@article {pmid41738762,
year = {2026},
author = {Irfan, M and Duran-Pinedo, A and Solbiati, J and Rocha, FG and Gibson, FC and Frias-Lopez, J},
title = {A CRISPR array orchestrates virulence and host response in Porphyromonas gingivalis.},
journal = {Microbiology spectrum},
volume = {},
number = {},
pages = {e0283425},
doi = {10.1128/spectrum.02834-25},
pmid = {41738762},
issn = {2165-0497},
abstract = {CRISPR-Cas systems are primarily recognized for their defensive role against foreign DNA. Recent studies, however, indicate involvement in regulatory functions. The persistence of a large spacer array without apparent phage targets in Porphyromonas gingivalis ATCC 33277 remains unexplained. This study demonstrates that deletion of the non-coding CRISPR array 30.1 in P. gingivalis ATCC 33277 results in increased biofilm formation, elevated virulence in a Galleria mellonella infection model, and significant alterations in the macrophage transcriptomic response. The ΔCRISPR-30.1 mutant forms twice as much biofilm as the wild type, induces 50% mortality in Galleria larvae within 130 h compared to 200 h for the wild type (P < 0.0001), and elicits a cytokine profile characterized by increased IL-6, CXCL1, CXCL2, and CXCL9 secretion. Dual RNA sequencing of THP-1 macrophages infected with wild-type and ΔCRISPR 30.1 strains reveals that the loss of the array activates bacterial metabolic and secretion pathways while suppressing host innate and adaptive immune signaling. Single-primer amplification (SPA) identifies numerous self-genome loci bound by individual 30.1 spacers, supporting a direct, spacer-mediated regulatory mechanism. These findings establish CRISPR array 30.1 as a previously unrecognized regulator of P. gingivalis physiology and host-pathogen interactions.IMPORTANCECRISPR-Cas systems are established as adaptive immune elements, yet spacer arrays without known targets are frequently observed in bacteria and often lack a defined function. In P. gingivalis, a keystone periodontal pathogen, a non-coding CRISPR array has been shown to regulate biofilm formation, virulence in an invertebrate model, and the macrophage transcriptional response. This expands the recognized functions of CRISPR arrays to include the direct regulation of bacterial physiology and the modulation of host immune responses, identifying CRISPR spacers as potential targets for antimicrobial interventions. Furthermore, elucidating the role of CRISPR arrays in P. gingivalis may have broader clinical implications, given the established associations between periodontal health and systemic inflammatory diseases. Targeting spacer arrays to modulate bacterial virulence could influence the management of these conditions and enhance the translational relevance of such therapeutic strategies.},
}

@article {pmid41738557,
year = {2026},
author = {Girard, V and Sorge, S and Kurth, J and Alexandre, C and Gould, A},
title = {ShineGAL4 drivers for tissue and cell-type specific optogenetics in Drosophila.},
journal = {Development (Cambridge, England)},
volume = {153},
number = {4},
pages = {},
doi = {10.1242/dev.204981},
pmid = {41738557},
issn = {1477-9129},
support = {FC001088/CRUK_/Cancer Research UK/United Kingdom ; FC001088/MRC_/Medical Research Council/United Kingdom ; FC001088/WT_/Wellcome Trust/United Kingdom ; 223760/WT_/Wellcome Trust/United Kingdom ; 104566/WT_/Wellcome Trust/United Kingdom ; 543-2022//EMBO/ ; //Francis Crick Institute/ ; },
mesh = {Animals ; *Optogenetics/methods ; *Drosophila Proteins/genetics/metabolism ; Transcription Factors/genetics/metabolism ; *Drosophila melanogaster/genetics ; Organ Specificity/genetics ; CRISPR-Cas Systems/genetics ; Neurons/metabolism ; *Drosophila/genetics ; },
abstract = {An optogenetic split-GAL4 system, ShineGAL4, allows genes to be manipulated with unprecedented spatiotemporal precision. Here, we convert a panel of 14 GAL4 drivers widely used in Drosophila research into their ShineGAL4 counterparts. Homology assisted CRISPR knock-in (HACK) is used to replace GAL4 with the GAL4 DNA binding domain fused to a Magnet photoswitch. We show that the resulting ShineGAL4 drivers enable gene expression to be rapidly induced by light specifically in fat body, muscles, enterocytes, oenocytes, Malpighian tubules, neurons, neuroblast lineages, glial subtypes or in all glia. We also develop an optogenetic cassette for photoactivation of GAL4 in 'silent' FLP-out clones. This panel of optogenetic tools will enable precise spatiotemporal control of gene expression in a wide range of different Drosophila tissues and cell-types.},
}

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

@article {pmid41736546,
year = {2026},
author = {Zhang, C and Ye, K and Shang, Y and Song, Y and Li, P and Jiang, X and Yang, C and Liang, A and Zhang, J and Meng, F and Zhang, M},
title = {Spatially concentrated adenine base editors efficiently correct PLP1 mutations in oligodendrocytes.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
doi = {10.1093/nar/gkag156},
pmid = {41736546},
issn = {1362-4962},
support = {32370853//National Natural Science Foundation of China/ ; },
mesh = {*Oligodendroglia/metabolism ; *Gene Editing/methods ; Animals ; *Myelin Proteolipid Protein/genetics/metabolism ; *Adenine/metabolism ; Mutation ; Mice ; *Pelizaeus-Merzbacher Disease/genetics/therapy/pathology ; Humans ; CRISPR-Cas Systems ; Adenosine Deaminase/metabolism/genetics ; },
abstract = {Oligodendrocytes (OLs), the myelinating cells of the central nervous system, are particularly prone to pathogenic G-to-A mutations, such as PLP1A243V, which causes Pelizaeus-Merzbacher disease (PMD), a lethal hypomyelinating disorder lacking effective therapy. Although adenine base editors (ABEs) can in principle correct such mutations, their application in OLs is limited by inefficient on-target editing. Here, we develop a spatially concentrated ABE (cABE) strategy that enhances editing by promoting nuclear translocation of tRNA adenosine deaminase (TadA*) from the cytoplasm. Using a SunTag-based multivalent recruitment system, TadA* is locally enriched at genomic targets (cABE-1.0), achieving robust editing in vitro. To enable in vivo delivery while preserving high efficiency and fidelity, SpCas9 is replaced with compact eNme2-C Cas9, generating an AAV-compatible variant (cABE-2.0). Notably, cABE-2.0 forms dynamic nuclear puncta with properties of liquid-liquid phase separation, enhancing on-target editing while substantially reducing transcriptome-wide RNA off-target effects. Functionally, cABE-2.0 efficiently corrects the PLP1A243V mutation in OLs, restores Plp subcellular localization, and rescues myelination-related phenotypes. These findings demonstrate that spatial reorganization, rather than increasing intrinsic catalytic activity of TadA*, provides a distinct principle for improving base editing in difficult-to-edit cell types, such as OLs, offering a mechanistic and technical framework for gene therapy of PMD and related myelin disorders.},
}

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

@article {pmid40561247,
year = {2025},
author = {Damaskou, A and Wilson, R and Gozdecka, M and Giotopoulos, G and Asby, R and Eleftheriou, M and Gu, M and Récher, C and Mansat-De Mas, V and Vergez, F and Sahal, A and Vick, B and Papachristou, EK and Sawle, A and Yankova, E and Dudek, M and Liu, X and Russell, J and Rak, J and Hilcenko, C and D'Santos, C and Jeremias, I and Sarry, JE and Tzelepis, K and Huntly, BJP and Warren, AJ and Tavana, O and Vassiliou, GS},
title = {Posttranscriptional depletion of ribosome biogenesis factors engenders therapeutic vulnerabilities in NPM1-mutant AML.},
journal = {Blood},
volume = {146},
number = {10},
pages = {1239-1252},
doi = {10.1182/blood.2024026113},
pmid = {40561247},
issn = {1528-0020},
mesh = {*RNA Processing, Post-Transcriptional/drug effects/genetics ; *Ribosomes/genetics/metabolism ; *Leukemia, Myeloid, Acute/drug therapy/genetics/pathology ; Cytoplasm ; Dactinomycin/pharmacology ; Nuclear Proteins/metabolism ; Mice, Knockout ; Hematopoietic Stem Cells ; Ribosome Subunits, Small, Eukaryotic/chemistry ; CRISPR-Cas Systems ; Humans ; Animals ; Mice ; *Nucleophosmin/genetics/physiology ; Sulfonamides ; Tumor Suppressor Protein p53 ; Bridged Bicyclo Compounds, Heterocyclic ; },
abstract = {NPM1 is a multifunctional phosphoprotein with key roles in ribosome biogenesis among its many functions. NPM1 gene mutations drive 30% of acute myeloid leukemia (AML) cases. The mutations disrupt a nucleolar localization signal and create a novel nuclear export signal, leading to cytoplasmic displacement of the protein (NPM1c). NPM1c mutations prime hematopoietic progenitors to leukemic transformation, but their precise molecular consequences remain elusive. Here, we first evaluate the effects of isolated NPM1c mutations on the global proteome of preleukemic hematopoietic stem and progenitor cells (HSPCs) using conditional knockin Npm1cA/+ mice. We discover that many proteins involved in ribosome biogenesis are significantly depleted in these murine HSPCs, but also importantly in human NPM1-mutant AMLs. In line with this, we found that preleukemic Npm1cA/+ HSPCs display higher sensitivity to RNA polymerase I inhibitors, including actinomycin D (ActD), compared with Npm1+/+ cells. Combination treatment with ActD and venetoclax inhibited the growth and colony-forming ability of preleukemic and leukemic NPM1c+ cells, whereas low-dose ActD treatment was able to resensitize resistant NPM1c+ cells to venetoclax. Furthermore, using data from CRISPR dropout screens, we identified and validated TSR3, a 40S ribosomal maturation factor whose knockout preferentially inhibited the proliferation of NPM1c+ AML cells by activating a p53-dependent apoptotic response. Similarly, to low-dose ActD treatment, TSR3 depletion could partially restore sensitivity to venetoclax in therapy-resistant NPM1c+ AML models. Our findings propose that targeted disruption of ribosome biogenesis should be explored as a therapeutic strategy against NPM1-mutant AML.},
}

*RNA Processing, Post-Transcriptional/drug effects/genetics
*Ribosomes/genetics/metabolism
*Leukemia, Myeloid, Acute/drug therapy/genetics/pathology
Cytoplasm
Dactinomycin/pharmacology
Nuclear Proteins/metabolism
Mice, Knockout
Hematopoietic Stem Cells
Ribosome Subunits, Small, Eukaryotic/chemistry
CRISPR-Cas Systems
Humans
Animals
Mice
*Nucleophosmin/genetics/physiology
Sulfonamides
Tumor Suppressor Protein p53
Bridged Bicyclo Compounds, Heterocyclic

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

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

@article {pmid41733973,
year = {2026},
author = {van der Meulen, SA and Roemhild, K and Driessen, M and van den Akker, E and Nethe, M},
title = {In vivo modeling of stress erythropoiesis through targeted gene editing of rat hematopoietic stem cells.},
journal = {Blood advances},
volume = {10},
number = {4},
pages = {1281-1292},
doi = {10.1182/bloodadvances.2025017433},
pmid = {41733973},
issn = {2473-9537},
mesh = {Animals ; *Erythropoiesis/genetics ; Rats ; *Gene Editing/methods ; *Hematopoietic Stem Cells/metabolism/cytology ; CRISPR-Cas Systems ; Anemia/etiology/genetics/metabolism ; Cadherins/genetics/metabolism ; Disease Models, Animal ; *Stress, Physiological ; },
abstract = {In response to anemia, the erythroid lineage significantly expands. This growth is driven by extramedullary erythropoiesis in mice, but is additionally regulated within the bone marrow (BM) of rats, a process likely conserved in humans due to similar BM architecture. This process is, however, mostly elusive. We identified E-cadherin to mark the expansion of the erythroid lineage in BM from anemic rats. To explore the regulation of erythropoiesis in the BM in response to anemia, we studied the role of E-cadherin in the erythroid lineage of rats. As genetic methods to model erythropoiesis in rats are limited, we established a rat BM transplant model that, combined with CRISPR/Cas9 genome editing, enabled us to examine the control of E-cadherin in BM in response to anemia. We identified CD90+CD44+CD45R- cells to contain hematopoietic stem and progenitor cells (HSPCs) in rats. CD90+CD44+CD45R--enriched HSPCs can be efficiently edited using CRISPR/Cas9, which, upon transplant, induce high BM chimerism. Importantly, we identified that recovery from irradiation-induced anemia involves 2 phases. Phase 1 is marked by expansion of erythroid precursors in the BM, supported by extramedullary erythropoiesis in the spleen. This phase is followed by a second phase, characterized by accelerated terminal differentiation, which is selectively controlled in the BM. Finally, we discovered that genetic inactivation of hematopoietic-expressed E-cadherin delays recovery from radiation-induced anemia. Our work provides novel means to expand our knowledge on hematology, and the opportunity to dissect the molecular regulation underlying the erythroid response(s) to anemia in BM, using rat models.},
}

Animals
*Erythropoiesis/genetics
Rats
*Gene Editing/methods
*Hematopoietic Stem Cells/metabolism/cytology
CRISPR-Cas Systems
Anemia/etiology/genetics/metabolism
Cadherins/genetics/metabolism
Disease Models, Animal
*Stress, Physiological

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

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

@article {pmid41732093,
year = {2026},
author = {Neupane, S and Pfrender, ME and Wang, L and Xu, S},
title = {Detection of CRISPR-Cas-induced mutations in Daphnia.},
journal = {G3 (Bethesda, Md.)},
volume = {},
number = {},
pages = {},
doi = {10.1093/g3journal/jkag050},
pmid = {41732093},
issn = {2160-1836},
abstract = {CRISPR-Cas9 has established itself as a robust tool for conducting loss of function gene research in emerging model species including the freshwater zooplankton Daphnia. However, sensitive detection of mutations, especially in genetic mosaic and pooled samples, remains a challenge. In this study we evaluate two of the most widely used mutation screening techniques, the T7 Endonuclease I (T7EI) assay and Fragment Analysis (FA) for their sensitivity, accuracy, and practical use in detecting CRISPR-induced indels in four targeted genes, DNMT3A, DNMT3B, PERIOD2, and DMRT1 in Daphnia magna. Here, we show that T7EI, although it offers a quick and cost-effective screening method, often produces false positives, especially when examining pooled samples. Conversely, FA facilitates detecting allele size differences at a fine resolution, reproducibility in detecting indels, and distinguishing zygosity and is more reliable as a method to detect mutation. Our comparative analyses convey the importance of carefully selecting the appropriate screening methods depending on research questions.},
}

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

@article {pmid41725298,
year = {2026},
author = {Chew, YP and Ferenczi, A and Dannay, M and Ponce-Lilly, C and Kovac, A and Tóth, D and Tóth, SZ and Molnar, A},
title = {Enhancing CRISPR/Cas-Mediated Gene Knockout With Short Non-Homologous Oligonucleotides.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/pbi.70548},
pmid = {41725298},
issn = {1467-7652},
support = {BB/W003538/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; //Darwin Trust of Edinburgh/ ; //UK Research and Innovation/ ; },
abstract = {Chlamydomonas reinhardtii is a model green microalga that has great industrial potential as a sustainable bio-factory for recombinant protein and high-value chemical production. Efficient genome editing tools are required to redesign this organism for synthetic biology applications. CRISPR-Cas editing technologies have already been adapted for gene knockout, transgene knock-in, and precise gene editing in C. reinhardtii. However, the efficacy of CRISPR/Cas-mediated gene knockout (KO) is low, which hampers pathway engineering and functional genomic studies. Here we report that co-delivery of CRISPR-Cas gene editing reagents with short double-stranded non-homologous oligodeoxynucleotides (dsNHO) increases gene knockout efficacy up to 100-fold in C. reinhardtii. This phenomenon, referred to as non-homologous oligonucleotide enhancement (NOE), is heavily affected by the length, structure, and chemical modifications of dsNHO, and is largely mediated by the DNA double-stranded break sensor KU70/80 (KU) heterodimer in a Cas nuclease-, locus-, and strain-independent manner. Our data suggest that dsNHOs disrupt the cell's double-stranded break (DSB) sensing pathways, consequently shifting the balance of DNA repair from canonical non-homologous end joining (c-NHEJ) towards the more error-prone, microhomology-mediated end joining (MMEJ), which could be harnessed as a strategy for improving gene KO efficiency in Chlamydomonas and beyond.},
}

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

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

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

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

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

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

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

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

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

@article {pmid41686483,
year = {2026},
author = {Tanaka, PP and Cotta-Almeida, V and Donadi, EA and Westerberg, L and Passos, GA},
title = {Distinct mutations in the autoimmune regulator gene differentially affect transcriptional and functional properties of medullary thymic epithelial cells.},
journal = {Human molecular genetics},
volume = {},
number = {},
pages = {},
doi = {10.1093/hmg/ddag004},
pmid = {41686483},
issn = {1460-2083},
support = {17/10780-4//São Paulo Research Foundation/ ; 311304/2021//National Council for Scientific and Technological Development/ ; 302060/2019-7//National Council for Scientific and Technological Development/ ; 88887.642780/2021-00//CAPES-STINT/ ; 001//Coordenação de Aperfeiçoamento de Pessoal de Nível Superior/ ; },
abstract = {Autoimmune Polyendocrine Syndrome Type 1 (APS-1) is a rare monogenic disorder caused by mutations in the autoimmune regulator (AIRE) gene. Although AIRE is essential for central immune tolerance, how distinct APS-1-associated mutations differentially affect medullary thymic epithelial cell (mTEC) biology remains incompletely understood. Here, we investigated the molecular and functional consequences of three Aire/AIRE variants using complementary murine mTEC models. To define transcriptional effects, we performed single-cell RNA sequencing (scRNA-seq) on mTECs carrying a heterozygous genomic Aire c.735delG mutation generated by CRISPR-Cas9. This analysis revealed reduced transcriptional heterogeneity, decreased expression of tissue-restricted antigens (TRAs) mRNAs (including Col4a3, Col7a1, and Neto2), and downregulation of key mTEC lineage markers (Epcam, Cldn4, Krt14). Mutant cells also displayed altered expression of mRNAs involved in chemokine-mediated migration (Ccl25, Cxcl16), extracellular matrix and cell adhesion (Fn1, Lama5, Col4a1, Nectin1, Cdh1), and actin cytoskeleton organization (Gsn, Rac1, Wasl, Actn1), indicating broad disruption of pathways governing mTEC identity and cell-cell interactions. Guided by these findings, we assessed mutation-specific functional outcomes using a CRISPR-derived Aire functional knockout and lentiviral expression of the human AIRE missense variants p.G229W and p.C313Y in wild-type mTECs. Functional assays revealed mutation-dependent alterations in mTEC morphology, thymocyte migration, and adhesion, with the p.C313Y variant exerting the strongest effects. Together, these data demonstrate that heterozygous and missense AIRE mutations exert distinct yet convergent effects on mTEC transcriptional programs and cellular behavior, providing mechanistic insight into AIRE-dependent immune tolerance failure in APS-1.},
}

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

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

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

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

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

@article {pmid41720308,
year = {2026},
author = {Shi, Q and Huang, W and Hu, D and Zhang, P and Chen, X and Hu, H and Wang, Y and Zhou, J and Weng, R and Quan, J and Zhao, D and Du, X and Yu, Y and Jiang, Y},
title = {The nationwide genomic characteristics and phylogenetic evolution of ST23-K1 hypervirulent Klebsiella pneumoniae in relation to virulence and antimicrobial resistance acquisition.},
journal = {The Journal of infection},
volume = {},
number = {},
pages = {106709},
doi = {10.1016/j.jinf.2026.106709},
pmid = {41720308},
issn = {1532-2742},
abstract = {OBJECTIVES: Hypervirulent Klebsiella pneumoniae (hvKp) ST23-K1 poses a global health threat due to its high virulence and increasing antimicrobial resistance. This study aimed to characterize the genomic feature and phylogenetic evolution of ST23-K1 in China.

