@article {pmid40049660,
year = {2025},
author = {Liu, ZX and Zou, TT and Liu, HH and Jia, HB and Zhang, XQ},
title = {Knockout of the fcsk gene in zebrafish causes neurodevelopmental defects.},
journal = {Zoological research},
volume = {46},
number = {2},
pages = {313-324},
doi = {10.24272/j.issn.2095-8137.2024.229},
pmid = {40049660},
issn = {2095-8137},
mesh = {Animals ; *Zebrafish/genetics ; Phosphotransferases (Alcohol Group Acceptor)/genetics/metabolism ; Neurodevelopmental Disorders/genetics ; Gene Knockout Techniques ; Zebrafish Proteins/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Congenital disorders of glycosylation (CDG) are a cluster of monogenic disorders resulting from defects in glycosylation. FCSK encodes fucokinase, an enzyme that catalyzes the phosphorylation of L-fucose to generate fucose-1-phosphate, an important step in fucosylation. Mutations in FCSK lead to CDG with an autosomal recessive inheritance pattern, primarily manifesting as developmental delay, hypotonia, and brain abnormalities. However, no fcsk mutant animal models have yet been established. This study constructed the first fcsk knockout (fcsk [-/-]) zebrafish model using CRISPR/Cas9 technology. Notably, fcsk [-/-] zebrafish exhibited impaired growth, characterized by delayed epiboly and DNA accumulation during early embryonic development, as well as brain atrophy in adulthood. Larval-stage fcsk [-/-] zebrafish displayed locomotor deficits and increased susceptibility to pentylenetetrazole-induced seizures. In adulthood, fcsk [-/-] zebrafish showed neurodevelopmental abnormalities, including increased anxiety, decreased aggression, reduced social preference, and impaired memory. Additionally, total protein fucosylation was markedly reduced in fcsk [-/-] zebrafish, accompanied by decreased expression of pofut2, which encodes protein O-fucosyltransferase 2, an enzyme involved in the fucosylation salvage pathway. Apoptotic activity was elevated in the midbrain-hindbrain boundary (MHB) of fcsk [-/-] zebrafish. Supplementation with GDP-L-fucose or the human FCSK gene restored developmental defects and total protein fucosylation in fcsk [-/-] zebrafish. RNA sequencing revealed dysregulated gene expression associated with glycosylation, apoptosis, and neurodegenerative diseases. These findings suggest that fcsk [-/-] zebrafish exhibit neurodevelopmental disorders, providing the first fcsk gene knockout animal model and offering a platform for investigating the molecular underpinnings of the disease and facilitating drug screening efforts.},
}

Animals
*Zebrafish/genetics
Phosphotransferases (Alcohol Group Acceptor)/genetics/metabolism
Neurodevelopmental Disorders/genetics
Gene Knockout Techniques
Zebrafish Proteins/genetics/metabolism
CRISPR-Cas Systems

@article {pmid40048448,
year = {2025},
author = {Du, Y and Yang, Y and Zhang, Y and Zhang, F and Wu, J and Yin, J},
title = {Unraveling enhanced liver regeneration in ALPPS: Integrating multi-omics profiling and in vivo CRISPR in mouse models.},
journal = {Hepatology communications},
volume = {9},
number = {3},
pages = {},
pmid = {40048448},
issn = {2471-254X},
mesh = {Animals ; *Liver Regeneration/genetics/physiology ; *Hepatectomy ; Mice ; *Portal Vein/surgery ; *Hepatocytes/metabolism ; *MicroRNAs/genetics/metabolism ; Ligation ; *Mice, Knockout ; CRISPR-Cas Systems ; Cell Proliferation/genetics ; Male ; Polo-Like Kinase 1 ; Mice, Inbred C57BL ; Gene Editing/methods ; Liver/surgery ; Protein Serine-Threonine Kinases/genetics/metabolism ; Disease Models, Animal ; Gene Expression Profiling ; Cell Cycle Proteins/genetics/metabolism ; Multiomics ; Hydrolases ; },
abstract = {BACKGROUND: Postoperative liver failure due to insufficient liver cell quantity and function remains a major cause of mortality following surgery. Hence, additional investigation and elucidation are required concerning suitable surgeries for promoting in vivo regeneration.

METHODS: We established the portal vein ligation (PVL) and associated liver partition and portal vein ligation for staged hepatectomy (ALPPS) mouse models to compare their in vivo regeneration capacity. Then, RNA-seq and microRNA-seq were conducted on the livers from both mouse models. Weighted gene co-expression network analysis algorithm was leveraged to identify crucial gene modules. ScRNA-seq analysis was used to understand the distinctions between Signature30high hepatocytes and Signature30low hepatocytes. Moreover, in vivo, validation was performed in fumarylacetoacetate hydrolase knockout mice with gene editing using the CRISPR-cas9 system. A dual luciferase report system was carried out to further identify the regulatory mechanisms.

RESULTS: RNA-seq analysis revealed that ALPPS could better promote cell proliferation compared to the sham and portal vein ligation models. Moreover, a Plk1-related 30-gene signature was identified to predict the cell state. ScRNA-seq analysis confirmed that signature30high hepatocytes had stronger proliferative ability than signature30low hepatocytes. Using microRNA-seq analysis, we identified 53 microRNAs that were time-dependently reduced after ALPPS. Finally, miR-30a-3p might be able to regulate the expression of Plk1, contributing to the liver regeneration of ALPPS.

CONCLUSIONS: ALPPS could successfully promote liver regeneration by activating hepatocytes into a proliferative state. Moreover, a Plk1-related 30-gene signature was identified to predict the cell state of hepatocytes. miR-30a-3p might be able to regulate the expression of Plk1, contributing to the liver regeneration of ALPPS.},
}

Animals
*Liver Regeneration/genetics/physiology
*Hepatectomy
Mice
*Portal Vein/surgery
*Hepatocytes/metabolism
*MicroRNAs/genetics/metabolism
Ligation
*Mice, Knockout
CRISPR-Cas Systems
Cell Proliferation/genetics
Male
Polo-Like Kinase 1
Mice, Inbred C57BL
Gene Editing/methods
Liver/surgery
Protein Serine-Threonine Kinases/genetics/metabolism
Disease Models, Animal
Gene Expression Profiling
Cell Cycle Proteins/genetics/metabolism
Multiomics
Hydrolases

@article {pmid39680504,
year = {2025},
author = {Schmidt, ENC and Evert, BO and Pregler, BEF and Melhem, A and Hsieh, MC and Raspe, M and Strobel, H and Roos, J and Pietsch, T and Schuss, P and Fischer-Posovszky, P and Westhoff, MA and Hölzel, M and Herrlinger, U and Vatter, H and Waha, A and Schneider, M and Potthoff, AL},
title = {Tonabersat enhances temozolomide-mediated cytotoxicity in glioblastoma by disrupting intercellular connectivity through connexin 43 inhibition.},
journal = {Molecular oncology},
volume = {19},
number = {3},
pages = {878-898},
doi = {10.1002/1878-0261.13786},
pmid = {39680504},
issn = {1878-0261},
support = {70113307//Mildred-Scheel School of Oncology (German Cancer Aid)/ ; },
mesh = {*Glioblastoma/drug therapy/pathology/metabolism/genetics ; *Connexin 43/metabolism/genetics ; Humans ; *Temozolomide/pharmacology ; Cell Line, Tumor ; *Gap Junctions/metabolism/drug effects ; Brain Neoplasms/drug therapy/pathology/metabolism/genetics ; Drug Synergism ; Cell Communication/drug effects ; CRISPR-Cas Systems/genetics ; },
abstract = {Glioblastoma cells rely on connexin 43 (Cx43)-based gap junctions (GJs) for intercellular communication, enabling them to integrate into a widely branched malignant network. Although there are promising prospects for new targeted therapies, the lack of clinically feasible GJ inhibitors has impeded their adoption in clinical practice. In the present study, we investigated tonabersat (TO), a blood-brain-barrier-penetrating drug with GJ-inhibitory properties, in regard to its potential to disassemble intercellular connectivity in glioblastoma networks. Fluorescence-guided measurements of calcein cell-to-cell transfer were used to study functional intercellular connectivity. Specific DNA fragmentation rates of propidium iodide-stained nuclei were measured as a surrogate readout for cell death using flow cytometry. CRISPR/Cas9-mediated gene editing of Cx43 served as a validation tool of cellular effects related to Cx43 GJ inhibition. 3' mRNA sequencing was performed for molecular downstream analysis. We found that TO reduced intercellular GJ-mediated cytosolic traffic and yielded a significant reduction of tumor microtube (TM) length. TO-mediated inhibition of cellular tumor networks was accompanied by a synergistic effect for temozolomide-induced cell death. CRISPR/Cas9 Cx43-knockout revealed similar results, indicating that TO-mediated inhibitory effects rely on the inhibition of Cx43-based GJs. Gene set enrichment analyses found that GJ-mediated synergistic cytotoxic effects were linked to a significant upregulation of cell death signaling pathways. In conclusion, TO disrupts TM-based network connectivity via GJ inhibition and renders glioblastoma cells more susceptible to cytotoxic therapy. Given its previous use in clinical trials for migraine therapy, TO might harbor the potential of bridging the idea of a GJ-targeted therapeutic approach from bench to bedside.},
}

*Glioblastoma/drug therapy/pathology/metabolism/genetics
*Connexin 43/metabolism/genetics
Humans
*Temozolomide/pharmacology
Cell Line, Tumor
*Gap Junctions/metabolism/drug effects
Brain Neoplasms/drug therapy/pathology/metabolism/genetics
Drug Synergism
Cell Communication/drug effects
CRISPR-Cas Systems/genetics

@article {pmid40048272,
year = {2025},
author = {Lin, M and Du, Z and Guo, D and Zhang, Y and Dan, J},
title = {Dux cluster duplication ensures full activation of totipotent genes.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {10},
pages = {e2421594122},
doi = {10.1073/pnas.2421594122},
pmid = {40048272},
issn = {1091-6490},
support = {32270845//MOST | National Natural Science Foundation of China (NSFC)/ ; 202201BE070001-006//Yunnan Fundamental Research Projects/ ; 202301AT070304//Yunnan Fundamental Research Projects/ ; KKRD202273103//Xingdian Talent Support Program of Yunnan Province/ ; 202102AA100053//| Natural Science Foundation of Yunnan Province (Yunnan Natural Science Foundation)/ ; },
mesh = {Animals ; Mice ; *Gene Duplication ; *Homeodomain Proteins/genetics/metabolism ; *Mouse Embryonic Stem Cells/metabolism ; *Multigene Family ; Gene Expression Regulation, Developmental ; Embryonic Development/genetics ; Zygote/metabolism ; CRISPR-Cas Systems ; },
abstract = {Zygotic genome activation (ZGA) confers to the mouse two-cell (2C) embryo a unique transcriptional profile characterized by transient up-regulation of many totipotency-related genes and MERVL retrotransposons. Intriguingly, those genes are duplicated and clustered in the genome during evolution, including Dux cluster, Obox, and Zscan4 family members in mice. However, the contribution and biological significance of the totipotency-related gene duplication events in early embryo development remain poorly understood. Here, we focus on Dux cluster, the master regulator of ZGA that is necessary and sufficient for the induction of 2C-like cells and activation of totipotency-related genes in mouse embryonic stem cells (mESCs). By reducing Dux gene copies from 31 to 0 or 1 through CRISPR-Cas9 technology, we generate Dux-KO and Dux (n = 1) mESC lines, respectively. We uncover that the totipotency-related gene transcriptional profile is awakened to a much lesser extent in Dux (n = 1) mESCs compared to wild type mESCs following global DNA demethylation reprogramming or induction of DNA damage, mimicking the intrinsic events in preimplantation development. Together, Dux cluster duplication is critically required for full activation of ZGA transcripts.},
}

