HCV JFH1 plasmid (genotype 2) is 250 plasmid copies/mL (approximately 100 IU/mL). For clinical samples, we determined the LLOD for genotypes 1 and 3, the most common in North America. Using 500 μL of plasma, genotype 1 RNA ≥100 IU/mL was detected within 40-45 minutes, while genotype 3 had an LLOD of 5000 IU/mL. The clinical sensitivity was 100% in 72 HCV patient samples, including acute HCV and HCV/hepatitis B virus (HBV) coinfection. The specificity was 100%, with no false-positives in 33 HCV-negative samples, including those with HBV or human immunodeficiency virus/HBV coinfection.

CONCLUSIONS: Our assay shows high specificity and sensitivity to detect HCV RNA directly from plasma within 45 minutes and hence could be used for efficient screening and diagnosis of HCV infection globally.},
}

Humans
*Hepacivirus/genetics/isolation & purification
*Hepatitis C/diagnosis/virology
*RNA, Viral/genetics/blood
Sensitivity and Specificity
*Nucleic Acid Amplification Techniques/methods
*CRISPR-Cas Systems
*Molecular Diagnostic Techniques/methods
Genotype
Limit of Detection

@article {pmid41604419,
year = {2026},
author = {Martínez Rivas, FJ and Smith, MA and Zangishei, Z and Alseekh, S and Usadel, B and Plaxton, WC and Fernie, AR},
title = {Malate matters: disrupting bacterial-type phosphoenolpyruvate carboxylase (BTPC) rewires tomato fruit development.},
journal = {Plant physiology},
volume = {201},
number = {1},
pages = {},
pmid = {41604419},
issn = {1532-2548},
support = {452682775//Deutsche Forschungsgemeinschaft/ ; 390686111//Deutsche Forschungsgemeinschaft/ ; //PlantaSyst/ ; 664621//European Union's Horizon 2020 Research and Innovation Programme/ ; 739582//European Union's Horizon 2020 Research and Innovation Programme/ ; 664620//European Union's Horizon 2020 Research and Innovation Programme/ ; RGPIN-2024-06467//Natural Sciences and Engineering Research Council of Canada/ ; //Queen's Research Chair/ ; },
mesh = {*Solanum lycopersicum/growth & development/genetics/enzymology/metabolism ; *Malates/metabolism ; *Fruit/growth & development/genetics/enzymology/metabolism ; *Phosphoenolpyruvate Carboxylase/metabolism/genetics ; *Plant Proteins/metabolism/genetics ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; },
abstract = {Plant phosphoenolpyruvate carboxylases (PEPCs) are ubiquitously expressed as cytosolic Class-1 PEPC homotetramers composed of 107 kDa plant-type PEPC (PTPC) subunits that are highly sensitive to allosteric inhibition by malate. Class-2 PEPC heterooctameric complexes that are desensitized to malate inhibition also exist in certain sink tissues due to the interaction of a Class-1 PEPC with unrelated 118 kDa bacterial-type PEPC (BTPC) polypeptides. Class-2 PEPCs dynamically associate with the mitochondrial outer envelope and have been hypothesized to support sustained anaplerotic flux and respiratory CO2 refixation in malate-rich sink tissues, including immature tomato fruit. The current study generated CRISPR-Cas9-edited tomato lines with targeted disruption of the BTPC gene and investigated the impact on fruit development, metabolism, and transcriptional regulation. Immunoblotting and co-immunoprecipitation confirmed the absence of BTPC polypeptides and Class-2 PEPC complexes in the edited lines. Fruits from the edited plants were 25% smaller and 40% lighter and required up to 10 additional days to complete ripening compared to the WT. Metabolomic analysis across ripening stages revealed substantial reductions in malate and citrate, with elevated sugars and amino acids, indicating reprogrammed carbon flux. RNA-seq data showed downregulation of genes for cell wall remodeling, sugar transport, and ethylene-responsive transcription factors. These results provide direct evidence that BTPC is essential for organic acid balance, sugar metabolism, and ripening regulation in tomato. Its absence perturbs metabolic homeostasis and developmental progression, positioning BTPC as a strategic target for enhancing fruit quality traits through genetic engineering.},
}

*Solanum lycopersicum/growth & development/genetics/enzymology/metabolism
*Malates/metabolism
*Fruit/growth & development/genetics/enzymology/metabolism
*Phosphoenolpyruvate Carboxylase/metabolism/genetics
*Plant Proteins/metabolism/genetics
Gene Expression Regulation, Plant
CRISPR-Cas Systems