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

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

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

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

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

@article {pmid41661055,
year = {2026},
author = {Bu, L and Yuan, A and Zhao, M and Pang, B and Li, J and Shang, Y and Xie, W and Peng, H},
title = {An Asymmetric Stem-Loop Translator Enhances Specificity of One-Pot Isothermal CRISPR/Cas12a Assay.},
journal = {Analytical chemistry},
volume = {98},
number = {7},
pages = {5513-5522},
doi = {10.1021/acs.analchem.5c07049},
pmid = {41661055},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; *MicroRNAs/genetics/analysis ; Limit of Detection ; Humans ; *CRISPR-Associated Proteins/metabolism ; Aptamers, Nucleotide/chemistry ; *Endodeoxyribonucleases/metabolism/genetics ; Bacterial Proteins ; },
abstract = {Sensitive and specific detection of molecular biomarkers is fundamental to clinical diagnostics and biomedical research, yet existing CRISPR-based assays often suffer from nonspecific activation and cross-reactivity. Here, we introduce an asymmetric stem-loop translator that markedly enhances the specificity of a one-pot isothermal strand displacement amplification (SDA)-CRISPR/Cas12a assay. The asymmetric probe enables precise molecular recognition and controlled signal transduction, converting diverse targets into amplifiable DNA intermediates. Within the integrated one-tube system, SDA-generated DNA products directly trigger Cas12a trans-cleavage, yielding rapid fluorescence responses without thermal cycling or manual intervention. Systematic optimization of reaction parameters effectively mitigated enzyme inhibition and aerosol contamination. The resulting assay achieves highly specific and sensitive detection of miRNA with a detection limit of 500 fM, accurately distinguishing single- and double-base mutations. Owing to its modular design, the asymmetric stem-loop translator is readily adaptable to other analytes. By coupling to an aptamer-based recognition element, the system enables sensitive aflatoxin B1 detection. This work establishes a generalizable framework for enhancing CRISPR/Cas12a specificity through asymmetric molecular translation, offering a versatile platform for rapid nucleic acid and small-molecule diagnostics in clinical and point-of-care settings.},
}

*CRISPR-Cas Systems/genetics
*Nucleic Acid Amplification Techniques/methods
*MicroRNAs/genetics/analysis
Limit of Detection
Humans
*CRISPR-Associated Proteins/metabolism
Aptamers, Nucleotide/chemistry
*Endodeoxyribonucleases/metabolism/genetics
Bacterial Proteins

@article {pmid41643679,
year = {2026},
author = {Ferrández-Roldán, A and Piulachs, MD},
title = {Using DIPA-CRISPR for simple and efficient endogenous protein tagging in insects.},
journal = {Cell reports methods},
volume = {6},
number = {2},
pages = {101297},
doi = {10.1016/j.crmeth.2025.101297},
pmid = {41643679},
issn = {2667-2375},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Insect Proteins/genetics/metabolism ; Genes, Reporter ; *Insecta/genetics ; *Blattellidae/genetics/metabolism ; },
abstract = {CRISPR-Cas9 is rapidly expanding across diverse organisms. Among these advances, in-frame knockins of reporter genes have become essential for studying gene expression and protein localization. However, in hemimetabolan insects such as the German cockroach Blattella germanica, a phylogenetically basal and relevant pest species, functional fusion proteins have remained technically difficult to obtain. We present a streamlined gene-editing strategy to knock in a reporter gene in-frame with the distal-less gene, generating a functional fusion protein in B. germanica. By combining direct parental CRISPR with donor constructs designed for homology-directed repair carrying the mCherry gene, we successfully achieved targeted integration at the distal-less locus. The resulting fusion protein was functional and heritable and enabled live visualization of Distal-less protein distribution, showing fluorescence in developing appendages and the nervous system. This simple and robust methodology opens the door to generating fusion proteins in non-model insects, providing a valuable molecular tool for ecological, developmental, and pest-management research.},
}

Animals
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Insect Proteins/genetics/metabolism
Genes, Reporter
*Insecta/genetics
*Blattellidae/genetics/metabolism

@article {pmid41247804,
year = {2026},
author = {Li, A and Yang, C and Zhao, Z and Lu, T and Yin, C and Xu, C and Lv, C and Liu, Y and Zhan, Y and Li, S and Cao, C},
title = {Strategic engineering of DNA aptamers as precision modulators in CRISPR-driven oncogene control.},
journal = {International journal of surgery (London, England)},
volume = {112},
number = {2},
pages = {3015-3027},
doi = {10.1097/JS9.0000000000003837},
pmid = {41247804},
issn = {1743-9159},
mesh = {*Aptamers, Nucleotide/genetics ; *CRISPR-Cas Systems/genetics ; Humans ; Animals ; Mice ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Oncogenes/genetics ; Cell Line, Tumor ; *Neoplasms/genetics/therapy ; *CRISPR-Associated Protein 9/genetics ; Gene Editing/methods ; *Genetic Therapy/methods ; },
abstract = {OBJECTIVE: The goal of this study was to address the limited availability of intracellular protein-binding aptamers that restrict the potential of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based signal conductor systems. We aimed to develop a programmable CRISPR-SaCas9 platform using DNA aptamers to achieve precise and dynamic gene regulation for cancer therapy.

METHODS: We identified and characterized five DNA aptamers with high specificity for the SaCas9 protein. Functional assays were performed to evaluate their effects on the transcriptional regulatory activity of SaCas9. Some aptamers were engineered to associate with promoter elements, enabling transcription factor-responsive control of downstream gene expression. To test therapeutic efficacy, the system was delivered in vivo using In vivo-jetPEI reagents, and tumor progression and metastasis were monitored.

RESULTS: The identified aptamers exhibited distinct functionalities - some partially inhibited SaCas9 activity, while others enabled precise gene expression control in response to endogenous transcription factors. The aptamer-CRISPR complexes formed ternary assemblies with SaCas9 and single-guide RNAs, enabling logic-gated responses. In vivo , the system significantly suppressed tumor growth and metastasis.

CONCLUSION: We developed a modular CRISPR signal conductor platform that leverages SaCas9-binding DNA aptamers for transcription factor-mediated logic operations and precise gene control. This system expands the toolkit for genetic circuit design and offers a promising strategy for targeted cancer gene therapy.},
}

*Aptamers, Nucleotide/genetics
*CRISPR-Cas Systems/genetics
Humans
Animals
Mice
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*Oncogenes/genetics
Cell Line, Tumor
*Neoplasms/genetics/therapy
*CRISPR-Associated Protein 9/genetics
Gene Editing/methods
*Genetic Therapy/methods

@article {pmid41224955,
year = {2026},
author = {Gil, JS and Lee, S and Koo, T},
title = {Therapeutic in vivo genome editing: innovations and challenges in rAAV vector-based CRISPR delivery.},
journal = {Gene therapy},
volume = {33},
number = {1},
pages = {97-106},
pmid = {41224955},
issn = {1476-5462},
mesh = {*Dependovirus/genetics ; *Gene Editing/methods ; *Genetic Vectors/genetics ; *Genetic Therapy/methods ; Humans ; *CRISPR-Cas Systems ; Animals ; Gene Transfer Techniques ; },
abstract = {The integration of CRISPR systems with recombinant adeno-associated virus (rAAV) vectors has opened new possibilities for therapeutic genome editing, offering potential treatments for both genetic and non-genetic disorders. rAAV vectors have emerged as promising vehicles for in vivo gene therapy due to their favorable safety profile, high tissue specificity, and ability to induce sustained transgene expression. However, their limited packaging capacity has been a significant challenge for delivering large CRISPR molecules. To overcome this limitation, innovative strategies have been developed, including the use of compact Cas orthologs, dual rAAV vector systems, and trans-splicing rAAV vectors. These approaches have significantly improved the efficiency of genome editing for therapeutic applications. This review presents recent advancements in rAAV-CRISPR-mediated in vivo gene therapy, highlighting key technological innovations, current challenges, and the therapeutic potential of these strategies in the development of next-generation gene therapies.},
}

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

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

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

@article {pmid41713039,
year = {2026},
author = {Zhang, H and Cui, C and Wang, X and Liu, S and Wang, X and Wang, Y and Ge, S and Cai, Y and Bao, J and Wang, Z},
title = {Development of a one-pot RT-RAA/CRISPR-Cas13a assay for rapid genotyping of Nipah virus in pigs.},
journal = {Diagnostic microbiology and infectious disease},
volume = {115},
number = {2},
pages = {117316},
doi = {10.1016/j.diagmicrobio.2026.117316},
pmid = {41713039},
issn = {1879-0070},
abstract = {INTRODUCTION: Nipah virus (NiV) is a highly pathogenic zoonotic virus transmitted from bats to humans through pigs as a key intermediate host. Given the existence of two distinct NiV genotypes, which differ in clinical manifestations and transmission patterns in both humans and pigs, rapid and sensitive method for detection and genotyping is crucial for effective disease control. Isothermal amplification combined with CRISPR/Cas-based assay provides a promising approach to meet this need.

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

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

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

@article {pmid41712626,
year = {2026},
author = {Sünderhauf, D and Ringger, JR and Payne, LJ and Pinilla-Redondo, R and Gaze, WH and Brown, SP and van Houte, S},
title = {CRISPR-Cas is beneficial in plasmid competition, but limited by competitor toxin-antitoxin activity when horizontally transferred.},
journal = {PLoS biology},
volume = {24},
number = {2},
pages = {e3003658},
doi = {10.1371/journal.pbio.3003658},
pmid = {41712626},
issn = {1545-7885},
abstract = {Bacteria can encode dozens of different immune systems that protect them from infection by mobile genetic elements (MGEs). MGEs themselves may also carry immune systems, such as CRISPR-Cas, to target competitor MGEs. It is unclear when this is favored by natural selection, and whether toxin-antitoxin (TA) systems-common competitive mechanisms carried by plasmids-can alter their efficacy. Here, we develop and test novel theory to analyze the outcome of competition between plasmids when one carries a CRISPR-Cas system that targets the other plasmid. Our mathematical model and experiments using Escherichia coli and competing IncP plasmids reveal that plasmid-borne CRISPR-Cas is beneficial to the plasmid carrying it when the plasmid has not recently transferred to a new host. However, CRISPR-Cas is selected against when the plasmid carrying it transfers horizontally, if a resident competitor plasmid encodes a TA system that elicits post-segregational killing. Consistent with a TA barrier to plasmid-borne CRISPR-Cas, a bioinformatic analysis reveals that naturally occurring CRISPR-Cas-bearing plasmids avoid targeting other plasmids with TA systems across bacterial genera. Our work shows how the benefit of plasmid-borne CRISPR-Cas is severely reduced against TA-encoding competitor plasmids, but only when plasmid-borne CRISPR-Cas is horizontally transferred. These findings have key implications for the distribution of prokaryotic defenses and our understanding of their role in competition between MGEs, and the utility of CRISPR-Cas as a tool to remove plasmids from pathogenic bacteria.},
}

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

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

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

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

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

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

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

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

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

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

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

@article {pmid41707086,
year = {2026},
author = {Banerjee, S and Banerjee, A and Ray, S and Ray, A and Paul, D and Dastidar, SG and Willard, B and Biswas, K},
title = {dCas9 Targeted Proteome Profiling Reveals p300-Mediated Reciprocal Regulation of SMAD and SP1 as a Driver of GM2-synthase Transcription in Renal Cell Carcinoma.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {40},
number = {4},
pages = {e71597},
doi = {10.1096/fj.202502746R},
pmid = {41707086},
issn = {1530-6860},
support = {2019-0137-CMB/adhoc/BMS//MOHFW | DHR | Indian Council of Medical Research (ICMR)/ ; EMR/2016/001983//Department of Science and Technology, Ministry of Science and Technology, India (DST)/ ; CRG/2021/004623//Department of Science and Technology, Ministry of Science and Technology, India (DST)/ ; },
mesh = {Humans ; *Carcinoma, Renal Cell/genetics/metabolism/pathology ; *Sp1 Transcription Factor/metabolism/genetics ; *Kidney Neoplasms/genetics/metabolism/pathology ; *Smad Proteins/metabolism/genetics ; *Sialyltransferases/genetics/metabolism ; Cell Line, Tumor ; *E1A-Associated p300 Protein/metabolism/genetics ; *Proteome/metabolism/genetics ; Gene Expression Regulation, Neoplastic ; Transcription, Genetic ; CRISPR-Cas Systems ; *p300-CBP Transcription Factors/metabolism ; CRISPR-Associated Protein 9/genetics/metabolism ; },
abstract = {Glycolipids constitute an important component of the plasma membrane based on both abundance as well as function. Gangliosides, being a class of structurally diverse and functionally varied glycolipids, can act both as a receptor as well as a ligand and therefore are established as a crucial player in several normal cellular processes. In certain diseases, and in particular cancer, select gangliosides are over-expressed often leading to disease manifestation. GM2-synthase, the enzyme responsible for the formation of a pro-tumorigenic ganglioside, GM2, is well reported to be over-expressed across various cancer tissues and cell lines. This over-expression of GM2-synthase has been linked with increased migration, invasion, and epithelial to mesenchymal transition (1) as well as induction of a local and systemic host immune suppression in cancer. Despite only a handful of studies demonstrating an epigenetic regulation underlying the transcriptional regulation of the GM2-synthase (B4GalNT1) gene, the detailed mechanism still remains unclear. Here we identified the total proteome associated with the GM2-synthase promoter through a two-step CRISPR-dCas9 based proteome profiling approach by categorizing all the identified proteins leading to a detailed elucidation of the molecular drivers behind GM2-synthase transcription. While the previous study identified an acetylation-dependent de-repression of the transcription factor SP1 causing GM2-synthase activation, the underlying molecular mechanism driving its activation wasn't clear. This study demonstrated that the histone acetyl transferase p300, acts as a pivotal factor which on one hand causes acetylation-mediated degradation of SP1, and on the other hand activates SMAD2/4 to have a direct positive impact on GM2-synthase gene transcription. We identified p300 to have an activator role in GM2-synthase gene transcription through knock out, knock down, and over-expression experiments. Furthermore, SP1 degradation, SMAD activation, and their DNA binding patterns show the reciprocal role of p300 on SP1 and SMAD complexes. Altogether we have identified SMAD2/4 as an activator complex, p300 as a positive regulator, and uncovered a critical p300-SMAD-SP1 regulatory axis in GM2-synthase transcriptional regulation.},
}

Humans
*Carcinoma, Renal Cell/genetics/metabolism/pathology
*Sp1 Transcription Factor/metabolism/genetics
*Kidney Neoplasms/genetics/metabolism/pathology
*Smad Proteins/metabolism/genetics
*Sialyltransferases/genetics/metabolism
Cell Line, Tumor
*E1A-Associated p300 Protein/metabolism/genetics
*Proteome/metabolism/genetics
Gene Expression Regulation, Neoplastic
Transcription, Genetic
CRISPR-Cas Systems
*p300-CBP Transcription Factors/metabolism
CRISPR-Associated Protein 9/genetics/metabolism

@article {pmid41706678,
year = {2026},
author = {Verhezen, T and Van Den Eynde, A and Verstraelen, P and Gehrcken, L and Palmiotto, G and Lau, HW and De Vos, WH and Van Der Heijden, S and Brants, L and Melis, J and Van Audenaerde, J and Rodrigues Fortes, F and Le Compte, M and Roeyen, G and Prenen, H and Campillo-Davo, D and Lion, E and Argüello, RJ and Van Laere, S and Lardon, F and Deben, C and Wouters, A and Smits, E and De Waele, J},
title = {DRP1 depletion protects NK cells from hypoxia-induced dysfunction.},
journal = {Redox report : communications in free radical research},
volume = {31},
number = {1},
pages = {2626181},
pmid = {41706678},
issn = {1743-2928},
mesh = {*Killer Cells, Natural/metabolism ; Humans ; *Dynamins/metabolism/genetics ; Reactive Oxygen Species/metabolism ; Mitochondria/metabolism ; Cell Line, Tumor ; Cell Hypoxia ; Membrane Potential, Mitochondrial ; *Hypoxia/metabolism ; CRISPR-Cas Systems ; Tumor Microenvironment ; },
abstract = {OBJECTIVES: The efficacy of cellular therapies has been disappointing in solid tumors. A major barrier that contributes to the low success rate, is hypoxia within the tumor microenvironment. In this study, we investigated the influence of hypoxia on natural killer (NK) cell function and to evaluated a strategy to restore their activity in hypoxia.

METHODS: Unarmed or CAR NK cells were placed in normoxia (21% O2) or hypoxia (1% O2) prior to experimental readouts. Mitochondrial content and morphology were assessed by confocal microscopy, membrane potential and reactive oxygen species (ROS) by flow cytometry, and global transcriptional changes by RNA sequencing. Cytotoxicity was evaluated against tumor cell lines and patient-derived cancer organoids, which were characterized by RNA sequencing. DRP1 function was inhibited pharmacologically or through CRISPR-Cas9-mediated knockout.

RESULTS: Hypoxia reduced NK cell mitochondrial content and membrane potential, while increasing mitochondrial ROS and inducing broad transcriptional changes in stress response pathways. Their cytotoxic activity was drastically impaired, which could not be prevented by CD70-CAR-IL-15 engineering. Pharmacological inhibition of DRP1 restored mitochondrial content and cytotoxic function. To confirm the role of DRP1, CRISPR-Cas9-mediated DRP1 knockout (KO) NK cells preserved mitochondrial load and membrane potential under hypoxia, and DRP1[KO] CAR NK cells retained cytotoxic activity under hypoxic conditions against cancer cell lines. Patient microtumor models with distinct transcriptomic profiles exhibited divergent responses to DRP1[WT] and DRP1[KO] CAR NK cells.