Animals
Mice
*Gene Duplication
*Homeodomain Proteins/genetics/metabolism
*Mouse Embryonic Stem Cells/metabolism
*Multigene Family
Gene Expression Regulation, Developmental
Embryonic Development/genetics
Zygote/metabolism
CRISPR-Cas Systems

@article {pmid40047422,
year = {2025},
author = {Layton, AM and McCauley, C and Redding, KE},
title = {Deletion of Re-citrate synthase allows for analysis of contributions of tricarboxylic acid cycle directionality to the growth of Heliomicrobium modesticaldum.},
journal = {Applied and environmental microbiology},
volume = {},
number = {},
pages = {e0177224},
doi = {10.1128/aem.01772-24},
pmid = {40047422},
issn = {1098-5336},
abstract = {UNLABELLED: Heliomicrobium modesticaldum, a phototrophic member of the phylum Firmicutes and family Clostridiales, possesses most of the enzymes specific to the reductive tricarboxylic acid (rTCA) cycle, except for the key enzyme, ATP-citrate lyase. It is thought to utilize a split TCA cycle when growing on pyruvate as a carbon source, in which the oxidative TCA (oTCA) direction generates most of the 2-ketoglutarate, but some can be produced in the reductive direction. Although a typical Si-citrate synthase gene is not found in the genome, it was suggested that gene HM1_2993, annotated as homocitrate synthase, actually encodes Re-citrate synthase, which would function as the initial enzyme of the oTCA cycle. We deleted this gene to test this hypothesis and, if true, see what effect severing access to the oTCA cycle would have on this organism. The endogenous CRISPR-Cas system was used to replace the open reading frame with a selectable marker. The deletion mutants could grow on pyruvate but were unable to grow phototrophically on acetate + CO2 as carbon source. Growth on acetate could be rescued by the addition of different electron sources (formate or ascorbate), suggesting that the oTCA cycle is used to oxidize acetate to generate electrons required to drive the carboxylation of acetyl-CoA. The deletion mutants were capable of growing in acetate minimal media without additional organic supplements beyond formate, demonstrating that the rTCA cycle can be employed to support sufficient 2-ketoglutarate production in this organism, unlike citrate synthase mutants in several chemoheterotrophic organisms utilizing the oTCA cycle.

IMPORTANCE: Heliobacteria are a unique group of phototrophic bacteria that are obligate anaerobes and possess a rudimentary system to use light as a source of energy. They do not make oxygen or fix carbon dioxide. Here, we explore their fundamental carbon metabolism to understand the role and operation of the central TCA cycle. This work shows both the role and operation of this cycle under different growth modes and explains how these organisms can obtain electrons to drive their biosynthetic metabolism. This foundational knowledge will be crucial in the future when attempts are made to use this organism as a platform for oxygen-sensitive synthesis of compounds in an anaerobe that can use light as its energy source.},
}

@article {pmid40045777,
year = {2025},
author = {Moser, TV and Bond, DM and Hore, TA},
title = {Variant ribosomal DNA is essential for female differentiation in zebrafish.},
journal = {Philosophical transactions of the Royal Society of London. Series B, Biological sciences},
volume = {380},
number = {1921},
pages = {20240107},
pmid = {40045777},
issn = {1471-2970},
support = {//Marsden Fund/ ; },
mesh = {Animals ; *Zebrafish/genetics ; Female ; *DNA, Ribosomal/genetics ; Sex Differentiation/genetics ; Male ; CRISPR-Cas Systems ; Gene Editing ; },
abstract = {The ribosome consists of protein and RNA components. Deletion of genes encoding specific ribosomal proteins has revealed that heterogeneity in the ribosome must exist in vertebrates; however, this has not been tested for ribosomal RNA (rRNA). In zebrafish (Danio rerio), the '45S-M' ribosomal RNA-encoding locus undergoes massive extrachromosomal amplification during oocyte growth and ovary differentiation and is distinct from the regular ribosomal DNA (rDNA) locus encoding somatic rRNA (45S-S). Although the 45S-M rDNA locus falls within the only described sex-linked region in multiple wild zebrafish strains, its role in sexual differentiation is unclear. We used CRISPR-Cas9 gene editing to alter 45S-M rDNA sequences in zygotes and found that although there was no effect on growth or male development, there was dramatic suppression of female differentiation. Males with edited 45S-M rDNA produced phenotypically normal sperm and were able to fertilize eggs from wild-type females, with resulting embryos once more displaying normal development. Our work supports the hypothesis that specialized 45S-M rDNA is the elusive apical sex-determining locus in zebrafish and that this region represents the most tractable genetic system to date for studying ribosomal RNA heterogeneity and function in a vertebrate.This article is part of the discussion meeting issue 'Ribosome diversity and its impact on protein synthesis, development and disease'.},
}

Animals
*Zebrafish/genetics
Female
*DNA, Ribosomal/genetics
Sex Differentiation/genetics
Male
CRISPR-Cas Systems
Gene Editing

@article {pmid40045573,
year = {2025},
author = {Li, F and Tan, X and Li, S and Chen, S and Liu, L and Huang, J and Li, G and Lu, Z and Wu, J and Zeng, D and Luo, Y and Dong, X and Ma, X and Zhu, Q and Chen, L and Liu, YG and Chen, C and Xie, X},
title = {SuperDecode: an integrated toolkit for analyzing mutations induced by genome editing.},
journal = {Molecular plant},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.molp.2025.03.002},
pmid = {40045573},
issn = {1752-9867},
abstract = {Genome editing using CRISPR/Cas or other systems has become a cornerstone of numerous biological and applied research fields. However, detecting the resulting mutations by analysis of sequencing data remains time-consuming and inefficient. In response to this issue, we designed SuperDecode, an integrated software toolkit for analyzing editing outcomes using a range of sequencing strategies. SuperDecode comprises three modules, DSDecodeMS, HiDecode, and LaDecode, each designed to automatically decode mutations from Sanger, high-throughput short-read (next-generation sequencing), and long-read sequencing data (third-generation sequencing), respectively, from targeted PCR amplicons. By leveraging specific strategies for constructing sequencing libraries of pooled multiple amplicons, HiDecode and LaDecode facilitate large-scale identification of mutations induced by single or multiplex target-site editing in a cost-effective manner. We demonstrate the efficacy of SuperDecode by analyzing mutations produced using different genome editing tools (CRISPR/Cas, base editing, prime editing) in different materials (diploid and tetraploid rice, protoplasts), underscoring its versatility in decoding genome editing outcomes across different applications. Furthermore, this toolkit can be used to analyze other genetic variations, as exemplified by its ability to estimate the C-to-U editing rate of the cellular RNA of a mitochondrial gene. SuperDecode offers both a standalone software package and a web-based version, ensuring its easy access and broad compatibility across diverse computer systems. Thus, SuperDecode provides a comprehensive platform for analyzing a wide array of mutations, advancing the utility of genome editing for scientific research and genetic engineering.},
}

@article {pmid40045211,
year = {2025},
author = {Kwon, DH and Gim, GM and Yum, SY and Eom, KH and Lee, SJ and Han, SE and Kim, HS and Kim, HJ and Lee, WS and Choi, WJ and Lee, JH and Kim, DY and Jung, DJ and Kim, DH and Yi, JK and Moon, BH and Lee, WY and Jang, G},
title = {Cas9-expressing cattle using the PiggyBac transposon all-in-one system.},
journal = {BMC genomics},
volume = {26},
number = {1},
pages = {217},
pmid = {40045211},
issn = {1471-2164},
support = {NRF-2021R1A5A1033157//Brain Korea 21 FOUR Future Veterinary Medicine Leading Education and Research Center/ ; },
mesh = {Animals ; Cattle ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *DNA Transposable Elements/genetics ; Male ; Female ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/metabolism/genetics ; Animals, Genetically Modified/genetics ; Prions/genetics ; },
abstract = {BACKGROUND: Livestock, particularly cattle, are crucial for biotechnology fields, such as genetic breeding, infectious diseases, bioreactors, and specific disease models. However, genetic engineering in cattle has lagged due to long gestation periods, single embryo pregnancies, and high rearing costs. Additionally, the slow validation of germline transmission and the absence of germline-competent embryonic stem cells hinder progress. With the development of genome editing technologies like ZFN, TALEN, and CRISPR-Cas9, recent advancements have shown that Cas9-expressing pigs and chickens have been successfully produced. We hypothesize that generating CRISPR/Cas9-expressing cattle and their resources will provide a powerful resource for bovine genome editing, advancing our understanding of bovine genetics and disease resistance.

RESULTS: In this study, two types of Cas9-expressing cattle were successfully produced: Cas9-RFP-fatty acid dehydrogenase I (FatI), Cas9-GFP-sgRNA for the prion protein (sgPRNP). Somatic cells from these cattle were induced to mutate multiple target genes when single-guide RNAs (sgRNAs) were transfected into the somatic cells. Additionally, semen from Cas9 expressing male cattle was frozen and used to fertilize wild-type oocytes, successfully transmitting the transgene (Cas9, reporter genes, FatI), and sgPRNP) to the next generation. Furthermore, the gene editing capabilities of Cas9, including knockout and high-efficiency knock-in, were confirmed in embryos derived from F1 semen through in vitro production.