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

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

@article {pmid41882360,
year = {2026},
author = {Smith, QM and Whittle, S and Aramayo, RJ and Rollins, DE and Jalal, ASB and Egharevba, DI and Morris, KL and Pyne, ALB and Rueda, DS},
title = {Structural basis of supercoiling-induced CRISPR-Cas9 off-target activity.},
journal = {Nature},
volume = {653},
number = {8114},
pages = {627-635},
pmid = {41882360},
issn = {1476-4687},
mesh = {*CRISPR-Associated Protein 9/metabolism/chemistry/ultrastructure ; *CRISPR-Cas Systems/genetics ; Cryoelectron Microscopy ; DNA, Circular/chemistry/ultrastructure/genetics/metabolism ; *DNA, Superhelical/chemistry/metabolism/ultrastructure/genetics ; *Gene Editing/methods ; Models, Molecular ; Protein Domains ; RNA/chemistry/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {CRISPR-Cas9 is a powerful genome-editing tool[1], but genome-wide off-target activity can hinder therapeutic applications. Negative supercoiling ((-)SC) has been implicated in off-target activity, but a molecular-level understanding is lacking. Here, using (-)SC DNA minicircles, we observe supercoiling-driven structural defects in the DNA that are resolved by Cas9 binding. Cryo-electron microscopy structures of Cas9 bound in both the on-target and off-target configurations highlight that the Cas9 HNH domain is poised in a more catalytically competent conformation. New DNA-RNA mismatch geometries are accommodated across the protospacer and structural plasticity in the protospacer adjacent motif distal region of the protospacer is topology dependent. Together, our study reveals the molecular basis for (-)SC-induced Cas9 targeting and provides a framework for the design of next-generation high-fidelity CRISPR effectors with topological context.},
}

*CRISPR-Associated Protein 9/metabolism/chemistry/ultrastructure
*CRISPR-Cas Systems/genetics
Cryoelectron Microscopy
DNA, Circular/chemistry/ultrastructure/genetics/metabolism
*DNA, Superhelical/chemistry/metabolism/ultrastructure/genetics
*Gene Editing/methods
Models, Molecular
Protein Domains
RNA/chemistry/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems

@article {pmid41913646,
year = {2026},
author = {Streiber, M and Liu, N and Simon, L and Adermann, F and Bachmann, V and Gath, L and Hoeppener, S and Schubert, S and Werz, O and Lapinte, V and Morille, M and Bauer, M and Press, AT and Schubert, US and Traeger, A},
title = {Extrahepatic Gene Editing In Vivo Using Organic Solvent-Free Lipid Nanoparticles.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {22},
number = {27},
pages = {e11489},
pmid = {41913646},
issn = {1613-6829},
support = {13XP5034A//Bundesministerium für Bildung und Forschung/ ; 03RU2U071H//Bundesministerium für Bildung und Forschung/ ; 2021 FGI 0005//Thüringer Aufbaubank/ ; 2023FGR0077//Thüringer Aufbaubank/ ; //Joachim Herz Stiftung/ ; P2024-02-016//Carl-Zeiss-Stiftung/ ; 57604510//German Academic Exchange Service/ ; 2018FGI0025//European Regional Development Fund/ ; ANR- 20-CE09-0011-01//Agence Nationale de la Recherche/ ; 514006196//Deutsche Forschungsgemeinschaft/ ; 316213987//Deutsche Forschungsgemeinschaft/ ; },
mesh = {*Nanoparticles/chemistry/ultrastructure ; Humans ; Animals ; *Lipids/chemistry ; *Gene Editing/methods ; Mice ; Solvents/chemistry ; CRISPR-Cas Systems/genetics ; Polyethylene Glycols/chemistry ; Transfection ; Liposomes ; },
abstract = {Targeted therapy, which modifies genes and their expression, holds great promise for treating a variety of diseases, including cancer, inborn errors of metabolism, and acute and chronic inflammatory and infectious conditions. However, it also presents challenges related to RNA delivery, immune responses, side effects of delivery vectors, and the need for individualized formulations. To overcome these limitations, the choice of lipids and formulation processes might be re-evaluated, with a focus on eliminating critical components, such as poly(ethylene glycol) (PEG) and ethanol. Thus, a purely water-based formulation for lipid nanoparticles was developed, offering a material-efficient, time-saving process with high reproducibility. Initially, a stealth lipid containing poly(2-methyl-2-oxazoline) (PMeOx) was used, and the formulation was later expanded to include approved lipids. These nanoparticles not only efficiently transfect primary human immune cells but also effectively deliver multiple nucleotides in CRISPR-Cas9 applications. Moreover, an in vivo comparison revealed that the nanoparticles exhibited preferential transfection in extrahepatic tissues. This distinguishes them from conventional cholesterol-rich lipid nanoparticles, which primarily target the liver regardless of the application route.},
}