CONCLUSION: These findings indicate that DRP1 inactivation supports NK cell function in hypoxia and metabolic engineering may enhance CAR-NK efficacy in solid tumors.},
}

*Killer Cells, Natural/metabolism
Humans
*Dynamins/metabolism/genetics
Reactive Oxygen Species/metabolism
Mitochondria/metabolism
Cell Line, Tumor
Cell Hypoxia
Membrane Potential, Mitochondrial
*Hypoxia/metabolism
CRISPR-Cas Systems
Tumor Microenvironment

@article {pmid41644992,
year = {2026},
author = {Oishi, S and Doi, S and Sekida, T and Yamashita, K and Yamada, Y and Muramoto, T},
title = {Genome editing across Dictyostelia species enables comparative functional genetics of social amoebas.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {41644992},
issn = {2045-2322},
support = {23KJ1977//Japan Society for the Promotion of Science/ ; 23K05785//Japan Society for the Promotion of Science/ ; },
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems ; *Dictyostelium/genetics ; *Genome, Protozoan ; Phylogeny ; },
abstract = {Gene manipulation is essential for understanding biological mechanisms, yet genetic modification in the social amoebas (Dictyostelia) has been largely limited to Dictyostelium discoideum. Here, we aimed to establish a CRISPR/Cas9-based genome-editing system applicable across the phylogenetic breadth of Dictyostelia, spanning Groups 1-4. Using an extrachromosomal CRISPR/Cas9 vector from D. discoideum, we disrupted stlA and pkaC in Polysphondylium violaceum and pkaC in two early-branching species, Heterostelium pallidum and Cavenderia fasciculata. In D. discoideum, co-introduction of donor oligos with the CRISPR vector enabled selection-free knockout generation of pkaC with 28.6% efficiency. In H. pallidum, where genome editing is typically inefficient, co-electroporation of donor oligos with the CRISPR/Cas9 vector followed by 4 days of drug selection increased the frequency of pkaC disruption from 0.9% to 8.3%. These results demonstrated that the D. discoideum CRISPR/Cas9 system can be extended across Dictyostelia, providing a versatile platform for comparative genetic and evolutionary developmental studies.},
}

*Gene Editing/methods
CRISPR-Cas Systems
*Dictyostelium/genetics
*Genome, Protozoan
Phylogeny

@article {pmid41554885,
year = {2026},
author = {Zhang, L and Cai, C and Chen, Q and Tan, X and Chen, S and Zhang, K and Cheng, F},
title = {A CRISPR-based sequence proximity binding protein labelling system for scanning upstream regulatory proteins.},
journal = {Nature plants},
volume = {12},
number = {2},
pages = {277-283},
pmid = {41554885},
issn = {2055-0278},
support = {31972411//National Science Foundation of China | National Natural Science Foundation of China-Yunnan Joint Fund (NSFC-Yunnan Joint Fund)/ ; 32302570//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Arabidopsis/genetics/metabolism ; Oryza/genetics/metabolism ; Promoter Regions, Genetic ; *DNA-Binding Proteins/genetics/metabolism ; Brassica/genetics/metabolism ; Gene Expression Regulation, Plant ; *CRISPR-Cas Systems ; *Plant Proteins/genetics/metabolism ; *Transcription Factors/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Arabidopsis Proteins/genetics/metabolism ; },
abstract = {Transcriptional regulation involves complex and dynamic protein-DNA interactions, which alter chromatin states and, consequently, regulate gene expression. In plants, current technologies face challenges in efficiently capturing dynamically DNA-binding proteins, especially transcription factors. Here, by leveraging the binding ability of dead Cas9 to specific DNA fragments and the labelling capacity of the TurboID protein for adjacent proteins, we have developed a CRISPR-based sequence proximity binding protein labelling system (CSPL) to detect promoter-binding proteins. Using this approach, we identified both known and novel upstream binding proteins on the PIF4 promoter in Arabidopsis, cabbage and rice. This demonstrates the powerful capabilities and broad potential applications of CSPL for detecting promoter-binding proteins in plants.},
}

*Arabidopsis/genetics/metabolism
Oryza/genetics/metabolism
Promoter Regions, Genetic
*DNA-Binding Proteins/genetics/metabolism
Brassica/genetics/metabolism
Gene Expression Regulation, Plant
*CRISPR-Cas Systems
*Plant Proteins/genetics/metabolism
*Transcription Factors/genetics/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats
Arabidopsis Proteins/genetics/metabolism

@article {pmid41354630,
year = {2026},
author = {Wang, W and Chen, K and Wang, Z},
title = {Genome-Wide CRISPR Screen Reveals PIK3CA Inhibition Enhances Lipid Nanoparticle-Mediated siRNA Delivery.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {11},
pages = {e17617},
doi = {10.1002/advs.202517617},
pmid = {41354630},
issn = {2198-3844},
support = {//International Society for Advancement of Cytometry/ ; //Chan Zuckerberg Biohub/ ; //Chan Zuckerberg Initiative/ ; },
mesh = {Humans ; *RNA, Small Interfering/genetics/administration & dosage ; *Nanoparticles/chemistry ; *Class I Phosphatidylinositol 3-Kinases/genetics/antagonists & inhibitors/metabolism ; Animals ; Mice ; Cell Line, Tumor ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Lipids/chemistry ; CRISPR-Cas Systems/genetics ; Liposomes ; },
abstract = {Lipid nanoparticles (LNPs) are useful carriers for therapeutic siRNA delivery, yet their clinical efficacy remains constrained by insufficient cellular uptake. Here, using a genome-wide CRISPR knockout screen, multiple genetic modulators of LNP uptake is uncovered, with PIK3CA emerging as a top druggable target. Pharmacologic inhibition of PIK3CA with BAY1082439 - a clinically evaluated small molecule - significantly enhances LNP uptake, siRNA delivery, and gene silencing across diverse epithelial cancer cell lines in vitro. Co-administration of BAY1082439 with siRNA-loaded LNPs also better suppressed tumor growth and reduced liver inflammation in vivo, respectively. These findings establish PIK3CA inhibition as a broadly applicable strategy to boost LNP-mediated RNA interference and highlight the promise of combining functional genomics with nanomaterials to advance RNA-based therapeutics.},
}

Humans
*RNA, Small Interfering/genetics/administration & dosage
*Nanoparticles/chemistry
*Class I Phosphatidylinositol 3-Kinases/genetics/antagonists & inhibitors/metabolism
Animals
Mice
Cell Line, Tumor
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Lipids/chemistry
CRISPR-Cas Systems/genetics
Liposomes

@article {pmid41705810,
year = {2026},
author = {Shin, K and Kim, ET},
title = {Viral genome editing methods and applications in the CRISPR era.},
journal = {Journal of virology},
volume = {},
number = {},
pages = {e0204825},
doi = {10.1128/jvi.02048-25},
pmid = {41705810},
issn = {1098-5514},
abstract = {CRISPR-Cas systems have transformed viral genetics by enabling precise and efficient manipulation of large DNA virus genomes. This review provides a practical framework for applying CRISPR technology to herpesviruses and other large DNA viruses as an alternative and complement to traditional BAC recombination. Key considerations include nuclease choice; sgRNA design that minimizes cut-to-edit distance and prevents re-cutting; donor template configuration and homology arm length; and synchronized delivery of Cas complexes and donor DNA. Strategies to promote HDR efficiency, such as the use of small-molecule modulators, are also summarized. In addition, practical workflows for clone selection, genotypic validation, and phenotypic confirmation are summarized. Case studies in herpes simplex virus type 1 and human cytomegalovirus illustrate how optimized CRISPR designs achieve reproducible, scarless knock-ins and conditional gene manipulation at essential loci without complementing cell lines. Together, these approaches establish CRISPR as a flexible, scalable platform for functional genomics, antiviral target discovery, and translational virology, enabling direct editing of clinical isolates previously inaccessible with bacterial artificial chromosome-based methods.},
}

@article {pmid41705504,
year = {2026},
author = {Sun, K and Wu, H},
title = {A highly-efficient isothermal nano-detection platform coupling CRISPR/Cas technology for detection of circRNA.},
journal = {The Analyst},
volume = {},
number = {},
pages = {},
doi = {10.1039/d6an00107f},
pmid = {41705504},
issn = {1364-5528},
abstract = {Triple-negative breast cancer (TNBC), an aggressive molecular subtype of breast cancer with poor prognosis, is characterized by a high rate of metastasis and proliferation, which makes early detection particularly challenging. Early diagnosis of TNBC through biomarkers and prompt development of treatment methods can lower its mortality rate. This work has designed a nano-detection platform for TNBC biomarker circRNA based on the CRISPR/Cas system and isothermal amplification strategy. Specifically, this detection system uses functional nucleic acid molecules for recognition of circCD44, as well as dual signal amplification using Klenow(3'-5'exo-) and Cas9n. Furthermore, it combines Cas12a and immunomagnetic beads for an extra signal boost and output. After confirming its feasibility and optimizing the conditions, the detection system achieved a 1.73-fold enhancement in sensitivity, offering a linear detection range of 1 pM to 100 nM, with the limit of detection lowered to 95.1 fM. It also showed good specificity through testing against 5 biomarkers. Therefore, this detection system provides a novel strategy for the early diagnosis of TNBC and other diseases.},
}

@article {pmid41705235,
year = {2026},
author = {Li, Z and Yu, T and Ge, L and Lv, S and Fu, Q and Shi, H},
title = {Genome-wide CRISPR/Cas9 screening identifies host factors critical for antiviral defense against equine herpesvirus type 1.},
journal = {Frontiers in immunology},
volume = {17},
number = {},
pages = {1764863},
pmid = {41705235},
issn = {1664-3224},
mesh = {Animals ; *CRISPR-Cas Systems ; *Herpesvirus 1, Equid/physiology/immunology ; Virus Replication/genetics ; Cell Line ; *Host-Pathogen Interactions/genetics/immunology ; *Herpesviridae Infections/immunology/virology/genetics/veterinary ; Horses ; Gene Knockout Techniques ; *Horse Diseases/virology/immunology/genetics ; Genome-Wide Association Study ; },
abstract = {INTRODUCTION: Equine herpesvirus type 1 (EHV-1) is a major veterinary pathogen causing significant economic losses in the livestock industry. Despite its impact, effective vaccines and targeted antiviral strategies remain limited, largely due to an incomplete understanding of host factors regulating viral replication and pathogenesis.

METHODS: To systematically identify host genes essential for EHV-1 infection, we established a BHK-21 cell line stably expressing Cas9 and performed a genome-wide CRISPR/Cas9 knockout screen using a pooled lentiviral single-guide RNA library. Significantly enriched candidate genes from positive selection were validated by generating knockout cell lines. Viral replication and protein expression were assessed using quantitative polymerase chain reaction and Western blot analysis. Pathway enrichment and protein interaction network analyses were subsequently conducted.

RESULTS: Genome-wide CRISPR/Cas9 screening identified multiple host factors critical for EHV-1 replication. Pathway enrichment analysis revealed that these genes were involved in key cellular signaling and regulatory networks associated with viral infection. Functional validation demonstrated that knockout of selected host genes significantly suppressed EHV-1 replication and viral protein synthesis.

DISCUSSION: These findings highlight essential host determinants required for EHV-1 replication and suggest that targeting host factors may represent a promising strategy for antiviral intervention. This study provides a foundation for the development of host-directed immunotherapeutic and antiviral approaches against EHV-1 infection.},
}

Animals
*CRISPR-Cas Systems
*Herpesvirus 1, Equid/physiology/immunology
Virus Replication/genetics
Cell Line
*Host-Pathogen Interactions/genetics/immunology
*Herpesviridae Infections/immunology/virology/genetics/veterinary
Horses
Gene Knockout Techniques
*Horse Diseases/virology/immunology/genetics
Genome-Wide Association Study

@article {pmid41704956,
year = {2026},
author = {Wang, Y and Diao, Y and Zhang, T and Zhang, F and Wang, W},
title = {Sensitive, specific, and rapid on-site detection of calf diarrhea pathogens using the RPA-CRISPR/Cas 12a assay.},
journal = {Frontiers in cellular and infection microbiology},
volume = {16},
number = {},
pages = {1734185},
pmid = {41704956},
issn = {2235-2988},
mesh = {Animals ; Cattle ; Sensitivity and Specificity ; *Diarrhea/veterinary/diagnosis/virology ; *CRISPR-Cas Systems ; *Cattle Diseases/diagnosis/virology/microbiology ; *Molecular Diagnostic Techniques/methods ; Diarrhea Viruses, Bovine Viral/isolation & purification/genetics ; *Nucleic Acid Amplification Techniques/methods ; Enterotoxigenic Escherichia coli/isolation & purification/genetics ; },
abstract = {Calf diarrhea is a common gastrointestinal disease that usually occurs within one month of birth. The disease causes the greatest economic losses to the cattle industry. Currently, a variety of diagnostic methods have been developed for calf diarrhea infections. However, existing methods are still unsatisfactory in terms of sensitivity, specificity, simplicity, cost, and speed.To provide a more sensitive, specific, simpler, and faster detection method, we recently developed an RPA-CRISPR/Cas12a assay that can detect BVDV, BCoV, BRV, and ETEC infections in cattle on-site. Testing for each pathogen is performed in a single test tube, without the need to open the tube in the middle, and can be completed in under 50 minutes.The RPA-CRISPR/Cas12a assay can detect BVDV, BCoV, BRV, and ETEC at concentrations of at least 10 copies/μL. The RPA-CRISPR/Cas12a assay does not produce false-positive results due to the presence of other pathogens. The sensitivity of BCoV, BRV, and ETEC in the RPA-CRISPR/Cas12a quadruple assay is equivalent to that of single qPCR. The sensitivity of BVDV in the quadruple assay is slightly lower than that of the single qPCR method.Due to its sensitivity, specificity, simplicity, and rapidity, the RPA-CRISPR/Cas12a assay is more practical for on-site detection of cattle diarrhea pathogens than any existing detection method.},
}

Animals
Cattle
Sensitivity and Specificity
*Diarrhea/veterinary/diagnosis/virology
*CRISPR-Cas Systems
*Cattle Diseases/diagnosis/virology/microbiology
*Molecular Diagnostic Techniques/methods
Diarrhea Viruses, Bovine Viral/isolation & purification/genetics
*Nucleic Acid Amplification Techniques/methods
Enterotoxigenic Escherichia coli/isolation & purification/genetics

@article {pmid41704216,
year = {2026},
author = {Khweis, S and Blackburn, M and Perdigao, C and Pierce, M and Lewis, C and Dunkle, J},
title = {Cas10 residues lining the target RNA binding channel regulate interference by distinguishing cognate target RNA from mismatched targets.},
journal = {RNA biology},
volume = {},
number = {},
pages = {},
doi = {10.1080/15476286.2026.2633385},
pmid = {41704216},
issn = {1555-8584},
abstract = {Type III CRISPR systems are defined by the presence of the Cas10 protein and are among the most abundant CRISPR systems in nature. Cas10 forms a complex with crRNA and several Cas proteins that surveils prokaryotic cells for foreign RNA molecules and when they are detected it activates a cascade of interference activities. The synthesis of the cyclic oligoadenylate signalling molecule by Cas10 is a key aspect of the interference cascade. Despite structures of the Cas10 complex bound to target RNAs, the molecular mechanism by which Cas10 senses the bound state to licence interference is lacking. We identified five residues in S. epidermidis Cas10, two in the Cas10 Palm2 domain and three in domain 4, that line the target RNA binding channel. We assessed the contribution of these residues to interference in the context of a cognate or mismatched target RNA. We found that the residues regulate whether a mismatched crRNA-target RNA duplex is able to activate interference in vivo. We purified two site-directed mutants of Cas10-Csm and show with in vitro cOA synthesis assays that they demonstrate enhanced discrimination of cognate versus mismatched target RNAs.},
}

@article {pmid41679604,
year = {2026},
author = {Li, L and Wang, Y and Wang, B and Shen, L and Gao, Y and Lin, W and Li, Z},
title = {A dCas9-integrated iLight9O system enables dynamic regulation for enhanced patchoulol biosynthesis in Saccharomyces cerevisiae.},
journal = {Bioresource technology},
volume = {446},
number = {},
pages = {134195},
doi = {10.1016/j.biortech.2026.134195},
pmid = {41679604},
issn = {1873-2976},
mesh = {*Saccharomyces cerevisiae/metabolism/genetics ; Metabolic Engineering/methods ; *CRISPR-Cas Systems/genetics ; Optogenetics/methods ; Light ; },
abstract = {Numerous organisms have evolved the ability to utilize light through photoreceptor proteins that mediate diverse biological processes. Currently, several optogenetic sensor systems are widely used in yeast. However, when these systems are applied for gene repression to regulate endogenous yeast gene expression, they typically require the insertion of corresponding target sites near the native promoter of the gene of interest to achieve precise modulation. To address these constraints, a novel blue light-inducible optogenetic tool designated iLight9 was developed, a single-component optogenetic biosensor integrated with the CRISPR-dCas9 platform. The stability of the iLight9 system was further enhanced by employing a strategy involving the addition of a protein degradation tag. The resulting system was designated as iLight9O, which facilitated programmable regulation of distinct genes through the introduction of specific sgRNAs. Subsequently, systematic metabolic engineering strategies were employed to construct an efficient patchoulol-producing cell factory in Saccharomyces cerevisiae. Moreover, a two-step isoprenol utilization (IU) pathway was introduced into the recombinant strain to enhance its capacity for patchoulol biosynthesis. Crucially, the iLight9O system was adopted to dynamically downregulate squalene synthase, a key enzyme in the competing squalene biosynthetic pathway. This optogenetic flux control strategy increased patchoulol titers by 66 % in the IU-optimized strain and 24 % in the MVAIU2 strain, demonstrating significant improvements over static engineering approaches.},
}

*Saccharomyces cerevisiae/metabolism/genetics
Metabolic Engineering/methods
*CRISPR-Cas Systems/genetics
Optogenetics/methods
Light

@article {pmid41672287,
year = {2026},
author = {Chaumont, L and Peruzzi, M and Huetz, F and Raffy, C and Le Hir, J and Minke, J and Leong, JC and Boudinot, P and Collet, B},
title = {Knockout of the antiviral genes mx1 or mx3 modulates the expression of paralogous genes in a salmonid cell line.},
journal = {Fish & shellfish immunology},
volume = {171},
number = {},
pages = {111204},
doi = {10.1016/j.fsi.2026.111204},
pmid = {41672287},
issn = {1095-9947},
mesh = {Animals ; *Myxovirus Resistance Proteins/genetics/immunology ; Cell Line ; *Fish Proteins/genetics/immunology ; Gene Knockout Techniques/veterinary ; *Fish Diseases/immunology ; *Immunity, Innate/genetics ; *Salmo salar/genetics/immunology ; *Gene Expression Regulation/immunology ; CRISPR-Cas Systems ; },
abstract = {Mx dynamin-like GTPases genes are Interferon Stimulated Genes (ISGs) encoding the Mx (myxovirus resistance) proteins that have an antiviral effect against a wide array of RNA viruses as well as some DNA viruses. In salmonids, the mx genes reside in four distinct chromosomal loci, that encode proteins clustering together into separate phylogenetic clades. To understand the contribution of Mx to the innate antiviral resistance, we knocked out the mx1 or mx3 gene by generating single mutant CHSE-derived cell lines by CRISPR/Cas9 genome editing. sgRNA were designed within the first coding exon of mx1 (LOC112247236), or mx3 (LOC112247235) genes, located 27 kb apart on the same chromosome LG02. Mx1, Mx2 and Mx3 proteins were quantified by western blotting. Only the Mx3 protein was found induced in the wild type EC cells after stimulation with recombinant Atlantic salmon interferon A2. mx1 gene knockout reduced the up-regulation of Mx3 protein expression, while mx3 gene knockout resulted in the induction of Mx1 and Mx2 proteins with and without additional stimulation. This was observed at the transcriptional level with the induction of mx1 gene was increased in mx3[-/-] cells compared to wild type cells. These results illustrate the complex interplay between mx1 and mx3 genes in salmonids.},
}