CONCLUSION: These data demonstrate, for the first time, that Cas9-expressing cattle were successfully born, and this transgene was transmitted to the next-generation calves (F1) and F2 embryos. In addition, somatic and germ cells derived from F0 and F1generations were used to evaluate the potential for gene editing (knockout and knock-in) in multiple genes. PRNP-mutated F1 cattle are currently being raised as a resistance model for bovine spongiform encephalopathy. These transgenic bovine models and their derivatives will serve as a valuable resource for both in vitro and in vivo genome editing, advancing our genetic understanding of bovine genomics and diseases.},
}

Animals
Cattle
*CRISPR-Cas Systems
*Gene Editing/methods
*DNA Transposable Elements/genetics
Male
Female
RNA, Guide, CRISPR-Cas Systems/genetics
CRISPR-Associated Protein 9/metabolism/genetics
Animals, Genetically Modified/genetics
Prions/genetics

@article {pmid40044943,
year = {2025},
author = {García-Tenorio, EM and Álvarez, M and Gallego-Bonhomme, M and Desviat, LR and Richard, E},
title = {Novel CRISPR-Cas9 iPSC knockouts for PCCA and PCCB genes: advancing propionic acidemia research.},
journal = {Human cell},
volume = {38},
number = {3},
pages = {64},
pmid = {40044943},
issn = {1749-0774},
support = {PAF113//Propionic Acidemia Foundation/ ; PID2022-137238OB-100//Ministerio de Ciencia e Innovación/ ; PID2022-137238OB-100//Ministerio de Ciencia e Innovación/ ; },
mesh = {*Propionic Acidemia/genetics/therapy ; *Induced Pluripotent Stem Cells ; *CRISPR-Cas Systems/genetics ; Humans ; *Gene Knockout Techniques/methods ; Methylmalonyl-CoA Decarboxylase/genetics ; Gene Editing/methods ; Frameshift Mutation/genetics ; Gene Expression/genetics ; Carbon-Carbon Ligases/genetics/deficiency ; Mutation/genetics ; Cell Differentiation/genetics ; },
abstract = {Propionic acidemia (PA) is a rare autosomal recessive metabolic disorder caused by mutations in the PCCA and PCCB genes, which encode subunits of the mitochondrial enzyme propionyl-CoA carboxylase (PCC). This enzyme deficiency leads to the accumulation of toxic metabolites, resulting in severe metabolic dysfunction. To create ideal in vitro disease models of PA with isogenic controls and provide a robust platform for therapeutic research, we generated two induced pluripotent stem cell (iPSC) lines with knockout (KO) mutations in the PCCA and PCCB genes using CRISPR-Cas9 gene editing in a healthy control iPSC line. The KO iPS cells were successfully established and characterized, confirming the presence of frameshift insertions and deletions in each target gene, as well as the loss of the corresponding transcript, protein expression, and activity. Additionally, the generated iPSC lines exhibit hallmark characteristics of pluripotency, including the potential to differentiate into all three germ layers. Our PCCA and PCCB KO iPSC models provide a valuable tool for studying the molecular mechanisms underlying PA and hold potential for advancing new therapeutic approaches.},
}

*Propionic Acidemia/genetics/therapy
*Induced Pluripotent Stem Cells
*CRISPR-Cas Systems/genetics
Humans
*Gene Knockout Techniques/methods
Methylmalonyl-CoA Decarboxylase/genetics
Gene Editing/methods
Frameshift Mutation/genetics
Gene Expression/genetics
Carbon-Carbon Ligases/genetics/deficiency
Mutation/genetics
Cell Differentiation/genetics

@article {pmid40044221,
year = {2025},
author = {Duan, X and Chen, C and Du, C and Guo, L and Liu, J and Hou, N and Li, P and Qi, X and Gao, F and Du, X and Song, J and Wu, S},
title = {Homozygous editing of multiple genes for accelerated generation of xenotransplantation pigs.},
journal = {Genome research},
volume = {},
number = {},
pages = {},
doi = {10.1101/gr.279709.124},
pmid = {40044221},
issn = {1549-5469},
abstract = {Although CRISPR-Cas based genome editing has made significant strides over the past decade, achieving simultaneous homozygous gene editing of multiple targets in primary cells remains a significant challenge. In this study, we optimized a coselection strategy to enhance homozygous gene editing rates in the genomes of primary porcine fetal fibroblasts (PFFs). The strategy utilizes the expression of a surrogate reporter (eGFP) to select for cells with the highest reporter expression, thereby improving editing efficiency. When applied to simultaneous multigene editing, we targeted the most challenging site for selection, while other target sites did not require selection. Using this approach, we successfully obtained single-cell PFF clones (3/10) with seven or more homozygously edited genes, including GGTA1, CMAH, B4GALNT2, CD46, CD47, THBD, and GHR Importantly, cells edited using this strategy were efficiently used for somatic cell nuclear transfer (SCNT) to generate healthy xenotransplantation pigs in less than five months, a process that previously required years of breeding or multiple rounds of SCNT.},
}

@article {pmid40044139,
year = {2025},
author = {Lee, HJ and Jeong, SH and Lee, SJ},
title = {Single nucleotide genome recognition and selective bacterial lysis using synthetic phages loaded with CRISPR-Cas12f1-truncated sgRNA.},
journal = {Journal of microbiology (Seoul, Korea)},
volume = {63},
number = {2},
pages = {e2501012},
doi = {10.71150/jm.2501012},
pmid = {40044139},
issn = {1976-3794},
support = {RS-2024-00342735//National Research Foundation of Korea/ ; //Ministry of Science and ICT/ ; RS-2024-00411263//Ministry of Education/ ; //Korea Health Industry Development Institute/ ; HI23C0041//Ministry of Health and Welfare/ ; },
mesh = {*CRISPR-Cas Systems ; *Escherichia coli/genetics/virology ; *Genome, Viral/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Bacteriophage lambda/genetics ; Lysogeny ; Bacteriolysis ; CRISPR-Associated Proteins/genetics ; Shiga Toxin 2/genetics ; Genome, Bacterial ; },
abstract = {Phage specificity primarily relies on host cell-surface receptors. However, integrating cas genes and guide RNAs into phage genomes could enhance their target specificity and regulatory effects. In this study, we developed a CRISPR-Cas12f1 system-equipped bacteriophage λ model capable of detecting Escherichia coli target genes. We demonstrated that synthetic λ phages carrying Cas12f1-sgRNA can effectively prevent lysogen formation. Furthermore, we showcased that truncating the 3(?)-end of sgRNA enables precise identification of single-nucleotide variations in the host genome. Moreover, infecting E. coli strains carrying various stx2 gene subtypes encoding Shiga toxin with bacteriophages harboring Cas12f1 and truncated sgRNAs resulted in the targeted elimination of strains with matching subtype genes. These findings underscore the ability of phages equipped with the CRISPR-Cas12f1 system to precisely control microbial hosts by recognizing genomic sequences with high resolution.},
}

*CRISPR-Cas Systems
*Escherichia coli/genetics/virology
*Genome, Viral/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Bacteriophage lambda/genetics
Lysogeny
Bacteriolysis
CRISPR-Associated Proteins/genetics
Shiga Toxin 2/genetics
Genome, Bacterial

@article {pmid40043077,
year = {2025},
author = {Zhou, Z and Xiao, J and Yin, S and Chen, Y and Yuan, Y and Zhang, J and Xiong, L and Xie, K},
title = {Cas9-Rep fusion tethers donor DNA in vivo and boosts the efficiency of HDR-mediated genome editing.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/pbi.70036},
pmid = {40043077},
issn = {1467-7652},
support = {2662023PY006//Fundamental Research Funds for the Central Universities/ ; B23YQ1516//Hainan Yazhou Bay Seed Laboratory and the China National Seed Group/ ; 2023ZD04062//Science and Technology Innovation 2030-Biological Breeding Major Projects/ ; 2023ZD0407403//Science and Technology Innovation 2030-Biological Breeding Major Projects/ ; 31821005//National Natural Science Foundation of China/ ; 32293243//National Natural Science Foundation of China/ ; },
abstract = {Genome editing based on the homology-directed repair (HDR) pathway enables scar-free and precise genetic manipulations. However, the low frequency of HDR hinders its application in plant genome editing. In this study, we engineered the fusion of Cas9 and a viral replication protein (Rep) as a molecular bridge to tether donor DNA in vivo, which enhances the efficiency of targeted gene insertion via the HDR pathway. This Rep-bridged knock-in (RBKI) method combines the advantages of rolling cycle replication of viral replicons and in vivo enrichment of donor DNA at the target site for HDR. Chromatin immunoprecipitation indicated that the Cas9-Rep fusion protein bound up to 66-fold more donor DNA than Cas9 did. We exemplified the RBKI method by inserting small- to middle-sized tags (33-519 bp) into 3 rice genes. Compared to Cas9, Cas9-Rep fusion increased the KI frequencies by 4-7.6-fold, and up to 72.2% of stable rice transformants carried in-frame knock-in events in the T0 generation. Whole-genome sequencing of 6 plants segregated from heterozygous KI lines indicated that the knock-in events were faithfully inherited by the progenies with neither off-target editing nor random insertions of the donor DNA fragment. Further analysis suggested that the RBKI method reduced the number of byproducts from nonhomologous end joining; however, HDR-mediated knock-in tended to accompany microhomology-mediated end joining events. Together, these findings show that the in vivo tethering of donor DNAs with Cas9-Rep is an effective strategy to increase the frequency of HDR-mediated genome editing.},
}

@article {pmid40041907,
year = {2025},
author = {Mao, K and Tan, H and Cong, X and Liu, J and Xin, Y and Wang, J and Guan, M and Li, J and Zhu, G and Meng, X and Lin, G and Wang, H and Han, J and Wang, M and Yang, YG and Sun, T},
title = {Optimized lipid nanoparticles enable effective CRISPR/Cas9-mediated gene editing in dendritic cells for enhanced immunotherapy.},
journal = {Acta pharmaceutica Sinica. B},
volume = {15},
number = {1},
pages = {642-656},
pmid = {40041907},
issn = {2211-3835},
abstract = {Immunotherapy has emerged as a revolutionary approach to treat immune-related diseases. Dendritic cells (DCs) play a pivotal role in orchestrating immune responses, making them an attractive target for immunotherapeutic interventions. Modulation of gene expression in DCs using genome editing techniques, such as the CRISPR-Cas system, is important for regulating DC functions. However, the precise delivery of CRISPR-based therapies to DCs has posed a significant challenge. While lipid nanoparticles (LNPs) have been extensively studied for gene editing in tumor cells, their potential application in DCs has remained relatively unexplored. This study investigates the important role of cholesterol in regulating the efficiency of BAMEA-O16B lipid-assisted nanoparticles (BLANs) as carriers of CRISPR/Cas9 for gene editing in DCs. Remarkably, BLANs with low cholesterol density exhibit exceptional mRNA uptake, improved endosomal escape, and efficient single-guide RNA release capabilities. Administration of BLANmCas9/gPD-L1 results in substantial PD-L1 gene knockout in conventional dendritic cells (cDCs), accompanied by heightened cDC1 activation, T cell stimulation, and significant suppression of tumor growth. The study underscores the pivotal role of cholesterol density within LNPs, revealing potent influence on gene editing efficacy within DCs. This strategy holds immense promise for the field of cancer immunotherapy, offering a novel avenue for treating immune-related diseases.},
}