*Nanoparticles/chemistry/ultrastructure
Humans
Animals
*Lipids/chemistry
*Gene Editing/methods
Mice
Solvents/chemistry
CRISPR-Cas Systems/genetics
Polyethylene Glycols/chemistry
Transfection
Liposomes

@article {pmid41931906,
year = {2026},
author = {Pedersen, A and Blay-Cadanet, J and Storgaard, J and Hernaez, B and Thyrsted, J and Bach-Nielsen, CS and Twayana, K and Jørgensen, SE and Rio-Bergé, C and Poulsen, C and Thielke, AL and Thomsen, EA and Kalucka, J and Olagnier, D and Luo, Y and Reggiori, F and Mogensen, TH and Alcamí, A and Hansen, AL and Holm, CK},
title = {NRF2 controls a diverse network of antiviral effectors with p62 acting as a central restriction factor effective across virus families.},
journal = {Redox biology},
volume = {93},
number = {},
pages = {104135},
pmid = {41931906},
issn = {2213-2317},
mesh = {*NF-E2-Related Factor 2/metabolism/genetics ; Humans ; *SARS-CoV-2/physiology ; Virus Replication/drug effects ; *Sequestosome-1 Protein/metabolism/genetics ; Influenza A virus/physiology ; Herpesvirus 1, Human/physiology ; *COVID-19/virology/genetics/metabolism ; Animals ; Autophagy ; Vaccinia virus/physiology ; Antiviral Agents/pharmacology ; CRISPR-Cas Systems ; Chlorocebus aethiops ; },
abstract = {The transcription factor erythroid 2 (NFE2)-related factor 2 (NRF2) is a key regulator of cellular homeostasis. Recent discoveries have identified agonists of NRF2 as inducers of broad cellular resistance to viral infection including SARS-CoV-2. Nevertheless, it is still unclear to what extent NRF2 itself is an inducer of anti-viral immunity and its downstream antiviral effectors have not been mapped. Here, we first demonstrate through specific genetic activation and silencing that NRF2 restricts SARS-CoV-2 replication. We then used a focused CRISPR-activation screen to map antiviral NRF2-inducible effector genes that restrict replication of SARS-CoV-2, Influenza A virus (IAV), Herpes Simplex virus 1 (HSV1) and Vaccinia virus (VACV). This approach allowed us to identify a range of antiviral effectors each of which restrict members of one or more virus families. Importantly, we identified the NRF2-inducible selective autophagy receptor p62/SQSTM1 as a broadly effective restriction factor across all the tested viruses. Importantly, p62 inhibited SARS-CoV-2 replication in cells treated with the lysosomal inhibitor bafilomycin A1, as well as in cells deficient in the autophagy protein ATG5. Similarly, p62 inhibited replication of HSV1 and IAV independently of ATG5 and ATG16L1 respectively. Thus, NRF2 restricts viral replication through a hitherto underappreciated network of antiviral restriction factors effective across multiple virus families. Importantly, we identify p62 as a broadly acting antiviral effector that restricts viral replication independently of canonical autophagy.},
}

*NF-E2-Related Factor 2/metabolism/genetics
Humans
*SARS-CoV-2/physiology
Virus Replication/drug effects
*Sequestosome-1 Protein/metabolism/genetics
Influenza A virus/physiology
Herpesvirus 1, Human/physiology
*COVID-19/virology/genetics/metabolism
Animals
Autophagy
Vaccinia virus/physiology
Antiviral Agents/pharmacology
CRISPR-Cas Systems
Chlorocebus aethiops