Animals
*Myxovirus Resistance Proteins/genetics/immunology
Cell Line
*Fish Proteins/genetics/immunology
Gene Knockout Techniques/veterinary
*Fish Diseases/immunology
*Immunity, Innate/genetics
*Salmo salar/genetics/immunology
*Gene Expression Regulation/immunology
CRISPR-Cas Systems

@article {pmid41671823,
year = {2026},
author = {Wang, Z and Yan, R and Lin, P and Yao, Y and He, H and Zhuang, W and Hu, J and Chen, Y and Xu, S and Hu, WW},
title = {CRISPR/Cas12a-nanozyme visual biosensor for detection of microRNA-21.},
journal = {Talanta},
volume = {303},
number = {},
pages = {129517},
doi = {10.1016/j.talanta.2026.129517},
pmid = {41671823},
issn = {1873-3573},
mesh = {*MicroRNAs/analysis/genetics ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; Humans ; DNA, Single-Stranded/chemistry ; Limit of Detection ; *Endodeoxyribonucleases/metabolism/chemistry ; *CRISPR-Associated Proteins/metabolism ; *Bacterial Proteins/metabolism ; Nucleic Acid Amplification Techniques ; Silver/chemistry ; },
abstract = {The precise diagnosis of aggressive tumors is crucial for guiding therapy, with microRNAs (miRNAs) increasingly recognized as pivotal biomarkers due to their regulatory roles in tumor development and dissemination. Herein, we report a CRISPR/Cas12a-nanozyme visual biosensor for direct and sensitive detection of tumor-associated miRNA-21. In this design, Ag/NiFe layered double hydroxide acts as a peroxidase-like nanozyme and is tethered to magnetic beads via single-stranded DNA (ssDNA) linkers. Upon recognition of miRNA-21, strand displacement amplification (SDA) can generate a DNA activator that activates CRISPR/Cas12a. The activated Cas12a then cleaves the ssDNA linkers, releasing the nanozyme to catalyze a chromogenic reaction that yields a visible color change. This approach enables sensitive detection of miRNA-21 with a detection limit of 420.0 fM, providing a direct visual readout and demonstrating strong potential for point-of-care nucleic acid diagnostics.},
}

*MicroRNAs/analysis/genetics
*Biosensing Techniques/methods
*CRISPR-Cas Systems
Humans
DNA, Single-Stranded/chemistry
Limit of Detection
*Endodeoxyribonucleases/metabolism/chemistry
*CRISPR-Associated Proteins/metabolism
*Bacterial Proteins/metabolism
Nucleic Acid Amplification Techniques
Silver/chemistry

@article {pmid41653624,
year = {2026},
author = {Sun, Y and Demachi-Okamura, A and Shinohara, S and Wang, Y and Guo, Z and Yamaguchi, R and Matsushita, H and Nabekura, T and Muraoka, D},
title = {Dual knockout of Fas and TCRα in Jurkat reporter cells enables highly sensitive identification of antigen-specific TCRs.},
journal = {Biochemical and biophysical research communications},
volume = {805},
number = {},
pages = {153400},
doi = {10.1016/j.bbrc.2026.153400},
pmid = {41653624},
issn = {1090-2104},
mesh = {Humans ; Jurkat Cells ; *fas Receptor/genetics/immunology ; CRISPR-Cas Systems ; *Gene Knockout Techniques ; CD8-Positive T-Lymphocytes/immunology ; Lung Neoplasms/immunology/genetics ; *Receptors, Antigen, T-Cell, alpha-beta/genetics/immunology ; *Receptors, Antigen, T-Cell/genetics ; },
abstract = {T-cell receptors (TCRs) that target tumor antigens are crucial for antitumor immunity; however, tumor-specific TCRs often exhibit low affinity for their cognate antigens, making the identification of functional TCRs challenging due to the limited sensitivity of current detection methods. In this study, we established a high-sensitivity TCR screening platform by generating Jurkat cell reporter clones with dual knockout (DKO) of endogenous Fas and TCRα via CRISPR-Cas9 system. In a viral antigen model system, these DKO Jurkat cells exhibited approximately 100-fold greater sensitivity to antigen stimulation compared with parental Jurkat cells. Notably, our DKO Jurkat-based platform enabled the identification of tumor-specific CD8[+] T cells from a lung cancer patient that could not be detected using parental Jurkat cells. Moreover, the identified tumor-specific T-cell clone exhibited a unique phenotype characterized by robust cytotoxic T lymphocyte (CTL) activity and natural killer-like properties. Together, these findings demonstrate that dual deletion of Fas and TCRα in Jurkat cells enables highly sensitive functional TCR screening. Integration of this platform with single-cell analysis facilitates the discovery of previously uncharacterized tumor-reactive TCRs and provides a powerful tool for advancing TCR-based cancer immunotherapy.},
}

Humans
Jurkat Cells
*fas Receptor/genetics/immunology
CRISPR-Cas Systems
*Gene Knockout Techniques
CD8-Positive T-Lymphocytes/immunology
Lung Neoplasms/immunology/genetics
*Receptors, Antigen, T-Cell, alpha-beta/genetics/immunology
*Receptors, Antigen, T-Cell/genetics

@article {pmid41288537,
year = {2026},
author = {He, J and Papa, G and Azizi, F and Kirsche, L and Artola-Boran, M and Ferreira Cassio, R and Hotz, AL and Geiger, G and Francas, B and Weber, A and Tzankov, A and Kontarakis, Z and Leary, P and Müller, A},
title = {Gastric Organoid-Based Ectopic and Orthotopic In Vivo CRISPR Screening for Tumor Suppressors in Gastric Cancer.},
journal = {Gastroenterology},
volume = {170},
number = {3},
pages = {495-510},
doi = {10.1053/j.gastro.2025.09.009},
pmid = {41288537},
issn = {1528-0012},
mesh = {Animals ; *Stomach Neoplasms/genetics/pathology/microbiology/metabolism ; *Organoids/pathology ; Mice ; *CRISPR-Cas Systems ; Helicobacter pylori ; Humans ; *Genes, Tumor Suppressor ; Helicobacter Infections/microbiology ; Signal Transduction/genetics ; PTEN Phosphohydrolase/genetics ; Gene Knockout Techniques ; Disease Models, Animal ; },
abstract = {BACKGROUND & AIMS: CRISPR-Cas9 screening is a powerful tool for the in vivo discovery of cancer dependencies. The aim of this study was to perform in vivo CRISPR knockout screening for gastric tumor suppressors using gastric murine organoids in a subcutaneous as well as a surgical model of orthotopic tumor growth.

METHODS: In vivo screening was performed using a custom library targeting 49 putative gastric tumor suppressor genes, as well as a "cancer genome-wide" library targeting 5000 genes, in immunocompetent and -deficient mice, and in the presence or absence of the gastric pathogen Helicobacter pylori. The top hits were selected for individual validation and mechanistic follow-up.

RESULTS: Our custom library knockout screens revealed single-guide RNAs targeting Pten, Fbxw7, and genes encoding several components of the transforming growth factor-ß signaling pathway (Smad4, Tgfbr1, Tgfbr2, and Acvr2a) to be recurrently enriched both in subcutaneously and orthotopically growing tumors. The same, and several additional genes were identified by cancer genome-wide CRISPR screening. Ten of our top hits could be validated individually in vivo. Pten inactivation resulted in large tumors characterized by increased neo-angiogenesis, neutrophil recruitment, and T-cell exclusion. Inactivation of Smad4, Tgfbr1, or Acvr2a all produced phenotypes that were reminiscent of early gastric cancer precursor lesions such as intestinal Alcian blue-positive metaplasia and compensatory hyperplasia. Helicobacter pylori infection failed to affect the mutational landscape of tumors; rather, we found that H pylori modulates the tumor microenvironment and recruits large numbers of tumor-promoting SiglecF[+] neutrophils.

CONCLUSIONS: In summary, we describe here a versatile model of gastric carcinogenesis that uncouples the genetics of the tumor and the host, and that faithfully recapitulates key risk factors of the malignancy.},
}

Animals
*Stomach Neoplasms/genetics/pathology/microbiology/metabolism
*Organoids/pathology
Mice
*CRISPR-Cas Systems
Helicobacter pylori
Humans
*Genes, Tumor Suppressor
Helicobacter Infections/microbiology
Signal Transduction/genetics
PTEN Phosphohydrolase/genetics
Gene Knockout Techniques
Disease Models, Animal

@article {pmid41703314,
year = {2026},
author = {Rocha, DC and Omoregbee, MO and Luo, W and Fang, H and Ye, Q and Liu, Y and Li, G and Mascoveto, J and de Souza, AA and Coleman, G and Culver, JN and Qi, Y},
title = {Transgene-free genome editing in citrus and poplar meristem tissues via biolistic ribonucleoprotein delivery of CRISPR-Cas9.},
journal = {Plant cell reports},
volume = {45},
number = {3},
pages = {58},
pmid = {41703314},
issn = {1432-203X},
support = {2020/07045-3, 2021/03466-7, and 2023/09068-9//Fundação de Amparo à Pesquisa do Estado de São Paulo/ ; DE-SC0023011//U.S. Department of Energy/ ; IOS-2132693 and IOS-2428015//Directorate for Biological Sciences/ ; 2020-70029-33161, 2021-67013-34554, 2024-33522-42755//National Institute of Food and Agriculture/ ; MD-PSLA-24014//McIntire Stennis Forest Research Program/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Meristem/genetics ; *Ribonucleoproteins/genetics/metabolism ; *Populus/genetics ; *Citrus/genetics ; *Biolistics/methods ; Plants, Genetically Modified ; Transgenes/genetics ; },
abstract = {Biolistic particle bombardment was used to deliver CRISPR-Cas9 ribonucleoprotein complexes (RNP) into the shoot apical meristem tissue of citrus and axillary meristem tissue of poplar, generating directed mutations in target genes. The use of meristematic tissues offers a strategic approach to genome editing in woody species, especially those that are recalcitrant to conventional tissue culture, as these regions contain totipotent, highly regenerative cells capable of giving rise to whole plants. Here, we employed biolistic delivery of genome-editing reagents into theshoot apical meristem (SAM) of citrus and the axillary meristems (AXM) of poplar. The system was first validated using a GFP expression construct and subsequently applied for targeted genome editing. In citrus, edited plants were obtained at the CsNPR3 locus exclusively through the delivery of CRISPR/Cas9 ribonucleoproteins (RNPs), whereas plasmid-based vectors were unsuccessful. Similarly, genome editing in poplar was achieved using RNPs targeting the Pt4CL1 gene. Although chimeric events were detected, this approach provides a feasible and innovative framework for producing transgene-free edited perennial plants.},
}

*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Meristem/genetics
*Ribonucleoproteins/genetics/metabolism
*Populus/genetics
*Citrus/genetics
*Biolistics/methods
Plants, Genetically Modified
Transgenes/genetics

@article {pmid41620608,
year = {2026},
author = {Pahlevan Kakhki, M and Rangani, F and Ewing, E and Starvaggi Cucuzza, C and Zheleznyakova, G and Kalomoiri, M and Kenny, L and Raghavan, A and Rao Prakash, C and van den Hoeven, G and Venkata S Badam, T and Covacu, R and Andreou, I and Needhamsen, M and Kular, L and Jagodic, M},
title = {Comprehensive profiling of CRISPR/dCas9 epigenome editors indicates a complex link between on and off target effects.},
journal = {Genome biology},
volume = {27},
number = {1},
pages = {},
pmid = {41620608},
issn = {1474-760X},
mesh = {*CRISPR-Cas Systems ; DNA Methylation ; *Gene Editing/methods ; Humans ; *Epigenome ; DNA Methyltransferase 3A ; DNA (Cytosine-5-)-Methyltransferases/metabolism/genetics ; Promoter Regions, Genetic ; RNA, Guide, CRISPR-Cas Systems/genetics ; Epigenesis, Genetic ; },
abstract = {BACKGROUND: CRISPR/dCas9-based epigenome editing systems, including DNA methylation epimodifiers, have greatly advanced molecular functional studies, revolutionizing their precision and applicability. Despite their promise, challenges such as the magnitude and stability of the on-target editing and unwanted off-target effects underscore the need for improved tool characterization and design.

RESULTS: We systematically compare specific targeting and genome-wide off-target effects of available and novel dCas9-based DNA methylation editing tools over time. We demonstrate that multimerization of the catalytic domain of DNA methyltransferase 3A enhances editing potency but also induces widespread, early methylation deposition at low-to-medium methylated promoter-related regions with specific gRNAs and also with non-targeting gRNAs. A small fraction of the methylation changes associated with transcriptional dysregulation and mapped predominantly to bivalent chromatin associating both with transcriptional repression and activation. Additionally, specific non-targeting control gRNAs cause pervasive and long-lasting methylation-independent transcriptional alterations particularly in genes linked to RNA and energy metabolism. CRISPRoff emerges as the most efficient tool for stable promoter targeting, with fewer and less stable off-target effects compared to other epimodifiers but with persistent transcriptome alterations.

CONCLUSIONS: Our findings highlight the delicate balance between potency and specificity of epigenome editing and provide critical insights into the design and application of future tools to improve their precision and minimize unintended consequences.},
}

*CRISPR-Cas Systems
DNA Methylation
*Gene Editing/methods
Humans
*Epigenome
DNA Methyltransferase 3A
DNA (Cytosine-5-)-Methyltransferases/metabolism/genetics
Promoter Regions, Genetic
RNA, Guide, CRISPR-Cas Systems/genetics
Epigenesis, Genetic

@article {pmid41527385,
year = {2026},
author = {Pang, Y and Duan, Y and Sun, Y and Zhou, T and Li, A and Ran, R and Hou, H and Liu, SM},
title = {One copy in one-pot for rapid and accurate SFTSV testing by LAC12b-2M.},
journal = {Clinical chemistry and laboratory medicine},
volume = {64},
number = {4},
pages = {957-967},
pmid = {41527385},
issn = {1437-4331},
mesh = {Humans ; *Phlebovirus/genetics/isolation & purification ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; RNA, Viral/genetics/blood ; CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; Sensitivity and Specificity ; Real-Time Polymerase Chain Reaction ; },
abstract = {OBJECTIVES: Severe fever with thrombocytopenia syndrome virus (SFTSV) is a tick-borne pathogen that can cause a fatality rate as high as 12-50 %, posing a significant threat to public health. SFTSV is prevalent in mountainous and hilly regions with relatively poor medical conditions. Therefore, there is an urgent need to develop a new convenient, rapid and sensitive method for SFTSV detection in low-resource environments.

METHODS: We developed a one-pot and visualized method for SFTSV detection using loop-mediated isothermal amplification assisted by CRISPR/Cas12b with G478A/K396A double mutations (LAC12b-2M). The specificity, sensitivity, accuracy and limit of detection (LOD) of LAC12b-2M were evaluated using clinical reverse transcription-quantitative PCR (RT-qPCR) as the reference method, with gradient dilutions of strong positive SFTSV RNA samples and 215 clinical serum samples from two cohorts.

RESULTS: LAC12b-2M is sensitive to detect SFTSV with a LOD of 1 copy/μL at 61 °C within 30 min. Compared to clinical RT-qPCR, LAC12b-2M demonstrated a sensitivity of 98.8 % (82/83), a specificity of 100.0 % (96/96), and an accuracy of 99.4 % (178/179) in cohort 1 (n=179), and an accuracy of 100.0 % in cohort 2 (n=36).

CONCLUSIONS: Our LAC12b-2M method holds promise for point-of-care SFTSV testing in different healthcare settings, particularly in low-resource region where SFTSV is prevalent.},
}

Humans
*Phlebovirus/genetics/isolation & purification
*Nucleic Acid Amplification Techniques/methods
Limit of Detection
RNA, Viral/genetics/blood
CRISPR-Cas Systems
*Molecular Diagnostic Techniques/methods
Sensitivity and Specificity
Real-Time Polymerase Chain Reaction

@article {pmid41702797,
year = {2026},
author = {Li, C and Mei, D and Cheng, H and Pan, X and Zhang, B},
title = {CRISPR genome editing in plants without tissue culture.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2025.12.017},
pmid = {41702797},
issn = {1879-3096},
abstract = {Conventional plant genome editing relies on tissue culture-mediated somatic cell regeneration, a technically demanding process that limits its application across diverse species. Emerging strategies now circumvent this bottleneck by enabling direct genome editing of meristematic or germline cells. Key advances include (i) genome editing via de novo meristem induction or dormant meristem activation; (ii) germline editing facilitated by graft-mobile tRNA-like sequence systems and haploid induction technologies; and (iii) optimized viral delivery platforms that exploit mobile RNA elements and compact editors such as TnpB to achieve efficient, transgene-free, heritable modifications across a broad range of genotypes and species. The development of robust, tissue culture-free editing platforms promises to revolutionize crop improvement pipelines and accelerate trait development for sustainable agriculture.},
}

@article {pmid41702404,
year = {2026},
author = {Li, Z and Li, Y and Kong, J and Wu, Q and Huang, P and Zhang, Y and Wu, W and Chen, M and Liu, Y and Lin, H and Hou, L and Liu, G and Zeng, T and He, Y and Hu, C and Yang, Z and Lu, M and Luo, M and Xiao, Y},
title = {Structural basis for Cas9-directed spacer acquisition in type II-A CRISPR-Cas systems.},
journal = {Molecular cell},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.molcel.2026.01.024},
pmid = {41702404},
issn = {1097-4164},
abstract = {CRISPR-Cas systems confer prokaryotic immunity by integrating foreign DNA (prespacers) into host arrays. Type II-A systems employ Cas9 for protospacer-adjacent motif (PAM) recognition and coordinate with Csn2 and the Cas1-Cas2 integrase during spacer acquisition, yet their structural basis remains unresolved. Here, we report cryo-electron microscopy (cryo-EM) structures of the Enterococcus faecalis Cas9-Csn2-Cas1-Cas2 supercomplex in apo and DNA-bound states. The apo state (Cas92-Csn28-Cas18-Cas24) is a resting complex, while DNA binding forms a prespacer-catching complex threading DNA through Csn2's channel, enabling Cas9 to interrogate the PAM sequence while sliding along the DNA. Cas9 and Csn2 jointly define a 30-bp DNA segment matching the prespacer length. Cas9 dissociation triggers structural reconfiguration of the Csn2-Cas1-Cas2 assembly. This exposes the PAM-proximal DNA, allowing Cas1-Cas2 to bind the exposed site for subsequent prespacer processing and directional integration. These findings reveal how Cas9, Csn2, and Cas1-Cas2 couple PAM recognition with prespacer selection, ensuring fidelity during adaptation.},
}