@article {pmid40040541,
year = {2025},
author = {Li, J and Lu, Y and Cheng, K and Zhu, G and Wang, X and Lin, T and Zhang, B and Ma, L and Qu, G and Zhu, B and Fu, D and Luo, Y and Zhu, H},
title = {ACS4 exerts a pivotal role in ethylene biosynthesis during the ripening of tomato fruits in comparison to ACS2.},
journal = {The Plant journal : for cell and molecular biology},
volume = {121},
number = {5},
pages = {e70043},
doi = {10.1111/tpj.70043},
pmid = {40040541},
issn = {1365-313X},
support = {32472803//National Natural Science Foundation of China/ ; 2022YFD2100101//National Key Research and Development Program of China/ ; },
mesh = {*Solanum lycopersicum/genetics/metabolism/growth & development ; *Ethylenes/metabolism/biosynthesis ; *Fruit/genetics/metabolism/growth & development ; *Plant Proteins/genetics/metabolism ; *Gene Expression Regulation, Plant ; *Lyases/metabolism/genetics ; CRISPR-Cas Systems ; Carbon-Carbon Lyases/metabolism/genetics ; },
abstract = {In the climacteric fruit tomato (Solanum lycopersicum), 1-aminocyclopropane-1-carboxylic acid (ACC) synthase 2 (ACS2) and ACS4 are jointly recognized as key enzymes in orchestrating System-2 ethylene biosynthesis during fruit ripening. However, the precise roles and individual contributions of ACS2 and ACS4 within this process remain elusive. Here, we generate acs2, acs4 single knockout, and acs2/4 double knockout mutants through the CRISPR/Cas9 system. Our results reveal that the knockout of ACS2 leads to a modest decrease in ethylene production, with minimal effects on fruit ripening. In contrast, the knockout of ACS4 unveils a severe ripening defect akin to that observed in the acs2/4 mutant, which stems from a profound disruption of ethylene autocatalytic biosynthesis, ultimately resulting in inadequate ethylene production vital for supporting fruit ripening. Transcriptome analysis, in conjunction with exogenous ethylene treatment, conclusively demonstrates a pronounced dose-dependent correlation between fruit ripening and ethylene, wherein varying doses of ethylene distinctly regulate the expression of a substantial number of ripening-related genes, eventually controlling both the ripening process and quality formation. These findings clarify the pivotal role of ACS4 in ethylene biosynthesis compared to ACS2 and deepen our understanding of the fine-tuned regulation of ethylene in climacteric fruit ripening.},
}

*Solanum lycopersicum/genetics/metabolism/growth & development
*Ethylenes/metabolism/biosynthesis
*Fruit/genetics/metabolism/growth & development
*Plant Proteins/genetics/metabolism
*Gene Expression Regulation, Plant
*Lyases/metabolism/genetics
CRISPR-Cas Systems
Carbon-Carbon Lyases/metabolism/genetics

@article {pmid40040244,
year = {2025},
author = {Cao, X and Wang, X and Chen, R and Chen, L and Liu, Y and Wang, M},
title = {Improving Bacillus subtilis as Biological Chassis Performance by the CRISPR Genetic Toolkit.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.4c00844},
pmid = {40040244},
issn = {2161-5063},
abstract = {Bacillus subtilis is the model Gram-positive and industrial chassis bacterium; it has blossomed as a robust and promising host for enzyme, biochemical, or bioflocculant production. However, synthetic biology and metabolic engineering technologies of B. subtilis have lagged behind the most widely used industrial chassis Saccharomyces cerevisiae and Escherichia coli. CRISPR (an acronym for clustered regularly interspaced short palindromic repeats) enables efficient, site-specific, and programmable DNA cleavage, which has revolutionized the manner of genome editing. In 2016, CRISPR technology was first introduced into B. subtilis and has been intensely upgraded since then. In this Review, we discuss recently developed key additions to CRISPR toolkit design in B. subtilis with gene editing, transcriptional regulation, and enzyme modulation. Second, advances in the B. subtilis chassis of efficient biochemicals and proteins with CRISPR engineering are discussed. Finally, we conclude with perspectives on the challenges and opportunities of CRISPR-based biotechnology in B. subtilis, wishing that B. subtilis can be comparable to traditional industrial microorganisms such as E. coli and S. cerevisiae someday soon.},
}

@article {pmid39987655,
year = {2025},
author = {Xu, H and Chen, Q and Meng, X and Yan, C and Yao, B and Chen, Z and Wang, Z and Chen, W},
title = {CRISPR/Cas12a-mediated cyclic signal amplification and electrochemical reporting strategy for rapid and accurate sensing of Vibrio parahaemolyticus in aquatic foods.},
journal = {Biosensors & bioelectronics},
volume = {277},
number = {},
pages = {117284},
doi = {10.1016/j.bios.2025.117284},
pmid = {39987655},
issn = {1873-4235},
mesh = {*Vibrio parahaemolyticus/isolation & purification/genetics ; *Biosensing Techniques/methods ; *Electrochemical Techniques/methods ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Animals ; Food Microbiology ; Limit of Detection ; Food Contamination/analysis ; Seafood/microbiology ; Bacterial Proteins/genetics ; Fishes/microbiology ; Vibrio Infections/microbiology/diagnosis ; CRISPR-Associated Proteins/genetics ; Humans ; Endodeoxyribonucleases ; },
abstract = {Rapid and accurate detection of target foodborne pathogenic bacteria is extremely important for preventing and controlling foodborne diseases. Vibrio parahaemolyticus (V. parahaemolyticus, Vp) is considered as a major cause of foodborne diseases, posing severe threat to food safety and public health. The efficiency and sensitivity of traditional protocols for Vp identification is time consuming and of poor precision. In this research, a simple electrochemical sensing method was developed for accurate detection of Vp in aquatic products. Target genes of Vp were rapid amplified with the designed recombinase polymerase amplification, which further activated the designed CRISPR/Cas12a system. The electrochemical active ssDNA probe on the sensing interface would be hydrolyzed by the activated trans-cleavage activity of Cas12a, inducing the release of active electrochemical tags from the sensing interface and the decreased sensing signals. Under the optimized conditions, this proposed RPA-mediated electrochemical-CRISPR (E-CRISPR) biosensor enabled sensitive detection of target Vp over a linear range from 10[1] to 10[6] CFU/mL, with limit of detection of 32 CFU/mL. Additionally, this E-CRISPR biosensor realized the successful determination of Vp in spiked fish samples with satisfied sensing performance. The isothermal amplification and the rapid electrochemical response of the E-CRISPR biosensor made it suitable for on-site screening. And this E-CRISPR biosensor could be well integrated with other isothermal protocols and extended to other target pathogens, showing great potential for practical applications in molecular diagnostics and other gene detection related fields.},
}

*Vibrio parahaemolyticus/isolation & purification/genetics
*Biosensing Techniques/methods
*Electrochemical Techniques/methods
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods
Animals
Food Microbiology
Limit of Detection
Food Contamination/analysis
Seafood/microbiology
Bacterial Proteins/genetics
Fishes/microbiology
Vibrio Infections/microbiology/diagnosis
CRISPR-Associated Proteins/genetics
Humans
Endodeoxyribonucleases

@article {pmid39980205,
year = {2025},
author = {Yang, K and Zhang, C and Wang, Z and Huang, Q and Qian, J and Shi, G and Sun, W and Wang, J and Ji, Y and Sun, Z and Song, Y and Han, X},
title = {CRISPR-dCas9-Mediated PTEN Activation via Tumor Cell Membrane-Coated Nanoplatform Enhances Sensitivity to Tyrosine Kinase Inhibitors in Nonsmall Cell Lung Cancer.},
journal = {ACS applied materials & interfaces},
volume = {17},
number = {9},
pages = {13605-13616},
doi = {10.1021/acsami.4c21740},
pmid = {39980205},
issn = {1944-8252},
mesh = {*PTEN Phosphohydrolase/genetics/metabolism ; *Carcinoma, Non-Small-Cell Lung/drug therapy/genetics/pathology/metabolism ; Humans ; *Lung Neoplasms/drug therapy/genetics/pathology/metabolism ; *CRISPR-Cas Systems/genetics ; *Protein Kinase Inhibitors/pharmacology/chemistry ; Animals ; *Cell Membrane/metabolism ; *Gefitinib/pharmacology ; Mice ; Cell Line, Tumor ; Antineoplastic Agents/pharmacology/chemistry ; Mice, Nude ; ErbB Receptors/genetics/metabolism/antagonists & inhibitors ; Drug Resistance, Neoplasm/drug effects/genetics ; Tyrosine Kinase Inhibitors ; },
abstract = {EGFR tyrosine kinase inhibitors (EGFR-TKIs) have garnered substantial clinical success in treating nonsmall cell lung cancer (NSCLC) harboring epidermal growth factor receptor (EGFR) mutations. However, the inevitable emergence of drug resistance, frequently attributed to activation, mutation, or deletion of multiple signaling pathways, poses a significant challenge. Notably, the loss of PTEN protein expression has emerged as a pivotal mechanism fostering resistance in EGFR mutant lung cancers. Consequently, strategies aimed at upregulating PTEN expression hold great promise for restoring drug sensitivity. Leveraging the versatility, precision, and efficacy of nuclease-deactivated Cas9 (dCas9) as a transcriptional activator, we designed a CRISPR-dCas9 system to stimulate PTEN expression. To further enhance target specificity and drug delivery efficiency, we innovatively harnessed the tumor cell membrane (CCM) as a homologous targeting surface coating for our vector, thereby creating a targeted activation nanoplatform. Comprehensive in vitro and in vivo evaluations demonstrated that the synergistic interplay between gefitinib and the CRISPR-dCas9 system significantly enhanced drug sensitivity. The finding underscores the potential of our approach in addressing the issue of lung cancer resistance, offering a promising avenue for personalized and effective cancer therapies.},
}

*PTEN Phosphohydrolase/genetics/metabolism
*Carcinoma, Non-Small-Cell Lung/drug therapy/genetics/pathology/metabolism
Humans
*Lung Neoplasms/drug therapy/genetics/pathology/metabolism
*CRISPR-Cas Systems/genetics
*Protein Kinase Inhibitors/pharmacology/chemistry
Animals
*Cell Membrane/metabolism
*Gefitinib/pharmacology
Mice
Cell Line, Tumor
Antineoplastic Agents/pharmacology/chemistry
Mice, Nude
ErbB Receptors/genetics/metabolism/antagonists & inhibitors
Drug Resistance, Neoplasm/drug effects/genetics
Tyrosine Kinase Inhibitors