@article {pmid41942711,
year = {2026},
author = {Wang, W and Li, Y and Dong, S and Liu, Y and Guo, C and Su, Y and Tian, W and Hu, X and Wang, Z},
title = {The exonic SNP rs11676272-C risk allele mediates diet-induced obesity and reduces enhancer activation.},
journal = {EMBO reports},
volume = {27},
number = {9},
pages = {2462-2490},
pmid = {41942711},
issn = {1469-3178},
support = {32470645//MOST | National Natural Science Foundation of China (NSFC)/ ; 32070567//MOST | National Natural Science Foundation of China (NSFC)/ ; 32202840//MOST | National Natural Science Foundation of China (NSFC)/ ; C2023201032//| Natural Science Foundation of Hebei Province ()/ ; 050001-5000019//Hebei University ()/ ; ZJ2025118//Zhejiang Province Human Resources and Social Security Department ()/ ; },
mesh = {Animals ; Humans ; *Obesity/genetics/etiology/metabolism ; HEK293 Cells ; Mice ; *Polymorphism, Single Nucleotide ; *Alleles ; *Adenylyl Cyclases/genetics/metabolism ; *Enhancer Elements, Genetic ; Diet, High-Fat/adverse effects ; Genetic Predisposition to Disease ; Promoter Regions, Genetic ; CRISPR-Cas Systems ; Gene Expression Regulation ; Male ; },
abstract = {Genome-wide association studies (GWASs) have identified hundreds of obesity-associated SNPs, but establishing their causality remains challenging. Here, we demonstrate that rs11676272, located in the ADCY3 gene, is a functional causal variant for obesity susceptibility. Bioinformatic analyses and dual-luciferase reporter assays indicate that the rs11676272 region may act as a human-gained enhancer regulating ADCY3 expression. In HEK293T cells, CRISPR-Cas9-mediated single-nucleotide editing of rs11676272 (T > C) reduces ADCY3 expression. Moreover, the rs11676272-T allele is preferentially bound by the transcription factor E2F3 to upregulate ADCY3 expression, whereas the rs11676272-C risk allele loses this binding. In vivo, the rs11676272 T > C variant in human ADCY3 (hADCY3) knock-in mice accelerates weight gain under high-fat diet conditions and shortens primary cilia in the ventromedial hypothalamus (VMH). CRISPRa-mediated activation of the hADCY3 promoter region rescues ciliary length in both the VMH and hypothalamic arcuate nucleus of Mut-hADCY3 mice. Our data reveal a causal role for rs11676272 in obesity, offering insight into potential therapeutic strategies.},
}

Animals
Humans
*Obesity/genetics/etiology/metabolism
HEK293 Cells
Mice
*Polymorphism, Single Nucleotide
*Alleles
*Adenylyl Cyclases/genetics/metabolism
*Enhancer Elements, Genetic
Diet, High-Fat/adverse effects
Genetic Predisposition to Disease
Promoter Regions, Genetic
CRISPR-Cas Systems
Gene Expression Regulation
Male

@article {pmid42020604,
year = {2026},
author = {Calvo Fernández, E and Tomassoni, L and Zhang, X and Wang, J and Obradovic, A and Laise, P and Griffin, AT and Vlahos, L and Minns, HE and Morales, DV and Simmons, C and Gallitto, M and Wei, HJ and Martins, TJ and Becker, PS and Crawford, JR and Tzaridis, T and Wechsler-Reya, RJ and Garvin, J and Gartrell, RD and Szalontay, L and Zacharoulis, S and Wu, CC and Zhang, Z and Califano, A and Pavisic, J},
title = {Systematic design of combination therapy by targeting master regulators of coexisting diffuse midline glioma cell states.},
journal = {Nature genetics},
volume = {58},
number = {5},
pages = {1112-1125},
pmid = {42020604},
issn = {1546-1718},
mesh = {Humans ; *Glioma/genetics/drug therapy/pathology ; Animals ; Mice ; Cell Line, Tumor ; Pyrazoles ; *Brain Neoplasms/genetics/drug therapy/pathology ; *Antineoplastic Combined Chemotherapy Protocols/therapeutic use/pharmacology ; Pyrimidines ; Gene Expression Regulation, Neoplastic/drug effects ; Nitriles ; Xenograft Model Antitumor Assays ; CRISPR-Cas Systems ; },
abstract = {Intratumor heterogeneity fundamentally challenges cancer treatment, as coexisting, molecularly distinct cell states with non-overlapping drug sensitivities can drive therapeutic resistance. We establish and validate a generalizable, network-based framework to systematically identify combination therapies targeting complementary tumor cell states. Applied to diffuse midline glioma (DMG)-a universally fatal pediatric malignancy-this approach identified master regulator protein dependencies in seven coexisting cell states, confirmed by pooled CRISPR-Cas9 assays. Perturbational transcriptional profiles for 372 clinically relevant drugs prioritized candidates predicted to invert state-specific master regulator activity. State-selective drug sensitivity was validated for eight out of nine (89%) drugs in vivo, including avapritinib, ruxolitinib and larotrectinib. Compared with monotherapy, co-administering drugs targeting complementary states significantly prolonged survival across virtually all combinations, with avapritinib plus ruxolitinib extending median survival nearly threefold versus vehicle and 1.5-fold versus avapritinib alone. These findings establish clinically actionable DMG combinations and a tumor-agnostic and mutation-agnostic framework for rational combination therapy design.},
}

Humans
*Glioma/genetics/drug therapy/pathology
Animals
Mice
Cell Line, Tumor
Pyrazoles
*Brain Neoplasms/genetics/drug therapy/pathology
*Antineoplastic Combined Chemotherapy Protocols/therapeutic use/pharmacology
Pyrimidines
Gene Expression Regulation, Neoplastic/drug effects
Nitriles
Xenograft Model Antitumor Assays
CRISPR-Cas Systems