@article {pmid41702403,
year = {2026},
author = {Gaizauskaite, U and Tamulaitiene, G and Silanskas, A and Gasiunas, G and Siksnys, V and Sasnauskas, G},
title = {Structural insights into Cas9-mediated prespacer selection in CRISPR-Cas adaptation.},
journal = {Molecular cell},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.molcel.2026.01.022},
pmid = {41702403},
issn = {1097-4164},
abstract = {During CRISPR-Cas adaptation, prokaryotic cells become immunized by the insertion of foreign DNA fragments, termed spacers, into the host genome to serve as templates for RNA-guided immunity. Spacer acquisition relies on the Cas1-Cas2 integrase and accessory proteins, which select DNA sequences flanked by the protospacer adjacent motif (PAM) and insert them into the CRISPR array. It has been shown that in type II-A systems, selection of PAM-proximal prespacers is mediated by the effector nuclease Cas9, which forms a "supercomplex" with the Cas1-Cas2 integrase and the Csn2 protein. Here, we present cryo-electron microscopy structures of the Streptococcus thermophilus type II-A prespacer selection supercomplex in the DNA-scanning and two distinct PAM-bound configurations, providing insights into the mechanism of Cas9-mediated prespacer selection in type II-A CRISPR-Cas systems. Repurposing Cas9 by the CRISPR adaptation machinery for prespacer selection, as characterized here, demonstrates Cas9 plasticity and expands our knowledge of Cas9 biology.},
}

@article {pmid41702027,
year = {2026},
author = {Galdikaite-Braziene, E and Krušnauskas, R and Henderson, E and Bujakowska, KM},
title = {CRISPR as a therapeutic tool for inherited retinal degenerations: Advances, challenges, and future directions.},
journal = {Molecular aspects of medicine},
volume = {108},
number = {},
pages = {101462},
doi = {10.1016/j.mam.2026.101462},
pmid = {41702027},
issn = {1872-9452},
abstract = {Inherited retinal diseases (IRDs) are a genetically diverse group of disorders characterized by progressive photoreceptor degeneration, leading to vision loss and blindness. With over 320 associated genes and significant phenotypic variability, effective treatment remains challenging. Recent advances in genome editing, particularly CRISPR/Cas-based technologies, have revolutionized therapeutic approaches by enabling precise and customizable DNA and RNA editing. This review explores the application of various CRISPR strategies-such as gene knockout via non-homologous end joining (NHEJ), exon skipping using dual-sgRNAs, homology-directed repair (HDR), base editing (BE), prime editing (PE), RNA editing with Cas13, and epigenetic modulation through CRISPRa/i-in preclinical models of IRDs. Emphasis is placed on allele-specific targeting, gene-agnostic approaches, and mutation-independent strategies to address dominant and recessive forms of disease. We also highlight recent clinical milestones, including the first human trial using CRISPR gene editing for CEP290-associated Leber congenital amaurosis. Finally, we discuss critical challenges, including delivery constraints, immune responses, and off-target effects, along with emerging solutions such as engineered Cas variants, split-intein systems, and advanced off-target detection methods. Together, these advances underscore the transformative potential of CRISPR technologies in treating IRDs and lay the foundation for future clinical translation.},
}

@article {pmid41699368,
year = {2026},
author = {Khalid, M and Ishaq, A and Arshad, M and Kaul, H and Majeed, M},
title = {Multiplex CRISPR/Cas9 editing of gliotoxin biosynthesis genes in Aspergillus fumigatus reduces pathogenicity in broilers.},
journal = {Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology]},
volume = {57},
number = {1},
pages = {55},
pmid = {41699368},
issn = {1678-4405},
support = {13224/2020//Higher Education Commision, Pakistan/ ; },
mesh = {Animals ; *Aspergillus fumigatus/genetics/pathogenicity/metabolism ; *Gliotoxin/biosynthesis ; Chickens ; *CRISPR-Cas Systems ; *Gene Editing ; *Aspergillosis/veterinary/microbiology/pathology ; *Poultry Diseases/microbiology/pathology ; Virulence ; Fungal Proteins/genetics/metabolism ; Spores, Fungal/genetics ; },
abstract = {Gliotoxin of Aspergillus fumigatus has been extensively studied for its role in pathogenesis in animals and humans. It triggers pathogenesis by its immunosuppressive and cytotoxic effects. Biosynthetic gene cluster (BGC) consisting of 13 genes regulates its biosynthesis. We targeted gliZ, gliP and gliA genes of this BGC using CRISPR/Cas9 system in a multigene editing approach to check the pathogenesis in broilers. crRNAs were designed using EuPaGDT and 3 single guide RNAs (sgRNA) were commercially synthesized. Each sgRNA was combined with Cas9 to form ribonucleoprotein complexes which were then used for simultaneously transfecting fungal protoplasts. Thin-layer chromatography showed the absence of gliotoxin on silica plate and DNA sequencing showed various indels in target genes. These indels caused amino acid substitutions in all three gene products but, the gliP mutation, since it was synonymous, was likely not functionally relevant. Regenerated protoplasts were matured to form fungal hyphae and spore production was induced. These spores were inoculated intra-air sac in broiler chicks. During one-week infection trial, birds infected with the wild-type spores (group 1) showed morbidity and their mortality rate was 30%. Birds inoculated with RNP-treated spores (group 2) showed mild clinical signs and no mortality. No morbidity or mortality was recorded in birds in negative control group (group 3). Histopathological analysis of lungs showed necrosis and congestion, and presence of mixed population of inflammatory cells in wild-type infected birds, while no such lesions were seen in birds infected with RNP-treated spores. These results show that multigene editing approach was successful in creating indels simultaneously in 3 gliotoxin genes which resulted in amino acid substitution which negatively impacted gliotoxin biosynthesis and export. In vivo experiment results show that RNP-treated fungal spores were unable to cause A. fumigatus pathogenicity in broiler. Targeting gliotoxin biosynthesis could thus be a promising approach to develop antifungal therapy.},
}

Animals
*Aspergillus fumigatus/genetics/pathogenicity/metabolism
*Gliotoxin/biosynthesis
Chickens
*CRISPR-Cas Systems
*Gene Editing
*Aspergillosis/veterinary/microbiology/pathology
*Poultry Diseases/microbiology/pathology
Virulence
Fungal Proteins/genetics/metabolism
Spores, Fungal/genetics

@article {pmid41669836,
year = {2026},
author = {Dai, YM and Zhao, L and Xu, T and Duan, JQ and Wang, YM and Yan, YX and Ge, LP and Liu, ZH and Sun, J and Zeng, X and Lai, SY and Ai, YR and Huang, JB and Zhu, L and Xu, ZW},
title = {Fast and Simplified CRISPR-Cas13d Assay for Sensitive Detection of Porcine Deltacoronavirus.},
journal = {ACS synthetic biology},
volume = {15},
number = {2},
pages = {834-845},
doi = {10.1021/acssynbio.5c00909},
pmid = {41669836},
issn = {2161-5063},
mesh = {Animals ; Swine ; *CRISPR-Cas Systems/genetics ; *Deltacoronavirus/genetics/isolation & purification ; RNA, Viral/genetics/analysis ; Sensitivity and Specificity ; Nucleic Acid Amplification Techniques/methods ; *Swine Diseases/diagnosis/virology ; },
abstract = {Rapid and portable diagnostic technologies are essential for controlling infectious diseases. Here, we describe RAPID (Rapid Automated Portable Integrated Detection), a single-step, extraction-free CRISPR-Cas13d-based assay for sensitive and specific detection of porcine deltacoronavirus (PDCoV) RNA. RAPID integrates isothermal recombinase polymerase amplification with EsCas13d-mediated collateral cleavage in a one-pot reaction, enabling sample-to-answer detection within 30 min. A brief room-temperature lysis step allows direct RNA release from unextracted samples, simplifying sample preparation and reducing equipment requirements. Lyophilized reagents enhance stability during refrigerated storage (≤4 °C) and facilitate simplified transportation using conventional cooling measures, thereby reducing reliance on strict cold-chain logistics. The assay operates optimally at 37 °C and remains functional under ambient (∼25 °C) conditions with reduced sensitivity, permitting instrument-free operation when temperature control is unavailable. Detection is achieved via in-tube fluorescence or lateral-flow readouts. Clinical validation using porcine samples showed complete concordance with RT-qPCR, achieving 100% sensitivity and specificity. RAPID provides a practical point-of-care diagnostic platform for on-farm surveillance and deployment in resource-limited settings.},
}

Animals
Swine
*CRISPR-Cas Systems/genetics
*Deltacoronavirus/genetics/isolation & purification
RNA, Viral/genetics/analysis
Sensitivity and Specificity
Nucleic Acid Amplification Techniques/methods
*Swine Diseases/diagnosis/virology

@article {pmid41645934,
year = {2026},
author = {Wang, Z and Wang, Y and Ji, Q},
title = {Protocol for Discovery and Characterization of Miniature Cas12 Systems.},
journal = {ACS chemical biology},
volume = {21},
number = {2},
pages = {401-409},
doi = {10.1021/acschembio.6c00016},
pmid = {41645934},
issn = {1554-8937},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Dependovirus/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; },
abstract = {Efficient delivery remains a major challenge for therapeutic genome editing because many widely used CRISPR nucleases are large and leave limited space for regulatory elements or additional payloads in a single adeno-associated virus (AAV) vector. Miniature Cas12 nucleases are particularly appealing, as their reduced size alleviates packaging constraints while preserving RNA-guided DNA cleavage. Here, we outline a workflow that links large-scale sequence mining with phylogenetic and structural filtering, followed by PAM profiling, in vitro cleavage, bacterial genome interference, and genome-editing assays in human cells to confirm activity. This protocol is intended to distill broad sequence collections into a small set of compact Cas12 nucleases with demonstrated functions that can serve as starting points for further engineering in delivery-limited settings.},
}

Humans
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Dependovirus/genetics
*CRISPR-Associated Proteins/genetics/metabolism

@article {pmid41639367,
year = {2026},
author = {Barreiro-Docío, E and Guerrero-Peña, L and Soni, P and Méndez-Martínez, L and Costas-Prado, C and Alvarado, MV and Vázquez, JA and Tort, L and Cerdá-Reverter, JM and Rotllant, J},
title = {Loss-of-function mutations in the melanocortin-2-receptor (mc2r) lead to skin hyperpigmentation in teleost fish.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {41639367},
issn = {2045-2322},
mesh = {Animals ; *Zebrafish/genetics/metabolism ; *Receptor, Melanocortin, Type 2/genetics/metabolism ; *Hyperpigmentation/genetics/metabolism ; *Loss of Function Mutation ; *Skin Pigmentation/genetics ; *Zebrafish Proteins/genetics/metabolism ; Melanophores/metabolism ; Melanins/biosynthesis ; CRISPR-Cas Systems ; },
abstract = {Melanocortins regulate pigmentation via melanocortin receptors (MCRs), which are highly conserved across vertebrates. Unlike other MCRs, the melanocortin 2 receptor (MC2R) is exclusively activated by ACTH; however, its role in pigmentation remains unclear. Using CRISPR/Cas9-generated mc2r knockout zebrafish, we demonstrated that the loss of mc2r in zebrafish results in impaired interrenal steroidogenesis and pronounced hyperpigmentation characterized by an increased number of melanophores and xanthophores while preserving normal patterning. Transcriptomic analyses revealed the upregulation of genes involved in melanosome formation, melanin synthesis, lipid metabolism, and carotenoid accumulation. These findings demonstrate that, in addition to controlling steroidogenesis, mc2r plays a key role in pigment cell development and metabolic regulation.},
}

Animals
*Zebrafish/genetics/metabolism
*Receptor, Melanocortin, Type 2/genetics/metabolism
*Hyperpigmentation/genetics/metabolism
*Loss of Function Mutation
*Skin Pigmentation/genetics
*Zebrafish Proteins/genetics/metabolism
Melanophores/metabolism
Melanins/biosynthesis
CRISPR-Cas Systems

@article {pmid41571527,
year = {2026},
author = {Kaneko, Y and Kawabe, Y and Nishijima, KI and Kamihira, M},
title = {Evaluation of cellular characteristics and genome editing responses in chicken primordial germ cell lines.},
journal = {Journal of bioscience and bioengineering},
volume = {141},
number = {4},
pages = {290-299},
doi = {10.1016/j.jbiosc.2025.12.010},
pmid = {41571527},
issn = {1347-4421},
mesh = {Animals ; *Chickens/genetics ; *Germ Cells/cytology/metabolism ; *Gene Editing/methods ; Male ; CRISPR-Cas Systems ; Female ; Cell Proliferation ; Cell Line ; },
abstract = {Chicken primordial germ cells (cPGCs) hold great potential for genetic modification and germ cell research in chickens. In this study, we evaluated the cellular characteristics of three cPGC lines: cPGC-1, cPGC-2, and cPGC-3. cPGC-1 and cPGC-2 were derived from male chickens, whereas cPGC-3 was derived from a female chicken. We analyzed and compared cell proliferation rates, marker gene expression, and gonadal colonization abilities. Three different cell culture temperatures were assessed (37 °C, 39 °C, and 41 °C) and proliferation rates were highest for all cPGC lines at 39 °C. Additionally, cPGC-1 demonstrated a higher proliferation rate than cPGC-2. No significant differences were observed between cPGC-1 and cPGC-2 with regard to the expression of germ cell and pluripotency marker genes (Cvh, Dazl, Pou5f3, and Nanog). To assess changes in cellular characteristics before and after genetic modification, we performed a green fluorescent protein (GFP) gene knock-in using the CRISPR/Cas9 system, followed by site-specific integration of the scFv-Fc gene using the Cre-loxP system. Transplantation experiments revealed that cPGC-2/GFP exhibited higher gonadal colonization efficiency than cPGC-1/GFP. This study demonstrates differences in cellular characteristics among established cPGC lines and highlights the impact of genetic modification on cPGC function. Our findings emphasize the importance of selecting appropriate cell lines and optimizing culture conditions based on cPGC traits to achieve efficient and reproducible production of transgenic chickens. These insights will aid in the conservation of poultry genetic resources and the advancement of transgenic chicken production for both research and industrial applications.},
}

Animals
*Chickens/genetics
*Germ Cells/cytology/metabolism
*Gene Editing/methods
Male
CRISPR-Cas Systems
Female
Cell Proliferation
Cell Line

@article {pmid41554741,
year = {2026},
author = {Li, S and Xu, K and Li, G and Jiang, H and Wu, Z and Gao, P and Yue, Y and Chen, Y and Liu, Z and Zhou, B and Zhou, M and Chen, Y and Liu, X and Wang, X and Wu, Z and Wei, Y},
title = {Engineering the MmeFz2-ωRNA system for efficient genome editing through an integrated computational-experimental framework.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41554741},
issn = {2041-1723},
support = {32441080, 32301251//National Natural Science Foundation of China (National Science Foundation of China)/ ; 22207074//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Gene Editing/methods ; Animals ; Humans ; Mice ; Muscular Dystrophy, Duchenne/genetics/therapy ; Male ; Dystrophin/genetics/metabolism ; HEK293 Cells ; Protein Engineering/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Disease Models, Animal ; Dependovirus/genetics ; CRISPR-Cas Systems ; },
abstract = {Eukaryotic Fanzor proteins are compact, programmable RNA-guided nucleases with substantial potential for genome editing, although their efficiency in mammalian cells remains suboptimal. Here, we present a combinatorial engineering strategy to optimize a representative Fanzor system, MmeFz2-ωRNA. AlphaFold3-powered rational redesign produced a minimized ωRNA scaffold that is 30% smaller while maintaining up to 82.2% efficiency. Synergistic structure-guided and AI-augmented protein engineering generated two variants, enMmeFz2 and evoMmeFz2, which exhibited an average ~32-fold increase in activity across 38 genomic loci. Moreover, fusion of the non-specific DNA-binding domain HMG-D further enhanced editing performance (enMmeFz2-HMG-D and evoMmeFz2-HMG-D). Notably, evoMmeFz2-HMG-D demonstrated robust in vivo genome editing activity, enabling dystrophin restoration in humanized male Duchenne muscular dystrophy mouse models via single adeno-associated virus (AAV) delivery. This study establishes Fanzor2 as a gene editing platform for genome engineering and therapeutic applications, and underscores the power of AI-guided engineering to accelerate genome editor development while reducing experimental burden.},
}

*Gene Editing/methods
Animals
Humans
Mice
Muscular Dystrophy, Duchenne/genetics/therapy
Male
Dystrophin/genetics/metabolism
HEK293 Cells
Protein Engineering/methods
RNA, Guide, CRISPR-Cas Systems/genetics
Disease Models, Animal
Dependovirus/genetics
CRISPR-Cas Systems

@article {pmid41538882,
year = {2026},
author = {Loubat, A and Wolfender, C and Calabre, M and Beaude, N and Tavares, P and Planson, AG and Jules, M},
title = {Advancing Fast-Track Genome Engineering in Bacillus subtilis Phages.},
journal = {ACS synthetic biology},
volume = {15},
number = {2},
pages = {687-700},
doi = {10.1021/acssynbio.5c00727},
pmid = {41538882},
issn = {2161-5063},
mesh = {*Bacillus subtilis/virology/genetics ; *Genome, Viral/genetics ; CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Bacillus Phages/genetics ; *Genetic Engineering/methods ; },
abstract = {Phage genome engineering methods accelerate the study of phage biology, the discovery of new functions, and the development of innovative genetic engineering tools. Here, we present QuickPhage, a rapid, technically accessible, precise, and cost-effective method for engineering Bacillus subtilis phages. Our approach uses CRISPR-Cas9 as a counter-selection system to isolate mutants of the model lytic siphovirus phage, SPP1. Efficient genome editing was achieved using homologous repair patches as short as 40 nucleotides, enabling streamlined patch construction and parallel engineering, resulting in highly accurate genome edits within a day. We applied QuickPhage to delete both essential and nonessential phage genes and to insert reporter genes. Protein production, such as GFP, was synthetically regulated using inducible systems without significantly affecting phage fitness, achieving induction levels of up to 400-fold. Time-series coinfection experiments with fluorescent protein expressing phages also revealed a highly efficient superinfection arrest mechanism that prevents reinfection as early as 13 min after initial infection. These findings highlight the potential of phages for protein production, opening new opportunities for metabolic engineering. This work also lays the foundation for systematic phage genome refactoring workflows and the development of phage-based tools for efficient DNA delivery, thereby expanding the synthetic biology toolbox for B. subtilis.},
}

*Bacillus subtilis/virology/genetics
*Genome, Viral/genetics
CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Bacillus Phages/genetics
*Genetic Engineering/methods