@article {pmid39905729,
year = {2025},
author = {Du, W and Noyan, F and McCallion, O and Drosdek, V and Kath, J and Glaser, V and Fuster-Garcia, C and Yang, M and Stein, M and Franke, C and Pu, Y and Weber, O and Polansky, JK and Cathomen, T and Jaeckel, E and Hester, J and Issa, F and Volk, HD and Schmueck-Henneresse, M and Reinke, P and Wagner, DL},
title = {Gene editing of CD3 epsilon to redirect regulatory T cells for adoptive T cell transfer.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {3},
pages = {997-1013},
doi = {10.1016/j.ymthe.2025.01.045},
pmid = {39905729},
issn = {1525-0024},
mesh = {*Gene Editing/methods ; Animals ; *T-Lymphocytes, Regulatory/immunology/metabolism ; *CD3 Complex/genetics/immunology/metabolism ; Humans ; Mice ; *Adoptive Transfer/methods ; CRISPR-Cas Systems ; Receptors, Chimeric Antigen/genetics/immunology/metabolism ; HLA-A2 Antigen/immunology/genetics/metabolism ; Immunotherapy, Adoptive/methods ; },
abstract = {Adoptive transfer of antigen-specific regulatory T cells (Tregs) is a promising strategy to combat immunopathologies in transplantation and autoimmune diseases. However, their low frequency in peripheral blood poses challenges for both manufacturing and clinical application. Chimeric antigen receptors have been used to redirect the specificity of Tregs, using retroviral vectors. However, retroviral gene transfer is costly, time consuming, and raises safety issues. Here, we explored non-viral CRISPR-Cas12a gene editing to redirect Tregs, using human leukocyte antigen (HLA)-A2-specific constructs for proof-of-concept studies in transplantation models. Knock-in of an antigen-binding domain into the N terminus of CD3 epsilon (CD3ε) gene generates Tregs expressing a chimeric CD3ε-T cell receptor fusion construct (TRuC) protein that integrates into the endogenous TCR/CD3 complex. These CD3ε-TRuC Tregs exhibit potent antigen-dependent activation while maintaining responsiveness to TCR/CD3 stimulation. This enables preferential enrichment of TRuC-redirected Tregs over CD3ε knockout Tregs via repetitive CD3/CD28 stimulation in a good manufacturing practice-compatible expansion system. CD3ε-TRuC Tregs retained their phenotypic, epigenetic, and functional identity. In a humanized mouse model, HLA-A2-specific CD3ε-TRuC Tregs demonstrate superior protection of allogeneic HLA-A2[+] skin grafts from rejection compared with polyclonal Tregs. This approach provides a pathway for developing clinical-grade CD3ε-TRuC-based Treg cell products for transplantation immunotherapy and other immunopathologies.},
}

*Gene Editing/methods
Animals
*T-Lymphocytes, Regulatory/immunology/metabolism
*CD3 Complex/genetics/immunology/metabolism
Humans
Mice
*Adoptive Transfer/methods
CRISPR-Cas Systems
Receptors, Chimeric Antigen/genetics/immunology/metabolism
HLA-A2 Antigen/immunology/genetics/metabolism
Immunotherapy, Adoptive/methods

@article {pmid39863930,
year = {2025},
author = {Verma, SS and Sen, CK and Srivastava, R and Gnyawali, SC and Katiyar, P and Sahi, AK and Kumar, M and Rustagi, Y and Liu, S and Pandey, D and Abouhashem, AS and Fehme, LNW and Kacar, S and Mohanty, SK and Faden-McCormack, J and Murphy, MP and Roy, S and Wan, J and Yoder, MC and Singh, K},
title = {Tissue nanotransfection-based endothelial PLCγ2-targeted epigenetic gene editing rescues perfusion and diabetic ischemic wound healing.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {3},
pages = {950-969},
doi = {10.1016/j.ymthe.2025.01.034},
pmid = {39863930},
issn = {1525-0024},
support = {R01 DK136814/DK/NIDDK NIH HHS/United States ; },
mesh = {*Phospholipase C gamma/genetics/metabolism ; Animals ; *Wound Healing/genetics ; Humans ; Mice ; *Ischemia/therapy/genetics ; *Epigenesis, Genetic ; *Promoter Regions, Genetic ; *Vascular Endothelial Growth Factor A/genetics/metabolism ; *Gene Editing/methods ; DNA Methylation ; Neovascularization, Physiologic/genetics ; Endothelial Cells/metabolism ; Diabetes Mellitus, Experimental/therapy/genetics ; Transfection ; CRISPR-Cas Systems ; Male ; Disease Models, Animal ; },
abstract = {Diabetic wounds are complicated by underlying peripheral vasculopathy. Reliance on vascular endothelial growth factor (VEGF) therapy to improve perfusion makes logical sense, yet clinical study outcomes on rescuing diabetic wound vascularization have yielded disappointing results. Our previous work has identified that low endothelial phospholipase Cγ2 (PLCγ2) expression hinders the therapeutic effect of VEGF on the diabetic ischemic limb. In this work, guided by single-cell RNA sequencing of human wound edge, we test the efficacy of gene-targeted therapeutic demethylation intending to improve VEGF-mediated neovascularization. PLCγ2 expression was diminished in all five identified diabetic wound-edge endothelial subclusters encompassing arterial, venous, and capillary cells. Such low expression was associated with hypermethylated PLCγ2 promoter. PLCγ2 promoter was also hypermethylated at murine diabetic ischemic wound edge. To specifically demethylate endothelial PLCγ2 promoter during VEGF therapy, a CRISPR-dCas9-based demethylation cocktail was delivered to the ischemic wound edge using tissue nanotransfection (TNT) technology. Demethylation-based upregulation of PLCγ2 during VEGF therapy improved wound tissue blood flow with an increased abundance of von Willebrand factor (vWF)[+]/PLCγ2[+] vascular tissue elements by activating p44/p42-mitogen-activated protein kinase (MAPK) → hypoxia-inducible factor [HIF]-1α pathway. Taken together, TNT-based delivery of plasmids to demethylate the PLCγ2 gene promoter activity led to significant improvements in VEGF therapy for cutaneous diabetic wounds, resulting in better perfusion and accelerated wound closure.},
}

*Phospholipase C gamma/genetics/metabolism
Animals
*Wound Healing/genetics
Humans
Mice
*Ischemia/therapy/genetics
*Epigenesis, Genetic
*Promoter Regions, Genetic
*Vascular Endothelial Growth Factor A/genetics/metabolism
*Gene Editing/methods
DNA Methylation
Neovascularization, Physiologic/genetics
Endothelial Cells/metabolism
Diabetes Mellitus, Experimental/therapy/genetics
Transfection
CRISPR-Cas Systems
Male
Disease Models, Animal

@article {pmid39815622,
year = {2025},
author = {Shankar, K and Zingler-Hoslet, I and Tabima, DM and Zima, S and Shi, L and Gimse, K and Forsberg, MH and Katta, V and Davis, SZ and Maldonado, D and Russell, BE and Murtaza, M and Tsai, SQ and Ayuso, JM and Capitini, CM and Saha, K},
title = {Virus-free CRISPR knockin of a chimeric antigen receptor into KLRC1 generates potent GD2-specific natural killer cells.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {3},
pages = {1014-1030},
doi = {10.1016/j.ymthe.2025.01.024},
pmid = {39815622},
issn = {1525-0024},
mesh = {Humans ; *Killer Cells, Natural/immunology/metabolism ; *Receptors, Chimeric Antigen/genetics/metabolism/immunology ; *CRISPR-Cas Systems ; *Gene Editing ; Gangliosides/metabolism ; NK Cell Lectin-Like Receptor Subfamily C/genetics/metabolism ; Gene Knock-In Techniques ; Cell Line, Tumor ; Immunotherapy, Adoptive/methods ; Cytotoxicity, Immunologic ; },
abstract = {Natural killer (NK) cells are an appealing off-the-shelf, allogeneic cellular therapy due to their cytotoxic profile. However, their activity against solid tumors remains suboptimal in part due to the upregulation of NK-inhibitory ligands, such as HLA-E, within the tumor microenvironment. Here, we utilize CRISPR-Cas9 to disrupt the KLRC1 gene (encoding the HLA-E-binding NKG2A receptor) and perform non-viral insertion of a GD2-targeting chimeric antigen receptor (CAR) within NK cells isolated from human peripheral blood. Genome editing with CRISPR-Cas9 ribonucleoprotein complexes yields efficient genomic disruption of the KLRC1 gene with 98% knockout efficiency and specific knockin of the GD2 CAR transgene as high as 23%, with minimal off-target activity as shown by CHANGE-seq, in-out PCR, amplicon sequencing, and long-read whole-genome sequencing. KLRC1-GD2 CAR NK cells display high viability and proliferation, as well as precise cellular targeting and potency against GD2[+] human tumor cells. Notably, KLRC1-GD2 CAR NK cells overcome HLA-E-based inhibition in vitro against HLA-E-expressing, GD2[+] melanoma cells. Using a single-step, virus-free genome editing workflow, this study demonstrates the feasibility of precisely disrupting inhibitory signaling within NK cells via CRISPR-Cas9 while expressing a CAR to generate potent allogeneic cell therapies against HLA-E[+] solid tumors.},
}

Humans
*Killer Cells, Natural/immunology/metabolism
*Receptors, Chimeric Antigen/genetics/metabolism/immunology
*CRISPR-Cas Systems
*Gene Editing
Gangliosides/metabolism
NK Cell Lectin-Like Receptor Subfamily C/genetics/metabolism
Gene Knock-In Techniques
Cell Line, Tumor
Immunotherapy, Adoptive/methods
Cytotoxicity, Immunologic

@article {pmid39814010,
year = {2025},
author = {Li, E and Benitez, C and Boggess, SC and Koontz, M and Rose, IVL and Martinez, D and Dräger, N and Teter, OM and Samelson, AJ and Pierce, N and Ullian, EM and Kampmann, M},
title = {CRISPRi-based screens in iAssembloids to elucidate neuron-glia interactions.},
journal = {Neuron},
volume = {113},
number = {5},
pages = {701-718.e8},
doi = {10.1016/j.neuron.2024.12.016},
pmid = {39814010},
issn = {1097-4199},
support = {U01 AG072464/AG/NIA NIH HHS/United States ; U54 NS123746/NS/NINDS NIH HHS/United States ; },
mesh = {*Neurons/metabolism/physiology ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; *Neuroglia/metabolism ; *Coculture Techniques ; NF-E2-Related Factor 2/metabolism/genetics ; Glycogen Synthase Kinase 3 beta/metabolism ; Cell Communication/physiology ; Apolipoprotein E4/genetics/metabolism ; CRISPR-Cas Systems ; Astrocytes/metabolism ; Oxidative Stress/physiology ; Cell Survival/physiology ; Cells, Cultured ; },
abstract = {The complexity of the human brain makes it challenging to understand the molecular mechanisms underlying brain function. Genome-wide association studies have uncovered variants associated with neurological phenotypes. Single-cell transcriptomics have provided descriptions of changes brain cells undergo during disease. However, these approaches do not establish molecular mechanism. To facilitate the scalable interrogation of causal molecular mechanisms in brain cell types, we developed a 3D co-culture system of induced pluripotent stem cell (iPSC)-derived neurons and glia, termed iAssembloids. Using iAssembloids, we ask how glial and neuronal cells interact to control neuronal death and survival. Our CRISPRi-based screens identified that GSK3β inhibits the protective NRF2-mediated oxidative stress response elicited by high neuronal activity. We then investigate the role of APOE-ε4, a risk variant for Alzheimer's disease, on neuronal survival. We find that APOE-ε4-expressing astrocytes may promote neuronal hyperactivity as compared with APOE-ε3-expressing astrocytes. This platform allows for the unbiased identification of mechanisms of neuron-glia cell interactions.},
}

*Neurons/metabolism/physiology
Humans
*Induced Pluripotent Stem Cells/metabolism
*Neuroglia/metabolism
*Coculture Techniques
NF-E2-Related Factor 2/metabolism/genetics
Glycogen Synthase Kinase 3 beta/metabolism
Cell Communication/physiology
Apolipoprotein E4/genetics/metabolism
CRISPR-Cas Systems
Astrocytes/metabolism
Oxidative Stress/physiology
Cell Survival/physiology
Cells, Cultured

@article {pmid39799395,
year = {2025},
author = {Zhou, H and Dai, J and Li, D and Wang, L and Ye, M and Hu, X and LoTurco, J and Hu, J and Sun, W},
title = {Efficient gene delivery admitted by small metabolites specifically targeting astrocytes in the mouse brain.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {3},
pages = {1166-1179},
doi = {10.1016/j.ymthe.2025.01.006},
pmid = {39799395},
issn = {1525-0024},
mesh = {Animals ; *Astrocytes/metabolism ; Mice ; *Gene Transfer Techniques ; *Brain/metabolism ; Glioma/therapy/genetics/metabolism ; Genetic Therapy/methods ; CRISPR-Cas Systems ; Humans ; PTEN Phosphohydrolase/genetics/metabolism ; Disease Models, Animal ; Cell Line, Tumor ; ErbB Receptors/metabolism/genetics ; Tumor Suppressor Protein p53/metabolism/genetics ; DNA/metabolism/genetics ; Brain Neoplasms/therapy/genetics/metabolism/pathology ; },
abstract = {The development of efficient and targeted methods for delivering DNA in vivo has long been a major focus of research. In this study, we introduce a gene delivery approach admitted by small metabolites (gDAM) for the efficient and targeted delivery of naked DNA into astrocytes in the adult brains of mice. gDAM uses a straightforward combination of DNA and small metabolites, including glycine, L-proline, L-serine, L-histidine, D-alanine, Gly-Gly, and Gly-Gly-Gly, to achieve astrocyte-specific delivery of naked DNA, resulting in transient and robust gene expression in these cells. Using gDAM, we successfully co-deliver the PiggyBac transposon and the CRISPR-Cas9 system to induce long-term overexpression of the oncogene EGFRvIII and knockout of tumor suppressor genes Nf1, Pten, and Trp53 in astrocytes, leading to the development of astrocyte-derived gliomas in immunocompetent mice. Furthermore, gDAM facilitates the delivery of naked DNA to peripheral glioma astrocytes. The overexpression of interferon-β and granulocyte-macrophage colony-stimulating factor in these peripheral glioma astrocytes significantly prolongs the overall survival of mice bearing 73C glioma cells. This approach offers a new perspective on developing gene delivery systems that specifically target astrocytes to meet the varied needs of both research and gene therapy. The innovative strategy behind gDAM is expected to provide fresh inspiration in the quest for DNA delivery to other tissues, such as skeletal muscle and skin.},
}

Animals
*Astrocytes/metabolism
Mice
*Gene Transfer Techniques
*Brain/metabolism
Glioma/therapy/genetics/metabolism
Genetic Therapy/methods
CRISPR-Cas Systems
Humans
PTEN Phosphohydrolase/genetics/metabolism
Disease Models, Animal
Cell Line, Tumor
ErbB Receptors/metabolism/genetics
Tumor Suppressor Protein p53/metabolism/genetics
DNA/metabolism/genetics
Brain Neoplasms/therapy/genetics/metabolism/pathology

@article {pmid39797401,
year = {2025},
author = {Carrozzo, I and Maule, G and Gentile, C and Umbach, A and Ciciani, M and Guidone, D and De Santis, M and Petris, G and Vicente Galietta, LJ and Arosio, D and Cereseto, A},
title = {Functional rescue of F508del-CFTR through revertant mutations introduced by CRISPR base editing.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {3},
pages = {970-985},
doi = {10.1016/j.ymthe.2025.01.011},
pmid = {39797401},
issn = {1525-0024},
mesh = {*Cystic Fibrosis Transmembrane Conductance Regulator/genetics/metabolism ; Humans ; *Gene Editing/methods ; *Cystic Fibrosis/genetics/therapy/metabolism ; *CRISPR-Cas Systems ; *Mutation ; Epithelial Cells/metabolism/drug effects ; Cell Line ; Protein Folding ; },
abstract = {Cystic fibrosis (CF) is a life-shortening autosomal recessive disease caused by mutations in the CFTR gene, resulting in functional impairment of the encoded ion channel. F508del mutation, a trinucleotide deletion, is the most frequent cause of CF, affecting approximately 80% of persons with CF (pwCFs). Even though current pharmacological treatments alleviate the F508del-CF disease symptoms, there is no definitive cure. Here, we leveraged revertant mutations (RMs) in cis with F508del to rescue CFTR protein folding and restore its function. We developed CRISPR base editing strategies to efficiently and precisely introduce the desired mutations in the F508del locus. Both editing and CFTR function recovery were verified in CF cellular models, including primary epithelial cells derived from pwCFs. The efficacy of the CFTR recovery strategy was validated in cultures of pseudostratified epithelia from pwCF cells showing full recovery of ion transport. Additionally, we observed an additive effect by combining our strategy with small molecules that enhance F508del activity, thus paving the way to combinatorial therapies.},
}

*Cystic Fibrosis Transmembrane Conductance Regulator/genetics/metabolism
Humans
*Gene Editing/methods
*Cystic Fibrosis/genetics/therapy/metabolism
*CRISPR-Cas Systems
*Mutation
Epithelial Cells/metabolism/drug effects
Cell Line
Protein Folding

@article {pmid39797397,
year = {2025},
author = {Hazel, K and Singh, D and He, S and Guertin, Z and Husser, MC and Helfield, B},
title = {Focused ultrasound and microbubble-mediated delivery of CRISPR-Cas9 ribonucleoprotein to human induced pluripotent stem cells.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {3},
pages = {986-996},
doi = {10.1016/j.ymthe.2025.01.013},
pmid = {39797397},
issn = {1525-0024},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems ; *Microbubbles ; *Ribonucleoproteins/metabolism/genetics ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Transfer Techniques ; Green Fluorescent Proteins/genetics/metabolism ; },
abstract = {CRISPR-Cas9 ribonucleoproteins (RNPs) have been heavily considered for gene therapy due to their high on-target efficiency, rapid activity, and lack of insertional mutagenesis relative to other CRISPR-Cas9 delivery formats. Genetic diseases such as hypertrophic cardiomyopathy currently lack effective treatment strategies and are prime targets for CRISPR-Cas9 gene editing technology. However, current in vivo delivery strategies for Cas9 pose risks of unwanted immunogenic responses. This proof-of-concept study aimed to demonstrate that focused ultrasound (FUS) in combination with microbubbles can be used to deliver Cas9-sgRNA (single-guide RNA) RNPs and functionally edit human induced pluripotent stem cells (hiPSCs) in vitro, a model system that can be expanded to cardiovascular research via hiPSC-derived cardiomyocytes. Here, we first determine acoustic conditions suitable for the viable delivery of large proteins to hiPSCs with clinical Definity microbubble agents using our customized experimental platform. From here, we delivered Cas9-sgRNA RNP complexes targeting the EGFP (enhanced green fluorescent protein) gene to EGFP-expressing hiPSCs for EGFP knockout. Simultaneous acoustic cavitation detection during treatment confirmed a strong correlation between microbubble disruption and viable FUS-mediated protein delivery in hiPSCs. This study shows for the first time the potential for an FUS-mediated technique for targeted and precise CRISPR-Cas9 gene editing in human stem cells.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*CRISPR-Cas Systems
*Microbubbles
*Ribonucleoproteins/metabolism/genetics
*Gene Editing/methods
RNA, Guide, CRISPR-Cas Systems/genetics
Gene Transfer Techniques
Green Fluorescent Proteins/genetics/metabolism

@article {pmid39792803,
year = {2025},
author = {Wu, Z and Zhao, C and Ai, H and Wang, Z and Chen, M and Lyu, Y and Tong, Q and Liu, L and Sun, H and Pu, J and Zhang, R and Hu, X and Liu, J and Ma, X and Sun, Y},
title = {A Susceptible Cell-Selective Delivery (SCSD) of mRNA-Encoded Cas13d Against Influenza Infection.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {9},
pages = {e2414651},
pmid = {39792803},
issn = {2198-3844},
support = {2022YFD1801005//National Key Research and Development Program of China/ ; 32171389//National Natural Science Foundation of China/ ; 32192450//National Natural Science Foundation of China/ ; WSJK2024MS235//Joint Program on Health Science & Technology Innovation of Hainan Province/ ; },
mesh = {Animals ; Mice ; *CRISPR-Cas Systems/genetics ; *Orthomyxoviridae Infections/virology/genetics ; *Disease Models, Animal ; Humans ; *RNA, Messenger/genetics/metabolism ; Influenza A virus/genetics ; Influenza, Human/genetics ; Antiviral Agents/pharmacology ; Swine ; Nanoparticles/chemistry ; },
abstract = {To bolster the capacity for managing potential infectious diseases in the future, it is critical to develop specific antiviral drugs that can be rapidly designed and delivered precisely. Herein, a CRISPR/Cas13d system for broad-spectrum targeting of influenza A virus (IAV) from human, avian, and swine sources is designed, incorporating Cas13d mRNA and a tandem CRISPR RNA (crRNA) specific for the highly conserved regions of viral polymerase acidic (PA), nucleoprotein (NP), and matrix (M) gene segments, respectively. Given that the virus targets cells with specific receptors but is not limited to a single organ, a Susceptible Cell Selective Delivery (SCSD) system is developed by modifying a lipid nanoparticle with a peptide mimicking the function of the hemagglutinin of influenza virus to target sialic acid receptors. The SCSD system can precisely deliver an all-RNA-based CRISPR/Cas13d system into potentially infected cells. This drug is shown to reduce the viral load in the lungs by 2.37 log10 TCID50 mL[-1] and protect 100% of mice from lethal influenza infection. The SCSD-based CRISPR/Cas13d system shows promise for the flexible and efficient therapy of infections caused by rapidly evolving and novel viruses.},
}

Animals
Mice
*CRISPR-Cas Systems/genetics
*Orthomyxoviridae Infections/virology/genetics
*Disease Models, Animal
Humans
*RNA, Messenger/genetics/metabolism
Influenza A virus/genetics
Influenza, Human/genetics
Antiviral Agents/pharmacology
Swine
Nanoparticles/chemistry

@article {pmid39753388,
year = {2025},
author = {Ahmad, N and Awan, MJA and Amin, I and Mansoor, S},
title = {Perfecting prime editing: achieving precise edits in dicots.},
journal = {Trends in plant science},
volume = {30},
number = {3},
pages = {232-234},
doi = {10.1016/j.tplants.2024.12.005},
pmid = {39753388},
issn = {1878-4372},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Genome, Plant/genetics ; Crops, Agricultural/genetics ; Magnoliopsida/genetics ; },
abstract = {Prime editing (PE), a precise CRISPR-based method, has worked well in some plants but faces challenges in dicots. Vu and colleagues developed new PE tools that greatly improve PE efficiency in dicots, enabling accurate, heritable genome edits. This advance marks a breakthrough that could revolutionize crop improvement and plant biotechnology.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
Genome, Plant/genetics
Crops, Agricultural/genetics
Magnoliopsida/genetics