@article {pmid41529994,
year = {2026},
author = {Ebe, S and Nakamura, H and Matsuda, M and Terauchi, Y and Akada, R and Hoshida, H},
title = {Identification and overexpression of genes encoding sugar alcohol transporter and metabolic enzymes for accelerated utilization in the yeast Kluyveromyces marxianus.},
journal = {Journal of bioscience and bioengineering},
volume = {141},
number = {4},
pages = {221-229},
doi = {10.1016/j.jbiosc.2025.12.003},
pmid = {41529994},
issn = {1347-4421},
mesh = {*Kluyveromyces/genetics/metabolism/enzymology/growth & development ; Mannitol/metabolism ; Sorbitol/metabolism ; CRISPR-Cas Systems ; *Fungal Proteins/genetics/metabolism ; L-Iditol 2-Dehydrogenase/genetics/metabolism ; },
abstract = {The yeast Kluyveromyces marxianus assimilates various sugars, including sorbitol and mannitol. However, the metabolic pathways for sugar utilization, including sugar transporters, remain to be elucidated. To identify these genes in this study, first 13 candidate transporter genes were disrupted using a newly developed non-homologous end joining (NHEJ)-mediated gene disruption method, combined with targeted digestion using the CRISPR-Cas9 system. While most disruptants exhibited no clear growth defects in various sugar media, a disruptant of the KmMLEV2025 gene (named KmSAT1) failed to grow in either sorbitol or mannitol media, suggesting that it encodes a sugar alcohol transporter. Next, we investigated the candidate dehydrogenase genes crucial for sugar alcohol metabolism, as they are converted to fructose by dehydrogenases. KmXyl2p, a known xylitol dehydrogenase, is a candidate sorbitol dehydrogenase. Disruption of KmXYL2 caused growth defects in sorbitol medium, but not in mannitol medium. We disrupted several genes to identify the mannitol dehydrogenase, revealing that the disruption of KmSOU2, annotated as a sorbose reductase, resulted in a growth defect in the mannitol medium. The identified genes were overexpressed for the efficient utilization of sugar alcohols. The strain overexpressing KmSAT1, but not the dehydrogenase genes, started growing immediately, whereas the wild-type strain exhibited a lag time of several days. Furthermore, the final cell optical densities in both the sorbitol and mannitol media were higher than those observed in the glucose medium. These results indicated that overexpression of a sugar alcohol transporter is a highly effective strategy for biotechnological applications.},
}

*Kluyveromyces/genetics/metabolism/enzymology/growth & development
Mannitol/metabolism
Sorbitol/metabolism
CRISPR-Cas Systems
*Fungal Proteins/genetics/metabolism
L-Iditol 2-Dehydrogenase/genetics/metabolism

@article {pmid41526975,
year = {2026},
author = {Hu, Q and Zhang, R and Liu, J and Zhang, W and Liao, X and Guo, Y and Lu, Q and Yang, B and Zhang, T and Zhai, X and Luo, Q},
title = {A rapid and field-deployable RAA-CRISPR/Cas12a platform for detection of Mycoplasma gallisepticum in poultry.},
journal = {BMC veterinary research},
volume = {22},
number = {1},
pages = {117},
pmid = {41526975},
issn = {1746-6148},
support = {2025BEB053//the Hubei Province Technology Innovation Plan Project/ ; 2024BBA004//the Hubei Province Technology Innovation Plan Project/ ; NYWSWZX2025-3432027-04//the Major Special Project for the Development of Agricultural Microbial Industry in Hubei Province/ ; 2023HBSTX4-04//the Hubei Province Modern Agricultural Industry Technology System/ ; CARS-41//the China Agriculture Research System/ ; },
mesh = {Animals ; *Mycoplasma gallisepticum/isolation & purification/genetics ; *Poultry Diseases/diagnosis/microbiology ; *Mycoplasma Infections/veterinary/diagnosis/microbiology ; *CRISPR-Cas Systems ; Chickens ; *Nucleic Acid Amplification Techniques/veterinary/methods ; Sensitivity and Specificity ; Recombinases ; Ducks ; Columbidae ; },
abstract = {BACKGROUND: Mycoplasma gallisepticum (MG) is a major pathogen that causes respiratory diseases 14in poultry, resulting in reduced production and severe economic losses. Current MG detection methods are time-consuming, labor-intensive, and expensive. Hence, the rapid and accurate detection of MG is critical for effective disease control. Therefore, this study aimed to develop a dual-mode diagnostic assay for sensitive and specific detection of MG by combining recombinase-aided amplification (RAA) with CRISPR/Cas12a technology. Conserved regions of the mgc2 gene were used for primer and CRISPR RNA design, and the reaction conditions were optimized to maximize detection efficiency.

RESULTS: The assay achieved a detection limit of 2 copies/µL and demonstrated high specificity against seven other common avian pathogens. Detection was visualized within 1 h using either fluorescence or lateral flow dipstick. Moreover, clinical validation of chicken samples showed complete concordance with quantitative real-time polymerase chain reaction results. Furthermore, an epidemiological investigation revealed that chickens had the highest positivity rate for MG among chickens, ducks, and pigeons in Hubei Province.

CONCLUSIONS: This simple, rapid, field-deployable method is valuable for timely MG surveillance and effective disease management in poultry production.},
}

Animals
*Mycoplasma gallisepticum/isolation & purification/genetics
*Poultry Diseases/diagnosis/microbiology
*Mycoplasma Infections/veterinary/diagnosis/microbiology
*CRISPR-Cas Systems
Chickens
*Nucleic Acid Amplification Techniques/veterinary/methods
Sensitivity and Specificity
Recombinases
Ducks
Columbidae

@article {pmid41486850,
year = {2026},
author = {Shi, M and Yu, P and Liu, L and Cheng, J and Shao, R and Sun, Y and Lv, J and Li, Y and Zheng, Z and Yu, J and Xu, B and Gan, L and Liang, Y and Zhang, Y and Fang, Y and Shen, W and Huang, J and Zhu, X and Hong, J and Ma, R and Wu, L and Cheng, Y and Zhao, C},
title = {Fluoropolymer-Mediated Delivery of a Dual TSHR/IGF1R-Targeting CRISPR-Cas9 System for Localized Therapy in Thyroid-Associated Ophthalmopathy.},
journal = {Advanced materials (Deerfield Beach, Fla.)},
volume = {38},
number = {11},
pages = {e11078},
doi = {10.1002/adma.202511078},
pmid = {41486850},
issn = {1521-4095},
support = {DGF828030-3/005//Clinical Scientist Foundation of Fudan University/ ; pp25014//Clinical Scientist Foundation of Fudan University/ ; U25A20258//National Natural Science Foundation of China/ ; 82271126//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Receptor, IGF Type 1/genetics/metabolism ; *Graves Ophthalmopathy/therapy/genetics/pathology ; *CRISPR-Cas Systems/genetics ; Humans ; Mice ; *Receptors, Thyrotropin/genetics/metabolism ; Gene Editing ; *Polymers/chemistry ; Fibroblasts/metabolism ; Disease Models, Animal ; Adipocytes/metabolism ; Genetic Therapy ; Organoids/metabolism ; Antibodies, Monoclonal, Humanized ; },
abstract = {Thyroid-associated ophthalmopathy (TAO), a vision-threatening and disfiguring autoimmune orbital disorder, remains a therapeutic challenge due to the lack of therapies with orbital specificity, sustained efficacy, and minimal side effects. Herein, we present G4F7-CRISPR, a fluoropolymer-based CRISPR-Cas9 delivery platform engineered for localized and efficient disruption of thyroid-stimulating hormone receptor (TSHR) and insulin-like growth factor 1 receptor (IGF1R), two key mediators of TAO pathogenesis. G4F7-CRISPR achieved high insertion/deletion frequencies in primary orbital fibroblasts (Tshr: 37.2%; Igf1r: 42.8%) and mature adipocytes (Tshr: 22.4%; Igf1r: 24.3%), and maintained robust editing efficiency in orbital adipose tissue of TAO mouse models (Tshr: 30.7%; Igf1r: 32.4%). In both TAO mouse models and 3D human orbital organoids, dual-gene editing of Tshr and Igf1r via G4F7-CRISPR significantly suppressed orbital adipogenesis, inflammation, and fibrosis, demonstrating superior therapeutic efficacy over either single-gene approaches. Comprehensive off-target analyses in both TAO mouse models and orbital organoids revealed minimal off-target activity. Furthermore, G4F7-CRISPR exhibited excellent short- and long-term ocular and systemic safety in TAO mouse models. Notably, it outperformed teprotumumab-the FDA-approved therapy for TAO-in both therapeutic efficacy and safety, highlighting its potential clinical advantages. Collectively, these findings highlight the translational promise of G4F7-CRISPR as a safe, precise, and clinically viable gene therapy for TAO.},
}

Animals
*Receptor, IGF Type 1/genetics/metabolism
*Graves Ophthalmopathy/therapy/genetics/pathology
*CRISPR-Cas Systems/genetics
Humans
Mice
*Receptors, Thyrotropin/genetics/metabolism
Gene Editing
*Polymers/chemistry
Fibroblasts/metabolism
Disease Models, Animal
Adipocytes/metabolism
Genetic Therapy
Organoids/metabolism
Antibodies, Monoclonal, Humanized

@article {pmid41254174,
year = {2026},
author = {Thomson, T and Li, G and Strilchuk, A and Cui, H and Wang, B and Li, B},
title = {Harnessing artificial intelligence to advance CRISPR-based genome editing technologies.},
journal = {Nature reviews. Genetics},
volume = {27},
number = {3},
pages = {212-230},
pmid = {41254174},
issn = {1471-0064},
mesh = {*Gene Editing/methods ; Humans ; *Artificial Intelligence ; *CRISPR-Cas Systems/genetics ; Animals ; Machine Learning ; Deep Learning ; },
abstract = {CRISPR-based genome editing technologies, including nuclease-based editing, base editing and prime editing, have revolutionized biological research and modern medicine by enabling precise, programmable modification of the genome and offering new therapeutic strategies for a wide range of genetic diseases. Artificial intelligence (AI), including machine learning and deep learning models, is now further advancing the field by accelerating the optimization of gene editors for diverse targets, guiding the engineering of existing tools and supporting the discovery of novel genome-editing enzymes. In this Review, we summarize key AI methodologies underlying these advances and discuss their recent noteworthy applications to genome editing technologies. We also discuss emerging opportunities, such as AI-powered virtual cell models, which can guide genome editing through target selection or prediction of functional outcomes. Finally, we identify key directions where the integration of AI methods is poised to have a substantial impact going forward.},
}

*Gene Editing/methods
Humans
*Artificial Intelligence
*CRISPR-Cas Systems/genetics
Animals
Machine Learning
Deep Learning

@article {pmid40738974,
year = {2026},
author = {Qu, Y and Huang, K and Yin, M and Zhan, K and Liu, D and Yin, D and Cousins, HC and Johnson, WA and Wang, X and Shah, M and Altman, RB and Zhou, D and Wang, M and Cong, L},
title = {CRISPR-GPT for agentic automation of gene-editing experiments.},
journal = {Nature biomedical engineering},
volume = {10},
number = {2},
pages = {245-258},
pmid = {40738974},
issn = {2157-846X},
support = {R35HG011316, 1R01GM141627//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; 1653435//National Science Foundation (NSF)/ ; },
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Artificial Intelligence ; Automation ; Cell Line, Tumor ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Performing effective gene-editing experiments requires a deep understanding of both the CRISPR technology and the biological system involved. Meanwhile, despite their versatility and promise, large language models (LLMs) often lack domain-specific knowledge and struggle to accurately solve biological design problems. We present CRISPR-GPT, an LLM agent system to automate and enhance CRISPR-based gene-editing design and data analysis. CRISPR-GPT leverages the reasoning capabilities of LLMs for complex task decomposition, decision-making and interactive human-artificial intelligence (AI) collaboration. This system incorporates domain expertise, retrieval techniques, external tools and a specialized LLM fine tuned with open-forum discussions among scientists. CRISPR-GPT assists users in selecting CRISPR systems, experiment planning, designing guide RNAs, choosing delivery methods, drafting protocols, designing assays and analysing data. We showcase the potential of CRISPR-GPT by knocking out four genes with CRISPR-Cas12a in a human lung adenocarcinoma cell line and epigenetically activating two genes using CRISPR-dCas9 in a human melanoma cell line. CRISPR-GPT enables fully AI-guided gene-editing experiment design and analysis across different modalities, validating its effectiveness as an AI co-pilot in genome engineering.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Artificial Intelligence
Automation
Cell Line, Tumor
RNA, Guide, CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid41699287,
year = {2026},
author = {Pindi, C and Palermo, G},
title = {Computation and deep-learning-driven advances in CRISPR genome editing.},
journal = {Nature structural & molecular biology},
volume = {33},
number = {2},
pages = {203-214},
pmid = {41699287},
issn = {1545-9985},
mesh = {*Deep Learning ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Humans ; Neural Networks, Computer ; Algorithms ; },
abstract = {Genome editing with CRISPR-Cas systems is revolutionizing medicine, molecular biology and biotechnology. In this Review, we discuss the contributions of deep learning-based structure prediction algorithms, physics-based simulations, neural networks, graph neural networks and generative models, including diffusion and large language models, in engineering and optimizing CRISPR systems and in understanding their mechanistic basis. We highlight the challenges and limitations to the transformative effects of computational modeling and tools in the context of the development of programmable genome editors for biomedicine and biotechnology.},
}

*Deep Learning
*Gene Editing/methods
*CRISPR-Cas Systems
Humans
Neural Networks, Computer
Algorithms

@article {pmid41696049,
year = {2026},
author = {Ahmadzadeh, M and Akbarian, F and Sanati, MH and Motaharirad, H and Farrokhi, F},
title = {Computational Optimization of CRISPR-Cas13a sgRNAs Targeting the SARS-CoV-2 Spike Gene for SHERLOCK-Based Diagnostics.},
journal = {Evolutionary bioinformatics online},
volume = {22},
number = {},
pages = {11769343251414318},
pmid = {41696049},
issn = {1176-9343},
abstract = {BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered a global health crisis, emphasizing the urgent need for accurate and rapid diagnostic tools. Modern molecular biology technologies, including CRISPR-Cas systems, provide highly efficient strategies for viral detection. Bioinformatic pipelines are essential for identifying conserved genomic regions and enabling rational single-guide RNA (sgRNA) design.

METHODS: This study aimed to design specific sgRNAs targeting the spike gene of SARS-CoV-2 isolates from Iranian patients using the SHERLOCK diagnostic platform. Complete genomes of the RefSeq virus and 470 SARS-CoV-2 isolates, representing all variants of concern (VOCs) detected in Iran, were retrieved from the NCBI and GISAID databases. Multiple sequence alignment with ClustalW identified conserved sequences within the receptor-binding domain (RBD) that differ from the RBD of SARS-CoV and MERS-CoV RefSeq genomes. Based on these regions, sgRNAs and isothermal amplification primers were designed using ADAPT, OLIGO7, and the UCSC Genome Browser to maximize diagnostic sensitivity and specificity. Secondary and tertiary structures of sgRNA-target complexes were analyzed via RNAfold and RNAup to select the most efficient sgRNA-amplicon combination.

RESULTS: Twenty-two-nucleotide sgRNA candidates were initially selected based on sequence alignment, showing high similarity to the SARS-CoV-2 RefSeq and low homology to SARS-CoV and MERS-CoV genomes. Analyses of secondary structures, RNA-RNA interactions, and free energy identified 6 sgRNAs with favorable 2-dimensional conformations and strong interaction profiles. Among these, the sgRNA1-Amplicon2 sequence exhibited the most stable 3-dimensional structure and a molecular docking score of -309.67, indicating high sensitivity and specificity for viral detection.

CONCLUSION: This study successfully designed an sgRNA with high sensitivity and specificity for rapid SARS-CoV-2 detection using the CRISPR-Cas13a system, informed by genomic analysis of Iranian isolates. The proposed approach provides an efficient framework for the rapid design and deployment of CRISPR-based diagnostic tools applicable to diverse viral pathogens.},
}

@article {pmid41695711,
year = {2026},
author = {Browne, TS and Edgell, DR and Gloor, GB},
title = {Better data for better predictions: data curation improves deep learning for sgRNA/Cas9 prediction.},
journal = {PeerJ},
volume = {14},
number = {},
pages = {e20706},
pmid = {41695711},
issn = {2167-8359},
mesh = {*Deep Learning ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems ; Escherichia coli/genetics ; *CRISPR-Associated Protein 9/genetics/metabolism ; *Gene Editing/methods ; },
abstract = {The Cas9 enzyme along with a single guide RNA molecule is a modular tool for genetic engineering and has shown effectiveness as a species-specific antimicrobial. The ability to accurately predict on-target cleavage is critical as activity varies by target. Using the sgRNA nucleotide sequence and an activity score, predictive models have been developed with the best performance resulting from deep learning architectures. Prior work has emphasized robust and novel architectures to improve predictive performance. Here, we explore the impact of a data-centric approach through optimization of the input target site adjacent nucleotide sequence length and the use of data filtering for read counts in the control conditions to improve input data utility. Using the existing crisprHAL architecture, we develop crisprHAL Tev, a bacterial SpCas9 prediction model with performance that generalizes across related species and across data types. During this process, we also rebuilt two prior Escherichia coli Cas9 datasets, demonstrating the importance of data quality, and resulting in the production of an improved bacterial eSpCas9 prediction model. The crisprHAL models are available through GitHub: https://github.com/tbrowne5/crisprHAL.},
}

*Deep Learning
*RNA, Guide, CRISPR-Cas Systems/genetics
*CRISPR-Cas Systems
Escherichia coli/genetics
*CRISPR-Associated Protein 9/genetics/metabolism
*Gene Editing/methods

@article {pmid41693565,
year = {2026},
author = {Sinnott, RW and Solanki, A and Govind, AP and Green, WN and Dickinson, BC},
title = {Engineering a human-based translational activator for targeted protein expression restoration.},
journal = {Nucleic acids research},
volume = {54},
number = {4},
pages = {},
pmid = {41693565},
issn = {1362-4962},
support = {FP106237//G. Harold and Leila Y. Mathers Charitable Foundation/ ; //Dr. Ralph and Marian Falk Medical Research Trust/ ; //Bank of America/ ; /EB/NIBIB NIH HHS/United States ; R01-EB035016/NH/NIH HHS/United States ; DGE-2022294368//National Science Foundation/ ; },
mesh = {Animals ; Humans ; Mice ; *Protein Biosynthesis ; *Epilepsies, Myoclonic/genetics/therapy ; Disease Models, Animal ; RNA, Messenger/genetics/metabolism ; *NAV1.1 Voltage-Gated Sodium Channel/genetics/metabolism ; CRISPR-Cas Systems ; *Protein Engineering/methods ; Haploinsufficiency ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Therapeutic modalities to programmably increase protein production are in critical need to address diseases caused by deficient gene expression via haploinsufficiency. Restoring physiological protein levels by increasing translation of their cognate messenger RNA (mRNA) would be an advantageous approach to correct gene expression but has not been evaluated in an in vivo disease model. Here, we investigated whether a translational activator could improve phenotype in a Dravet syndrome mouse model, a severe developmental and epileptic encephalopathy caused by SCN1a haploinsufficiency, by increasing translation of the SCN1a mRNA. We identify and engineer human proteins capable of increasing mRNA translation using the CRISPR-Cas-inspired RNA-targeting system (CIRTS) platform to enable programmable, guide RNA-directed translational activation with entirely engineered human proteins. We identify a compact (601 amino acid) CIRTS translational activator (CIRTS-4GT3) that can drive targeted, sustained translation increases up to 100% from three endogenous transcripts relevant to epilepsy and neurodevelopmental disorders. AAV-delivery of CIRTS-4GT3 targeting SCN1a mRNA to a Dravet syndrome mouse model led to increased SCN1a translation and improved survivability and seizure threshold-key phenotypic indicators of Dravet syndrome. This work validates a strategy to address SCN1a haploinsufficiency and emphasizes the preclinical potential of targeted translational activation to address neurological haploinsufficiency.},
}