@article {pmid40038432,
year = {2025},
author = {Zhang, J and Yin, W and Jiang, Q and Mao, W and Deng, W and Jin, S and Wang, X and He, R and Qiao, J and Liu, Y},
title = {Precise amplification-free detection of highly structured RNA with an enhanced SCas12a assay.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {366},
pmid = {40038432},
issn = {2399-3642},
mesh = {*CRISPR-Cas Systems ; *SARS-CoV-2/genetics/isolation & purification ; *COVID-19/diagnosis/virology ; Humans ; Polymorphism, Single Nucleotide ; RNA/genetics/analysis ; RNA, Viral/genetics ; CRISPR-Associated Proteins/genetics/metabolism ; Nucleic Acid Conformation ; Limit of Detection ; },
abstract = {The CRISPR/Cas12a system has revolutionized molecular diagnostics, yet the direct detection of RNA, particularly those with complex structures, remains a significant challenge. Here, we present an updated SCas12a system, termed SCas12aV2, which enables precise, amplification-free detection of highly structured RNA molecules. By optimizing the length of scaffold RNA, targeting asymmetric structures, and utilizing dsDNA-ssDNA hybrid activators, we have significantly reduced steric hindrance in the detection system, thereby markedly enhancing both sensitivity and kinetics compared to traditional DNA activators. The SCas12aV2 assay achieves a detection limit of 246 aM for pooled activators and 10 pM for single-site targeting, demonstrating high specificity for single nucleotide polymorphisms (SNPs). It successfully identifies viable bacteria and SARS-CoV-2 infections in clinical samples. The assay is versatile and can be applied to various Cas12a orthologs, including thermostable CtCas12a. This work advances molecular diagnostics by improving the accuracy and efficiency of RNA detection.},
}

*CRISPR-Cas Systems
*SARS-CoV-2/genetics/isolation & purification
*COVID-19/diagnosis/virology
Humans
Polymorphism, Single Nucleotide
RNA/genetics/analysis
RNA, Viral/genetics
CRISPR-Associated Proteins/genetics/metabolism
Nucleic Acid Conformation
Limit of Detection

@article {pmid40036851,
year = {2025},
author = {Nakamura, K and Kanou, M and Fujii, W and Kouzaki, K and Jimbo, T and Yamanouchi, K and Nakazato, K and Ueda, H and Hirata, J and Yamana, K},
title = {New selective androgen receptor modulator TEI-SARM2 improves muscle function in a Duchenne muscular dystrophy rat model.},
journal = {Human molecular genetics},
volume = {},
number = {},
pages = {},
doi = {10.1093/hmg/ddaf028},
pmid = {40036851},
issn = {1460-2083},
support = {//Teijin Limited and Teijin Pharma Limited/ ; },
abstract = {Duchenne muscular dystrophy (DMD) is a severe muscle wasting disease caused by a genetic mutation in the Dmd gene. Dystrophin mutant mice (mdx) have traditionally been used for DMD research as a disease model in the preclinical stage; however, mdx mice exhibit only very mild phenotypes to partially mimic muscle degeneration and regeneration. To overcome this limitation in preclinical studies, DMD mutant rats (DMD rats) generated by CRISPR/Cas were used as a DMD model to exhibit age-dependent progressive muscle degeneration and pathophysiological features similar to DMD patients and more severe than those displayed by mdx mice. TEI-SARM2 is a non-steroidal, orally available selective androgen receptor modulator (SARM) developed as a pharmaceutical candidate for the treatment of muscle wasting diseases based on its potent anabolic activity on skeletal muscle mass. In this study, long-term treatment of daily oral administration of TEI-SARM2 to DMD rats significantly improved muscle function (endurance and strength) assessed by grip and tetanic force measurements. TEI-SARM2 did not increase the muscle weight of hindlimbs in male DMD rats; moreover, long-term, weekly oral administration for 24 weeks improved muscle function with reduced side effects on the prostate and testes weight. Histological analysis showed that TEI-SARM2 significantly reduced adipose tissue infiltration in DMD muscle. In female DMD rats, both daily and weekly TEI-SARM2 treatment showed anabolic effects and enhanced muscle strength and endurance. Taken together, these results indicate that TEI-SARM2 has non-anabolic and anabolic effects that improve dystrophic muscle dysfunction and can be a supportive therapeutic option for DMD.},
}

@article {pmid40036508,
year = {2025},
author = {Karp, H and Zoltek, M and Wasko, K and Vazquez, AL and Brim, J and Ngo, W and Schepartz, A and Doudna, JA},
title = {Packaged delivery of CRISPR-Cas9 ribonucleoproteins accelerates genome editing.},
journal = {Nucleic acids research},
volume = {53},
number = {5},
pages = {},
doi = {10.1093/nar/gkaf105},
pmid = {40036508},
issn = {1362-4962},
support = {T32GM098218/NH/NIH HHS/United States ; 2203903//National Science Foundation/ ; PDF-578176-2023//Natural Sciences and Engineering Research Council of Canada/ ; /HHMI/Howard Hughes Medical Institute/United States ; //HHMI/ ; //Emerson Collective/ ; },
mesh = {*Gene Editing/methods ; *Ribonucleoproteins/genetics/metabolism ; *CRISPR-Cas Systems ; Humans ; CRISPR-Associated Protein 9/genetics/metabolism ; Electroporation ; HEK293 Cells ; Cell Nucleus/metabolism/genetics ; },
abstract = {Effective genome editing requires a sufficient dose of CRISPR-Cas9 ribonucleoproteins (RNPs) to enter the target cell while minimizing immune responses, off-target editing, and cytotoxicity. Clinical use of Cas9 RNPs currently entails electroporation into cells ex vivo, but no systematic comparison of this method to packaged RNP delivery has been made. Here we compared two delivery strategies, electroporation and enveloped delivery vehicles (EDVs), to investigate the Cas9 dosage requirements for genome editing. Using fluorescence correlation spectroscopy, we determined that >1300 Cas9 RNPs per nucleus are typically required for productive genome editing. EDV-mediated editing was >30-fold more efficient than electroporation, and editing occurs at least 2-fold faster for EDV delivery at comparable total Cas9 RNP doses. We hypothesize that differences in efficacy between these methods result in part from the increased duration of RNP nuclear residence resulting from EDV delivery. Our results directly compare RNP delivery strategies, showing that packaged delivery could dramatically reduce the amount of CRISPR-Cas9 RNPs required for experimental or clinical genome editing.},
}

*Gene Editing/methods
*Ribonucleoproteins/genetics/metabolism
*CRISPR-Cas Systems
Humans
CRISPR-Associated Protein 9/genetics/metabolism
Electroporation
HEK293 Cells
Cell Nucleus/metabolism/genetics

@article {pmid40030016,
year = {2025},
author = {Le, TT and Choi, HI and Kim, JW and Yun, JH and Lee, YH and Jeon, EJ and Kwon, KK and Cho, DH and Choi, DY and Park, SB and Yoon, HR and Lee, J and Sim, EJ and Lee, YJ and Kim, HS},
title = {Cas9-mediated gene-editing frequency in microalgae is doubled by harnessing the interaction between importin α and phytopathogenic NLSs.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {10},
pages = {e2415072122},
doi = {10.1073/pnas.2415072122},
pmid = {40030016},
issn = {1091-6490},
support = {RS-2024-00459155//National Research Foundation of Korea (NRF)/ ; 2021R1C1C1003425//National Research Foundation of Korea (NRF)/ ; },
mesh = {*Gene Editing/methods ; *Chlamydomonas reinhardtii/genetics/metabolism ; *alpha Karyopherins/metabolism/genetics ; *Nuclear Localization Signals/genetics ; *CRISPR-Cas Systems ; *Microalgae/genetics/metabolism ; CRISPR-Associated Protein 9/metabolism/genetics ; Bacterial Proteins/metabolism/genetics ; Cell Nucleus/metabolism ; Agrobacterium/genetics/metabolism ; },
abstract = {Pathogen-derived nuclear localization signals (NLSs) enable vigorous nuclear invasion in the host by the virulence proteins harboring them. Herein, inspired by the robust nuclear import mechanism, we show that NLSs originating from the plant infection-associated Agrobacterium proteins VirD2 and VirE2 can be incorporated into the Cas9 system as efficient nuclear delivery enhancers, thereby improving the low gene-editing frequency in a model microalga, Chlamydomonas reinhardtii, caused by poor nuclear localization of the bulky nuclease. Prior to evaluation of the NLSs, IPA1 (Cre04.g215850) was first defined in the alga as the nuclear import-related importin alpha (Impα) that serves as a counterpart adaptor protein of the NLSs, based on extensive in silico analyses considering the protein's sequence, tertiary folding behavior, and structural basis when interacting with a well-studied SV40TAg NLS. Through precursive affinity explorations, we reproducibly found that the NLSs mediated the binding between the Cas9 and Impα with nM affinities and visually confirmed that the fusion of the NLSs strictly localized the peptide-bearing cargoes in the microalgal nucleus without compensating for their cleavage ability. When employed in a real-world application, the VirD2 NLS increases the mutation frequency (~1.12 × 10[-5]) over 2.4-fold compared to an archetypal SV40TAg NLS (~0.46 × 10[-5]) when fused with Cas9. We demonstrate the cross-species versatility of the Impα-dependent strategy by successfully applying it to an industrial alga, Chlorella Sp. HS2. This work, focused on affinity augmentation, provides insights into increasing the frequency of gene editing, which can be advantageously used in programmable mutagenesis with broad applicability.},
}

*Gene Editing/methods
*Chlamydomonas reinhardtii/genetics/metabolism
*alpha Karyopherins/metabolism/genetics
*Nuclear Localization Signals/genetics
*CRISPR-Cas Systems
*Microalgae/genetics/metabolism
CRISPR-Associated Protein 9/metabolism/genetics
Bacterial Proteins/metabolism/genetics
Cell Nucleus/metabolism
Agrobacterium/genetics/metabolism

@article {pmid40011704,
year = {2025},
author = {Adler, BA and Al-Shimary, MJ and Patel, JR and Armbruster, EG and Colognori, D and Charles, EJ and Miller, KV and Lahiri, A and Cui, ML and Oromí-Bosch, A and Voelker, A and Trinidad, M and Lee, J and Beurnier, S and Boger, R and Nomburg, J and Barrangou, R and Mutalik, VK and Schoeniger, JS and Pogliano, JA and Savage, DF and Doudna, JA and Cress, BF},
title = {CRISPRi-ART enables functional genomics of diverse bacteriophages using RNA-binding dCas13d.},
journal = {Nature microbiology},
volume = {10},
number = {3},
pages = {694-709},
pmid = {40011704},
issn = {2058-5276},
mesh = {*Bacteriophages/genetics ; *Genomics/methods ; *CRISPR-Cas Systems ; *Genome, Viral/genetics ; Phylogeny ; RNA, Antisense/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; Genetic Fitness ; RNA-Binding Proteins/genetics/metabolism ; },
abstract = {Bacteriophages constitute one of the largest reservoirs of genes of unknown function in the biosphere. Even in well-characterized phages, the functions of most genes remain unknown. Experimental approaches to study phage gene fitness and function at genome scale are lacking, partly because phages subvert many modern functional genomics tools. Here we leverage RNA-targeting dCas13d to selectively interfere with protein translation and to measure phage gene fitness at a transcriptome-wide scale. We find CRISPR Interference through Antisense RNA-Targeting (CRISPRi-ART) to be effective across phage phylogeny, from model ssRNA, ssDNA and dsDNA phages to nucleus-forming jumbo phages. Using CRISPRi-ART, we determine a conserved role of diverse rII homologues in subverting phage Lambda RexAB-mediated immunity to superinfection and identify genes critical for phage fitness. CRISPRi-ART establishes a broad-spectrum phage functional genomics platform, revealing more than 90 previously unknown genes important for phage fitness.},
}