Animals
Humans
Mice
*Protein Biosynthesis
*Epilepsies, Myoclonic/genetics/therapy
Disease Models, Animal
RNA, Messenger/genetics/metabolism
*NAV1.1 Voltage-Gated Sodium Channel/genetics/metabolism
CRISPR-Cas Systems
*Protein Engineering/methods
Haploinsufficiency
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid41691452,
year = {2026},
author = {Li, T and Zeng, F and Zhang, J and Zhang, Y and Yin, W},
title = {Reversing Antibiotic Resistance: Strategies From Adjuvants to Innovative Therapeutics.},
journal = {MicrobiologyOpen},
volume = {15},
number = {1},
pages = {e70233},
pmid = {41691452},
issn = {2045-8827},
support = {H2024201044//Natural Science Foundation of Hebei Province/ ; 20231560//The Medical Scientific Research of Hebei Health Commission/ ; },
mesh = {*Anti-Bacterial Agents/pharmacology/therapeutic use ; Humans ; *Bacteria/drug effects/genetics ; *Drug Resistance, Bacterial/drug effects/genetics ; Gene Editing ; *Bacterial Infections/drug therapy/microbiology ; CRISPR-Cas Systems ; Photochemotherapy ; Nanotechnology ; },
abstract = {The escalating prevalence of antibiotic resistance has become a major threat to the effectiveness of conventional antibiotics. Meanwhile, the development of novel antibiotics faces substantial challenges, including lengthy research cycles, high costs, and the rapid emergence of bacterial tolerance, making it difficult for new drugs to keep pace with bacterial evolution. In this context, molecular reversal strategies targeting antibiotic resistance genes have emerged as a promising avenue to overcome this impasse. Among them, the use of antibiotic adjuvants, agents that enhance the efficacy of existing antibiotics by inhibiting resistance gene function, preventing their horizontal transfer or modulating host defense has gained considerable attention. Furthermore, innovative approaches such as CRISPR-Cas gene editing, photodynamic therapy, nanotechnology, and ecological competition strategies have shown great potential in reversing antimicrobial resistance. Collectively, these strategies offer novel insights into addressing the global crisis of antibiotic resistance, paving the way for more effective clinical interventions and ensuring the sustained efficacy of current antibiotic therapies.},
}

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

@article {pmid41691442,
year = {2026},
author = {Thevendran, R and Maheswaran, S},
title = {Molecular Genetics as the Leading-Edge Approach in Driving the Development of Live Attenuated Vaccines.},
journal = {Biotechnology journal},
volume = {21},
number = {2},
pages = {e70185},
doi = {10.1002/biot.70185},
pmid = {41691442},
issn = {1860-7314},
support = {//MOHE/ ; },
mesh = {*Vaccines, Attenuated/genetics/immunology ; Humans ; *Vaccine Development/methods ; Gene Editing ; *Molecular Biology/methods ; Animals ; CRISPR-Cas Systems ; Biotechnology ; },
abstract = {Molecular genetics has propelled advancements in scientific instrumentation, yielding transformative discoveries from CRISPR-mediated gene editing and detailed protein identification to sophisticated biosensor fabrications. This profound shift has also reshaped the landscape of live attenuated vaccine (LAV) development in contrast to conventional methods. Here we explore how modern molecular strategies have superseded previous empirical approaches, moving toward deliberate genetic modifications that both enhance and balance the aspects of LAV safety, stability, and potent immunogenicity. By detailing the transition from classical approaches to targeted, molecular-driven attenuation, our work reviews how advanced genetic methods address historical limitations and expand the potential for vaccine design. Hence, the current paper bridges the gap between foundational vaccine practices and cutting-edge biotechnology, offering a comprehensive perspective on the progression of molecular genetic strategies on LAV development and its future trajectory. The paper also elaborates on the key challenges of raising LAVs to clinical standards while describing in tandem the genetic approaches to overcome the limitations. Critical performance factors governing the LAV market and clinical deployment, alongside the pivotal role of artificial intelligence in refining LAV rational design, are also further discussed.},
}

*Vaccines, Attenuated/genetics/immunology
Humans
*Vaccine Development/methods
Gene Editing
*Molecular Biology/methods
Animals
CRISPR-Cas Systems
Biotechnology

@article {pmid41690735,
year = {2026},
author = {Li, G and Su, Z and Li, F and Liu, J and Shi, Y and Chen, J and Wang, P and Wang, R},
title = {Advances in rapid on-site detection techniques for food safety and authenticity.},
journal = {Advances in food and nutrition research},
volume = {118},
number = {},
pages = {43-87},
doi = {10.1016/bs.afnr.2025.08.004},
pmid = {41690735},
issn = {1043-4526},
mesh = {*Food Safety/methods ; *Food Contamination/analysis ; Biosensing Techniques/methods ; *Food Analysis/methods ; Humans ; Food Microbiology ; },
abstract = {Ensuring food safety and authenticity is a pressing global concern. This chapter provides a comprehensive overview of advanced rapid, on-site detection technologies targeting foodborne hazards, including pathogens, toxins, chemical contaminants, and authenticity issues such as adulteration and species fraud. Key aspects of these technologies include signal amplification strategies like catalytic hairpin assembly (CHA) and nanozyme-enhanced systems, as well as signal output platforms encompassing colorimetric, electrochemical, and fluorescence-based methods. The chapter also explores the emerging role of nanopore-based sensing as a novel signal output platform, emphasizing its single-molecule precision and broad applicability. By integrating laboratory innovations with practical field applications, this chapter underscores the potential of these technologies to address challenges in food safety and authenticity monitoring effectively.},
}

*Food Safety/methods
*Food Contamination/analysis
Biosensing Techniques/methods
*Food Analysis/methods
Humans
Food Microbiology

@article {pmid41679990,
year = {2026},
author = {Carota, AG and Spiaggia, F and Poma, N and Palladino, P and Cuffaro, D and Vivaldi, F and Ravelet, C and Di Francesco, F and Minunni, M},
title = {Highly fluorescent copper nanoclusters as programmable reporters for CRISPR/Cas12a-based detection of bacterial DNA.},
journal = {Biosensors & bioelectronics},
volume = {300},
number = {},
pages = {118492},
doi = {10.1016/j.bios.2026.118492},
pmid = {41679990},
issn = {1873-4235},
mesh = {*CRISPR-Cas Systems/genetics ; *Copper/chemistry ; *Biosensing Techniques/methods ; *DNA, Bacterial/isolation & purification/genetics ; Fluorescent Dyes/chemistry ; *Escherichia coli/genetics/isolation & purification ; *Metal Nanoparticles/chemistry ; Humans ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Early and accessible pathogen detection is crucial for global health security and demands diagnostic assays that are rapid, affordable, and suitable for Point-of-Care use. This study presents a cost-effective, rapid, one-pot fluorescence assay for bacterial DNA detection that exploits the unique optical properties of DNA-templated copper nanoclusters (CuNCs). These nanoclusters offer a sustainable alternative to conventional fluorophores, thanks to their eco-friendly synthesis, high photostability, and large Stokes shift. The assay integrates CuNCs with the CRISPR/Cas12a system to achieve programmable and highly specific target recognition. Upon target binding, activation of the Cas12a/gRNA complex triggers collateral cleavage of rationally designed DNA templates that normally support CuNCs formation, resulting in a marked fluorescence decrease. A panel of hairpin and poly-thymine DNA structures was systematically evaluated to maximize both CuNCs fluorescence and responsiveness to Cas12a/gRNA trans-cleavage, ultimately identifying an AT-rich stem-loop reporter that provided strong signal intensity and complete signal shutdown upon target recognition. The final CRISPR-CuNCs assay achieved picomolar sensitivity, accurately detected E. coli DNA from reference strains, clinical isolates, and serum-spiked samples, and required no fluorophore-quencher probes or multistep procedures. Overall, this work demonstrated that combining the programmability of CRISPR/Cas12a with the versatility and low-cost of DNA-templated CuNCs enables a robust and accessible platform for molecular diagnostics, with strong potential for Point-of-Care deployment.},
}

*CRISPR-Cas Systems/genetics
*Copper/chemistry
*Biosensing Techniques/methods
*DNA, Bacterial/isolation & purification/genetics
Fluorescent Dyes/chemistry
*Escherichia coli/genetics/isolation & purification
*Metal Nanoparticles/chemistry
Humans
Limit of Detection
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41679028,
year = {2026},
author = {Yao, F and Qi, X and Yongli, S and Xiaofen, Z},
title = {Generation of a SV2A knockout human embryonic stem cell line by CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {91},
number = {},
pages = {103924},
doi = {10.1016/j.scr.2026.103924},
pmid = {41679028},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism/cytology ; *Membrane Glycoproteins/genetics/metabolism/deficiency ; *Nerve Tissue Proteins/genetics/metabolism/deficiency ; *Gene Knockout Techniques ; Cell Line ; },
abstract = {Synaptic Vesicle Glycoprotein 2A (SV2A) is a ubiquitously expressed brain glycoprotein, localized to synaptic terminals. It regulates vesicle exocytosis, maintains neurotransmitter release, and serves as a receptor for both botulinum neurotoxins (e.g., BoNT/A) and tetanus neurotoxin (TeNT). It is a target for antiseizure drugs and implicated in epilepsy, Alzheimer's, and Parkinson's diseases. We generated a homozygous SV2A-knockout human embryonic stem cell (hESC) line WAe001-A-3F (H1-SV2A[-/-]), using CRISPR/Cas9 genome editing technology. The SV2A-knockout embryonic stem cell lines provide a precise in vitro model to dissect its roles in synaptic function and disease mechanisms.},
}

Humans
*CRISPR-Cas Systems/genetics
*Human Embryonic Stem Cells/metabolism/cytology
*Membrane Glycoproteins/genetics/metabolism/deficiency
*Nerve Tissue Proteins/genetics/metabolism/deficiency
*Gene Knockout Techniques
Cell Line

@article {pmid41655494,
year = {2026},
author = {Kantor, I and Wright, JL and Amor, DJ and Lockhart, PJ},
title = {Generation of two tetracycline-inducible NGN2 iN iPSC lines carrying a heterozygous floating-Harbor syndrome SRCAP truncating mutation.},
journal = {Stem cell research},
volume = {91},
number = {},
pages = {103922},
doi = {10.1016/j.scr.2026.103922},
pmid = {41655494},
issn = {1876-7753},
mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology/drug effects ; Humans ; *Nerve Tissue Proteins/genetics/metabolism ; *Mutation/genetics ; *Tetracycline/pharmacology ; Heterozygote ; Cell Line ; Cell Differentiation ; CRISPR-Cas Systems ; },
abstract = {Floating-Harbor syndrome (FHS) is a rare neurodevelopmental disorder caused by truncating variants in the last two exons of the gene encoding the chromatin remodeler SRCAP. We used CRISPR-Cas9 genome editing to introduce a monoallelic c.7330C > T (p.Arg2444*) truncating mutation into a published WTC11 iPSC line containing a tetracycline-inducible NGN2 transgene. We characterised two independent lines that maintained a normal karyotype, pluripotency and the ability to differentiate in vitro into all three embryonic germ layers. These lines can be rapidly differentiated into cortical neurons through the addition of doxycycline, making them a useful model for understanding the pathogenic mechanisms underlying FHS.},
}

*Induced Pluripotent Stem Cells/metabolism/cytology/drug effects
Humans
*Nerve Tissue Proteins/genetics/metabolism
*Mutation/genetics
*Tetracycline/pharmacology
Heterozygote
Cell Line
Cell Differentiation
CRISPR-Cas Systems

@article {pmid41653914,
year = {2026},
author = {Chandrasekaran, SS and Tau, C and Fu, BXH and Nemeth, M and Bartie, L and Pawluk, A and Konermann, S and Hsu, PD},
title = {Rewriting endogenous human transcripts with dual CRISPR-guided 3' trans-splicing.},
journal = {Cell systems},
volume = {17},
number = {2},
pages = {101487},
doi = {10.1016/j.cels.2025.101487},
pmid = {41653914},
issn = {2405-4720},
mesh = {Humans ; *Trans-Splicing/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; RNA, Messenger/genetics ; RNA Editing/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; HEK293 Cells ; RNA Precursors/genetics ; Exons/genetics ; },
abstract = {Unlike genome editing, RNA editing offers the ability to transiently alter cells with minimal risk from off-target effects. While exon-skipping technologies can influence splice site selection, many desired perturbations to the transcriptome require replacement or addition of exogenous exons to target mRNAs, such as replacing disease-causing exons, repairing truncated proteins, or engineering protein fusions. Here, we report the development of RNA-guided trans-splicing with Cas editor (RESPLICE). RESPLICE uses two orthogonal RNA-targeting CRISPR effectors to co-localize a trans-splicing pre-mRNA and to inhibit the cis-splicing reaction, respectively. We demonstrate efficient, specific, and programmable trans-splicing of RNA cargo (up to 2.1 kb) into 11 endogenous transcripts across 3 cell types, achieving up to 45% trans-splicing efficiency in bulk or 90% when sorting for high effector expression. Our results present RESPLICE as a mode of RNA editing that could provide fine-tuned and transient control of cellular programs.},
}

Humans
*Trans-Splicing/genetics
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
RNA, Guide, CRISPR-Cas Systems/genetics
RNA, Messenger/genetics
RNA Editing/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
HEK293 Cells
RNA Precursors/genetics
Exons/genetics

@article {pmid41649621,
year = {2026},
author = {Alavian, F and Ghasemi, S},
title = {CRISPR-Based Therapy for Ischemic Stroke: A Narrative Review.},
journal = {Cellular and molecular neurobiology},
volume = {46},
number = {1},
pages = {45},
pmid = {41649621},
issn = {1573-6830},
mesh = {Humans ; Animals ; *Ischemic Stroke/therapy/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Genetic Therapy/methods ; },
abstract = {Ischemic stroke (IS) is one of the most common neurological diseases worldwide and is caused by the blockage of cerebral blood vessels, leading to reduced blood flow and neuronal damage. Given the limitations of existing treatments, CRISPR gene-editing technology has emerged as a promising strategy to precisely target the molecular pathways underlying IS pathophysiology. By enabling intervention in genes regulating inflammation, apoptosis, and repair, CRISPR enables more precise and effective therapies. Various CRISPR delivery systems, including viral vectors, nanocarriers, and extracellular vesicles, play crucial roles in the effective access of this tool to neural cells. Studies have shown that the use of CRISPR-Cas9 to modulate key pathogenic pathways, including those governing inflammation, oxidative stress, and cell death, can prevent neuronal damage and improve neurological function. Additionally, targeting ncRNAs and RNA methylation with CRISPR-based systems plays a role in regulating oxidative stress and stress granule formation. The use of CRISPR to modulate cell communication and organelle transfer and correct mitochondrial mutations has also been considered a neuroprotective mechanism. Despite persistent challenges in targeted and safe delivery, substantial preclinical advances, primarily in rodent models, underscore the potential for CRISPR-based therapies to transform future stroke treatment. These findings suggest that CRISPR-based strategies could evolve into precision neurotherapeutics that address root molecular pathologies, potentially complementing or surpassing current stroke interventions.},
}

Humans
Animals
*Ischemic Stroke/therapy/genetics
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Genetic Therapy/methods

@article {pmid41645777,
year = {2026},
author = {Wu, H and Lin, S and Zuo, X and Hua, W and Sun, T and Shao, G and Li, F and Zhao, D and Feng, Z and Zhu, D and Wang, L},
title = {RAA-CRISPR-HCR cascade amplification for ultrasensitive visual detection of African swine fever virus DNA.},
journal = {Chemical communications (Cambridge, England)},
volume = {62},
number = {14},
pages = {4329-4332},
doi = {10.1039/d5cc06815k},
pmid = {41645777},
issn = {1364-548X},
mesh = {*African Swine Fever Virus/genetics/isolation & purification ; *DNA, Viral/analysis/genetics ; *Nucleic Acid Amplification Techniques/methods ; Animals ; Colorimetry/methods ; Limit of Detection ; Swine ; *CRISPR-Cas Systems ; African Swine Fever/diagnosis/virology ; },
abstract = {An RAA-CRISPR-HCR (RCH) cascade amplification system was developed, which generates an ultrasensitive colorimetric response to low concentrations of African swine fever virus (ASFV) DNA with a detection limit of 1 copy µL[-1]. It demonstrates excellent accuracy and specificity in real samples, facilitating efficient on-site visual early detection of ASFV.},
}

*African Swine Fever Virus/genetics/isolation & purification
*DNA, Viral/analysis/genetics
*Nucleic Acid Amplification Techniques/methods
Animals
Colorimetry/methods
Limit of Detection
Swine
*CRISPR-Cas Systems
African Swine Fever/diagnosis/virology

@article {pmid41643424,
year = {2026},
author = {Wang, S and Li, C and Zhu, L and Liu, K and Jing, L and Xie, L and Guo, H and Ge, S and Yu, J},
title = {Engineered DNA hydrogel paper chip biosensor by cascaded hybridization chain reaction-assisted CRISPR/Cas12a system for sensitive detection of miRNA 622.},
journal = {Biosensors & bioelectronics},
volume = {300},
number = {},
pages = {118482},
doi = {10.1016/j.bios.2026.118482},
pmid = {41643424},
issn = {1873-4235},
mesh = {*MicroRNAs/genetics/isolation & purification/analysis ; *Biosensing Techniques/instrumentation ; CRISPR-Cas Systems/genetics ; Hydrogels/chemistry ; Limit of Detection ; Nucleic Acid Hybridization ; Humans ; Paper ; *DNA/chemistry/genetics ; Endodeoxyribonucleases/chemistry/genetics ; Metal Nanoparticles/chemistry ; Gold/chemistry ; Electrochemical Techniques ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {The integration of DNA hydrogel with electrochemiluminescence (ECL) technology represents a synergistic enhancement through molecular-level precision design and nanoscale coordination. This strategic integration confers biosensors novel functionalities including intelligent responsiveness and environmental adaptability. In this study, a cascaded hybridization chain reaction (HCR) and CRISPR/Cas12a-integrated DNA hydrogel paper chip was engineered for ultrasensitive microRNA 622 (miRNA 622) detection. Target miRNA 622 triggered HCR amplification via hairpin DNA assembly, while Cas12a recognized protospacer adjacent motif (PAM) sequences within the HCR-generated double-stranded products to activate its trans-cleavage ability. The DNA hydrogel was constructed through copolymerization of acrylamide-modified DNA single strands (SA and SB) with Ru (II) complex-functionalized linker DNA. Activated Cas12a cleaved single-stranded DNA within the DNA hydrogel network, thereby releasing Ru (II) complexes. AuPd nanoparticles (AuPd NPs) served as the co-reactant accelerator, amplifying the cathodic ECL signals of the liberated Ru (II) complexes. The developed platform demonstrated a dynamic detection range from 0.001 to 500 pM with a detection limit of 0.33 fM, establishing a groundbreaking approach for detecting miRNA 622 in clinical diagnostics.},
}

*MicroRNAs/genetics/isolation & purification/analysis
*Biosensing Techniques/instrumentation
CRISPR-Cas Systems/genetics
Hydrogels/chemistry
Limit of Detection
Nucleic Acid Hybridization
Humans
Paper
*DNA/chemistry/genetics
Endodeoxyribonucleases/chemistry/genetics
Metal Nanoparticles/chemistry
Gold/chemistry
Electrochemical Techniques
Bacterial Proteins
CRISPR-Associated Proteins

@article {pmid41642051,
year = {2026},
author = {Yang, Y and Yang, L and Ma, H and Zhang, S and Zhu, Y and Zhang, S and Lin, X and La, H and Gu, X and Ma, J and Zhao, S and Yang, Y and Lei, H and Yang, Y},
title = {Precision detection of rifampicin-resistant rpoB_L378R mutation in Mycobacterium tuberculosis with CRISPR-Cas12a.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {7},
pages = {1442-1453},
doi = {10.1039/d5ay01718a},
pmid = {41642051},
issn = {1759-9679},
mesh = {*Rifampin/pharmacology ; *Mycobacterium tuberculosis/genetics/drug effects ; *CRISPR-Cas Systems/genetics ; *Drug Resistance, Bacterial/genetics ; *Bacterial Proteins/genetics ; *DNA-Directed RNA Polymerases/genetics ; Mutation ; Humans ; Antitubercular Agents/pharmacology ; Nucleic Acid Amplification Techniques/methods ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Rifampicin is one of the most effective anti-tuberculosis drugs. However, certain strains of Mycobacterium tuberculosis (MTB) have developed resistance to rifampicin, making it crucial to identify alternative drugs for treating rifampicin-resistant MTB infections. Mutations in the rpoB gene play a pivotal role in MTB's resistance to rifampicin. Therefore, identifying these mutations is essential for effectively managing rifampicin-resistant MTB strains. Here, we developed a CRISPR-Cas12a platform integrated with recombinase polymerase amplification (RPA) and fluorescence detection, which was specifically designed to identify the rpoB_L378R mutation associated with rifampicin resistance in MTB. Our findings indicated that this detection technique exhibited high specificity and did not cross-react with reference samples constructed from the genomes of MTB H37Rv, Mycobacterium smegmatis, Mycobacterium aurum, and Escherichia coli. The RPA-CRISPR-Cas12a-based platform established in this research was simple, sensitive, and specific for detecting the rifampicin-resistant MTB strain with the rpoB_L378R mutation. These results suggest its potential applicability in clinical diagnosis for identifying the MTB rpoB_L378R mutation.},
}

*Rifampin/pharmacology
*Mycobacterium tuberculosis/genetics/drug effects
*CRISPR-Cas Systems/genetics
*Drug Resistance, Bacterial/genetics
*Bacterial Proteins/genetics
*DNA-Directed RNA Polymerases/genetics
Mutation
Humans
Antitubercular Agents/pharmacology
Nucleic Acid Amplification Techniques/methods
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41638184,
year = {2026},
author = {Li, Z and Zhang, W and Feng, Z and Liu, Z and Feng, Z and Shi, Y and Zhan, J and Zhang, J},
title = {A turn-on CRISPR/Cas12a strategy featuring a sterically-hindered activator for in situ fluorescence imaging of H2O2 in vivo.},
journal = {Biosensors & bioelectronics},
volume = {300},
number = {},
pages = {118449},
doi = {10.1016/j.bios.2026.118449},
pmid = {41638184},
issn = {1873-4235},
mesh = {*Hydrogen Peroxide/isolation & purification/analysis/chemistry ; *Biosensing Techniques/methods ; Animals ; *CRISPR-Cas Systems/genetics ; Mice ; Humans ; *Optical Imaging/methods ; Limit of Detection ; *Endodeoxyribonucleases/chemistry/genetics ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Hydrogen peroxide (H2O2) serves as a key biomarker of oxidative stress in pathological processes such as cancer and inflammation. However, its in vivo visualization remains challenging due to the lack of sensitive, rapid, and bioorthogonal imaging methods. Here, we present a H2O2-activatable CRISPR/Cas12a strategy, termed A-BO-CRISPR, for real-time fluorescence imaging in living systems. This biosensing strategy employs a 4-bromomethylphenylboronic acid pinacol ester-caged DNA activator whose binding to crRNA is initially blocked by steric hindrance, effectively suppressing Cas12a trans-cleavage activity. Upon encountering endogenous H2O2, the boronate ester is selectively hydrolyzed, restoring activator/crRNA hybridization and triggering amplified fluorescent signal generation via Cas12a-mediated collateral cleavage of a ssDNA reporter. The system achieves a detection limit of 0.64 μM and responds within minutes, enabling real-time monitoring of H2O2 fluxes in living cells and tumor-bearing mice. It exhibits high selectivity and robust stability in complex biological environments. By integrating a chemical gating mechanism with CRISPR-based signal amplification, this work paves the way for potential applications in probing redox biology, imaging-guided diagnostics and therapeutic monitoring.},
}

*Hydrogen Peroxide/isolation & purification/analysis/chemistry
*Biosensing Techniques/methods
Animals
*CRISPR-Cas Systems/genetics
Mice
Humans
*Optical Imaging/methods
Limit of Detection
*Endodeoxyribonucleases/chemistry/genetics
Bacterial Proteins
CRISPR-Associated Proteins

@article {pmid41628878,
year = {2026},
author = {Gao, S and Wang, L and Hou, M and Zhang, M and Zhu, X and Luo, H and Yu, X and Lv, H and Chen, S and Huang, Y and Zhang, K and Wu, J},
title = {Gene insertion and transcriptional regulation of Escherichia coli based on CRISPR-associated transposases.},
journal = {International journal of biological macromolecules},
volume = {348},
number = {},
pages = {149850},
doi = {10.1016/j.ijbiomac.2025.149850},
pmid = {41628878},
issn = {1879-0003},
mesh = {*Escherichia coli/genetics ; *Gene Expression Regulation, Bacterial ; *Transposases/genetics/metabolism ; *CRISPR-Cas Systems ; *Transcription, Genetic ; Plasmids/genetics ; },
abstract = {Synthetic biology aims to construct robust microbial cell factories for sustainable biomanufacturing. A major obstacle lies in the difficulty of efficiently integrating large polycistronic expression cassettes into the genome and flexibly regulating gene expression. Here, a programmable tool MUSCULAR-CAST was developed based on type IF CRISPR-associated transposases (CAST) system Tn6677. Using MUSCULAR-CAST, we have achieved efficient genomic integration of various size polycistronic expression cassettes (1-10K). Among these, a human milk oligosaccharide 3-fucosyllactose (3-FL) chassis strain was successfully constructed, with similar yields and better growth compared with that of plasmid expression strain, and a plasmid-free cutinase recombinant expression strain was constructed, with enzyme activity higher than that of plasmid containing strain. Meanwhile, we developed a gene repression tool Tn-CRISPRi based on the targeting module of MUSCULAR-CAST, which achieved high single-gene repression across diverse PAM sequences and near-complete dual-gene suppression (98.6-99.8%). Applying Tn-CRISPRi to repress 17 genes competing with 3-FL biosynthesis or nonessential for growth revealed that knockdown of osmoregulated periplasmic glucans biosynthesis protein H (mdoH) and motility protein A (motA) increased 3-FL titers by 2.79- and 4.4-fold, respectively. This study establishes MUSCULAR-CAST and Tn-CRISPRi as efficient tools for genomic integration and transcriptional regulation, providing a scalable framework for advanced chassis strain engineering in synthetic biology.},
}

*Escherichia coli/genetics
*Gene Expression Regulation, Bacterial
*Transposases/genetics/metabolism
*CRISPR-Cas Systems
*Transcription, Genetic
Plasmids/genetics

@article {pmid41622828,
year = {2026},
author = {Sen, MK and Roy, A and Varshney, RK and Chakraborty, A},
title = {Engineering next-generation crops through CRISPR-mediated horizontal gene transfer.},
journal = {The New phytologist},
volume = {249},
number = {6},
pages = {2683-2689},
doi = {10.1111/nph.70951},
pmid = {41622828},
issn = {1469-8137},
mesh = {*Crops, Agricultural/genetics ; *Gene Transfer, Horizontal/genetics ; Gene Editing ; *CRISPR-Cas Systems/genetics ; *Genetic Engineering/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Crops increasingly face overlapping stresses such as heat, drought, salinity, and pathogens that conventional breeding or genome editing rarely overcome in combination. To address this, we propose CRISPR-enabled horizontal gene transfer (CRISPR-HGT) as a programmable framework that recreates the evolutionary process by which plants historically acquired adaptive microbial genes. Microbial genes, refined under extreme environments, provide a naturally preadapted resource for multi-trait resilience. By integrating tools such as Cas12a, CasΦ, RNA-targeting, and dCas-based epigenome editors with AI-guided microbial gene discovery, CRISPR-HGT enables modular and inducible stress regulation. This approach shifts genome editing from allelic modification to evolution-guided design. We outline a conceptual pipeline spanning microbial gene mining to adaptive field deployment, highlighting the ecological, biosafety, and regulatory dimensions, from the European Union's cautious oversight to the UK's product-based framework. CRISPR-HGT thus introduces an evolution-informed paradigm for engineering crops that anticipate stress and sustain yield under climate uncertainty.},
}

*Crops, Agricultural/genetics
*Gene Transfer, Horizontal/genetics
Gene Editing
*CRISPR-Cas Systems/genetics
*Genetic Engineering/methods
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid41620031,
year = {2026},
author = {Jiang, Y and Yang, Z and Yang, J and Li, Y and Liu, J and Zhao, L and Ge, J},
title = {Development of a rapid and portable detection method for canine distemper virus based on CRISPR-Cas13a.},
journal = {Journal of virological methods},
volume = {342},
number = {},
pages = {115355},
doi = {10.1016/j.jviromet.2026.115355},
pmid = {41620031},
issn = {1879-0984},
mesh = {Animals ; *Distemper Virus, Canine/isolation & purification/genetics ; Sensitivity and Specificity ; Dogs ; *CRISPR-Cas Systems ; *Distemper/diagnosis/virology ; *Nucleic Acid Amplification Techniques/methods ; *Molecular Diagnostic Techniques/methods ; RNA, Viral/genetics ; },
abstract = {Canine distemper virus (CDV) is a pathogenic microorganism that severely affects the respiratory, digestive, and nervous systems, causing multi-systemic symptoms. It infects nearly all terrestrial carnivores worldwide, particularly the Canidae and Mustelidae families, posing a serious threat to global socio-economic and public health security. Given the importance of etiological treatment and early diagnosis, developing novel detection methods with improved accuracy, rapidity, and user-friendliness is necessary for effective prevention and control of CDV infection. In this study, we established a novel testing method using recombinase-aid amplification (RAA) coupled with CRISPR-Cas13a and optimized the working concentration of CRISPR RNA (crRNA) and Cas13a for the lateral flow detection (LFD) of CDV. The RAA-CRISPR-Cas13a-LFD for CDV did not cross-react against other prevalent canine pathogens and the sensitivity can detect as little as 10[2] copies/μL of CDV cDNA plasmids. Additionally, combined with HUDSON this RAA-CRISPR-Cas13a-LFD method could be used to detect clinical samples within 1.5 h, with performance comparable to that of RT-PCR. The results for the RAA-CRISPR-Cas13a detection could be visualized using either fluorescence or lateral flow strips for in field-deployable viral diagnosis. Overall, our developed method showed good potential in point-of-care testing (POCT) to control and reduce the losses by CDV infection.},
}

Animals
*Distemper Virus, Canine/isolation & purification/genetics
Sensitivity and Specificity
Dogs
*CRISPR-Cas Systems
*Distemper/diagnosis/virology
*Nucleic Acid Amplification Techniques/methods
*Molecular Diagnostic Techniques/methods
RNA, Viral/genetics

@article {pmid41593699,
year = {2026},
author = {Zhu, J and Li, Y and Yu, C and Huang, W and Chen, J and Liu, X and Qin, R and Li, J and Xu, R and Wei, P},
title = {Engineering hypercompact IscB nucleases for efficient and versatile genome editing in rice.},
journal = {Genome biology},
volume = {27},
number = {1},
pages = {49},
pmid = {41593699},
issn = {1474-760X},
mesh = {*Oryza/genetics ; *Gene Editing/methods ; CRISPR-Cas Systems ; Genome, Plant ; *Endonucleases/genetics/metabolism ; },
abstract = {BACKGROUND: IscB (Insertion sequences Cas9-like OrfB) represents a novel class of RNA-guided nucleases, approximately one-third the size of Cas9 proteins. Despite the limited natural efficiency in eukaryotic cells, recent advances have led to the engineering of several IscBs for mammalian genome editing.

RESULTS: In this study, we screen and identify high-activity IscB variants for rice. A version of pIscB-v3, combining enOgeuIscB and ωRNA-v13, demonstrated superior mutagenesis efficiency compared to other systems. The average editing efficiency of pIscB-v3 is 17.61% from ten endogenous targets, and we obtain edited lines in up to 83.33% of T0 generation with 33.33% of homozygous and bi-allelic mutations. Further analysis reveals that pIscB-v3 exhibits high editing specificity and relaxed target-adjacent motif (TAM) compatibility in rice. Beyond gene knockout systems, we develop cytosine base editors (CBEs) and adenine base editors (ABEs) from pIscB-v3. We find that the ssDNA-targeting SCP1.201 family deaminase Sdd7 outperformed human APOBEC3A in IscB-CBEs for C-to-T conversions in rice. The Sdd7-nIscB achieves precise edits in 22.92% of lines on average, with a maximum frequency of 47.92%. Additionally, TadA8e-nIscB exhibits limited activity. However, fusing an extra copy of TadA-8e to either terminus of TadA8e-nIsc significantly enhances A-to-G conversions.

CONCLUSIONS: Collectively, our results demonstrate the robust capabilities of IscB to develop an efficient and versatile miniature plant genome editing toolkit to substantially facilitate crop breeding.},
}

*Oryza/genetics
*Gene Editing/methods
CRISPR-Cas Systems
Genome, Plant
*Endonucleases/genetics/metabolism

@article {pmid41579593,
year = {2026},
author = {Jovanovic, VM and Rausch, R and DeRosa, MC and Castellano, D and McKee, C and Sen, C and Daly, F and Doege, CA and Tristan, CA},
title = {Generation and characterization of POMC-tdTomato reporter human pluripotent stem cell lines.},
journal = {Stem cell research},
volume = {91},
number = {},
pages = {103905},
doi = {10.1016/j.scr.2026.103905},
pmid = {41579593},
issn = {1876-7753},
mesh = {Humans ; *Pro-Opiomelanocortin/metabolism/genetics ; *Pluripotent Stem Cells/metabolism/cytology ; Neurons/metabolism/cytology ; Cell Line ; Cell Differentiation ; Genes, Reporter ; CRISPR-Cas Systems ; Red Fluorescent Protein ; },
abstract = {Proopiomelanocortin (POMC) is a precursor polypeptide that undergoes extensive, tissue-specific post-translational processing. It is expressed in several tissues, including pituitary gland, hypothalamus, brain stem, and skin. The hypothalamic POMC neurons in the arcuate nucleus are major neuronal populations involved in the regulation of body weight. In these neurons, POMC is processed into several peptides, among them the anorexigenic alpha-melanocyte stimulating hormone. Thus, the POMC neurons in the ARC have been named "satiety" neurons and are highly desirable drug targets. Here, we performed CRISPR/Cas9-mediated insertion of tdTomato reporter at the endogenous POMC locus, enabling direct visualization of POMC expression through tdTomato fluorescence in human pluripotent stem cell (hPSC)-derived hypothalamic neurons. This reporter line enables real-time visualization of POMC neuron differentiation, and selective enrichment of these populations for molecular, functional, and pharmacological studies. This line is readily available as new alternative method (NAM) platform, to support disease modeling and drug discovery in metabolic and neuroendocrine disorders within a human context.},
}

Humans
*Pro-Opiomelanocortin/metabolism/genetics
*Pluripotent Stem Cells/metabolism/cytology
Neurons/metabolism/cytology
Cell Line
Cell Differentiation
Genes, Reporter
CRISPR-Cas Systems
Red Fluorescent Protein

@article {pmid41546923,
year = {2026},
author = {Patil, S and Das, A and Inamdar, MS},
title = {Generation of a Brachyury reporter cell line (BJNhem20 Brachyury (TBXT)-2A-EGFP) in human embryonic stem cells using CRISPR-Cas9 gene targeting.},
journal = {Stem cell research},
volume = {91},
number = {},
pages = {103907},
doi = {10.1016/j.scr.2026.103907},
pmid = {41546923},
issn = {1876-7753},
mesh = {Humans ; Brachyury Protein ; *T-Box Domain Proteins/genetics/metabolism ; *Fetal Proteins/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism/cytology ; Cell Line ; *Green Fluorescent Proteins/metabolism/genetics ; Genes, Reporter ; Cell Differentiation ; },
abstract = {Brachyury is a key transcription factor, which is required for mesoderm lineage development. Here, we have generated a Brachyury/TBXT-2A-EGFP knock-in Reporter line in the BJNhem20 human embryonic stem cell line, using CRISPR/Cas9-based gene editing. Successful gene editing was verified by DNA sequencing and comparing endogenous gene expression to reporter gene expression. This reporter line represents an important tool for tracking and assessing mesoderm differentiation.},
}

Humans
Brachyury Protein
*T-Box Domain Proteins/genetics/metabolism
*Fetal Proteins/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Human Embryonic Stem Cells/metabolism/cytology
Cell Line
*Green Fluorescent Proteins/metabolism/genetics
Genes, Reporter
Cell Differentiation

@article {pmid41539085,
year = {2026},
author = {Dong, T and Zhao, Y and Jin, HF and Pan, HM and Yue, LL and Lin, Y and Shen, L},
title = {The establishment of a GPD1L knockout human embryonic stem cell line (WAe009-A-80) using the CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {91},
number = {},
pages = {103910},
doi = {10.1016/j.scr.2026.103910},
pmid = {41539085},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism/cytology ; Cell Line ; *Glycerolphosphate Dehydrogenase/genetics/metabolism/deficiency ; *Gene Knockout Techniques ; Cell Differentiation ; },
abstract = {The GPD1L gene is located on 3p22.3. It encodes the glycerol phosphate dehydrogenase 1-like protein with homology to glycerol phosphate dehydrogenase (GPD1L), but the function of this enzyme is unclear. Mutations in GPD1L have been associated with BrS (Brugada syndrome) and SIDS (sudden infant death syndrome) and reduce Na[+] inward current through an unknown mechanism in human cardiomyocytes. Here, a GPD1L knockout human embryonic stem cell line was generated using CRISPR/Cas9 system. The GPD1L knockout human embryonic stem cell maintains the pluripotency, differentiation into three germ layers, forming normal EBs.},
}

Humans
*CRISPR-Cas Systems/genetics
*Human Embryonic Stem Cells/metabolism/cytology
Cell Line
*Glycerolphosphate Dehydrogenase/genetics/metabolism/deficiency
*Gene Knockout Techniques
Cell Differentiation