*Bacteriophages/genetics
*Genomics/methods
*CRISPR-Cas Systems
*Genome, Viral/genetics
Phylogeny
RNA, Antisense/genetics/metabolism
Clustered Regularly Interspaced Short Palindromic Repeats
Genetic Fitness
RNA-Binding Proteins/genetics/metabolism

@article {pmid39964191,
year = {2025},
author = {Chen, L and Wang, C and Zhu, Z and Yang, L},
title = {Field-Deployable Detection of Genetically Modified Organisms with an Integrated Method of Loop-Mediated Isothermal Amplification and CRISPR/FnCas12a.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {9},
pages = {5625-5634},
doi = {10.1021/acs.jafc.4c11373},
pmid = {39964191},
issn = {1520-5118},
mesh = {*Nucleic Acid Amplification Techniques/methods ; *Zea mays/genetics ; *CRISPR-Cas Systems ; *Glycine max/genetics ; *Plants, Genetically Modified/genetics ; Molecular Diagnostic Techniques ; },
abstract = {The detection of genetically modified organisms (GMOs) is crucial for regulatory compliance and consumer safety. This study presents a novel method combining loop-mediated isothermal amplification (LAMP) with CRISPR/Cas12a cleavage, termed Cas-pfLAMP, for sensitive and specific GMO detection. We developed assays for three GM events: maize DBN9936 and MON810 and soybean GTS40-3-2. By incorporating a universal protospacer adjacent motif (PAM) sequence into LAMP primers, we overcame the limitations of PAM site dependence. The Cas-pfLAMP assays demonstrated high specificity and sensitivity, with limits of detection as low as 10-12 copies per reaction. Furthermore, we developed a point-of-care testing platform integrating rapid DNA extraction, Cas-pfLAMP, and lateral flow strips for on-site GMO detection. This platform achieved comparable sensitivity to qPCR, detecting GM contents as low as 0.1% in simulated samples within 40 min. The Cas-pfLAMP method offers the advantages of PAM site independence, high specificity and sensitivity, and suitability for field testing without specialized equipment. This approach represents a promising new generation of GMO detection methods with potential applications in various scenarios.},
}

*Nucleic Acid Amplification Techniques/methods
*Zea mays/genetics
*CRISPR-Cas Systems
*Glycine max/genetics
*Plants, Genetically Modified/genetics
Molecular Diagnostic Techniques

@article {pmid40029720,
year = {2025},
author = {do Rêgo, RL and Neves, FPG and Miranda, FM and da Silva, AB and Cabral, AS and Dos Santos, BA and Lima, JLC and de Souza, ARV},
title = {CRISPR Elements and Their Association with Macrolide and Aminoglycoside Resistance Genes in Enterococci.},
journal = {Microbial drug resistance (Larchmont, N.Y.)},
volume = {},
number = {},
pages = {},
doi = {10.1089/mdr.2024.0236},
pmid = {40029720},
issn = {1931-8448},
abstract = {CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated) systems are common among enterococci and may prevent the acquisition of mobile genetic elements carrying antimicrobial resistance genes. In this study, we correlate the presence of CRISPR with genes associated with macrolide resistance and high-level resistance to aminoglycosides (HLR-A) among 216 Enterococcus faecalis and 82 Enterococcus faecium isolates. We used PCR to detect genes associated with macrolide resistance, HLR-A, and type II-A CRISPR elements. We used two-tailed Fisher's exact test to evaluate correlation between CRISPR and resistance genes. One hundred and seven (35.9%) isolates had at least one HLR-A gene; the prevalent genes were aac(6')-Ie-aph(2″)-Ia and ant(6)-Ia found in 61 (57%) and 46 (43%) isolates, respectively. The macrolide resistance genes erm(A) and erm(B) were found in 116 (38.9%) isolates. Overall, 174 (58.4%) isolates had at least one CRISPR element; the predominant one was CRISPR3-Cas (n = 117; 39.2%). The presence of three genes, two related to HLR-A [aph(2″)-Ic and ant(6)-Ia] and one macrolide resistance gene [erm(B)], was associated with the absence of CRISPR (p < 0.05), mainly in E. faecalis lacking CRISPR3-Cas. We observed the association between the absence of CRISPR and the presence of major aminoglycoside and macrolide resistance determinants, contributing to the understanding of the evolution of resistance in enterococci.},
}

@article {pmid40029521,
year = {2025},
author = {Berzosa, M and McShane, AN and Nanda, P and Williams, M and Malinova, D},
title = {CRISPR/Cas9 Methods for Identification and Validation of Genes Regulating BCR-Mediated Antigen Uptake.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2909},
number = {},
pages = {153-164},
pmid = {40029521},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Receptors, Antigen, B-Cell/genetics/metabolism ; Animals ; Humans ; Antigens/genetics/metabolism ; Mice ; B-Lymphocytes/metabolism/immunology ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Genome-wide CRISPR screens are a powerful tool to interrogate and identify gene function in a wide variety of applications and cell types. CRISPR-Cas9 technology using pooled CRISPR single guide RNA (sgRNA) libraries enables genetic editing in bulk in a large population of cells of interest. After selection of gene-edited cells, phenotyping effects can be evaluated by quantifying abundance (over- or under-representation) of individual sgRNAs using DNA sequencing. In addition to cell survival, these assays can be applied to investigations of drug sensitivity, as well as almost any cellular process with a clear phenotypic read out, for example, receptor internalization, migration, autophagy, and differentiation.Here, taking as an example the identification of molecular components governing B-cell antigen uptake through the B-cell receptor, we describe whole-genome, small-scale, and in vivo validation methods to identify and validate genes regulating BCR-mediated antigen uptake.},
}

*CRISPR-Cas Systems
*Gene Editing/methods
*Receptors, Antigen, B-Cell/genetics/metabolism
Animals
Humans
Antigens/genetics/metabolism
Mice
B-Lymphocytes/metabolism/immunology
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid40029520,
year = {2025},
author = {Kumar, M and Gentner-Göbel, E and Maity, PC},
title = {Facilitating Gene Editing in Human Lymphoma Cells Using Murine Ecotropic γ-Retroviruses.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2909},
number = {},
pages = {133-151},
pmid = {40029520},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems ; Animals ; Mice ; Cell Line, Tumor ; Genetic Vectors/genetics ; Lymphoma, B-Cell/genetics ; Lymphoma/genetics/therapy ; Lentivirus/genetics ; Retroviridae/genetics ; },
abstract = {Genetic modifications using CRISPR-Cas9 have revolutionized cancer research and other preclinical studies. Exceptionally, these efficient tools are inadequate in a few disease models and cell lines due to the aberrant differentiation states and the accumulation of excessive somatic mutations that compromise the robustness of viral gene delivery and stable transduction. A couple of B lymphoma cell lines fall into this category where lentiviral transfection becomes inefficient and exhibits variable efficiency. Additionally, lentiviral delivery requires high biosafety levels. To address this challenge, we have developed a two-step strategy that supports CRISPR-Cas9 through lentivirus and murine ecotropic γ-retrovirus. By engineering B lymphoma cell lines to express Cas9 and mCat1, a specific receptor for ecotropic retroviruses, we enable efficient and safe gene editing through ecotropic γ-retrovirus. We demonstrate the efficacy of this method by generating IgM-deficient B lymphoma cell lines. This innovative approach simplifies protocols, enhances accessibility, and paves the way for standardized gene manipulation of B cell lymphoma models for molecular cell biology research.},
}

*Gene Editing/methods
Humans
*CRISPR-Cas Systems
Animals
Mice
Cell Line, Tumor
Genetic Vectors/genetics
Lymphoma, B-Cell/genetics
Lymphoma/genetics/therapy
Lentivirus/genetics
Retroviridae/genetics

@article {pmid39982896,
year = {2025},
author = {Sui, Z and Chen, B and Zhao, J and Yang, H and Guo, L and Xu, J},
title = {Dual-Accelerated Signal Amplification in Biosensing via Spatial Confining Catalytic Hairpin Assembly-Activated Spherical CRISPR/Cas12a System for Trans-Cleavage of Hairpin DNA Reporters.},
journal = {Analytical chemistry},
volume = {97},
number = {8},
pages = {4668-4677},
doi = {10.1021/acs.analchem.4c07111},
pmid = {39982896},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; *MicroRNAs/analysis ; *CRISPR-Cas Systems/genetics ; Humans ; *DNA/chemistry/genetics ; Nucleic Acid Amplification Techniques/methods ; Inverted Repeat Sequences ; CRISPR-Associated Proteins/metabolism ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression and are implicated in various diseases, including cancer. Due to their critical role in diagnostics, there is a growing need for sensitive, specific, and rapid detection methods for miRNAs. In this study, we present a dual-accelerated signal amplification platform for miRNA biosensing, which integrates spatial confining catalytic hairpin assembly (SC-CHA) with spherical CRISPR/Cas12a (S-CRISPR/Cas12a) system for (SC-CHA@S-CRISPR/Cas12a) trans-cleavage of hairpin DNA reporters. The method employs a biotinylated palindrome-rich assembly sequence (PAS) to form DNA nanoballs, which serve as a scaffold for the operation of SC-CHA upon miRNA binding. The SC-CHA products bind with crRNA and Cas 12a protein, activating S-CRISPR/Cas12a system to cleave the hairpin DNA reporter and generate a detectable fluorescence signal. The uniqueness of this system lies in the combined use of DNA nanoballs and hairpin DNA reporters, both of which significantly accelerate reaction kinetics, resulting in rapid signal generation. Additionally, the spherical DNA nanostructure, integrated with the S-CRISPR/Cas12a system, greatly enhances biostability and accelerating reaction kinetics. These features enable the platform to exhibit high sensitivity, with a limit of detection (LOD) as low as 13.75 fM, and excellent specificity, successfully distinguishing miRNA-21 from other miRNAs. The assay is also biostable, demonstrating reliable performance in complex biological samples such as human serum. This dual-acceleration approach offers a promising solution for sensitive, rapid, and specific miRNA biosensing, with potential applications in early cancer diagnosis and clinical monitoring.},
}

*Biosensing Techniques/methods
*MicroRNAs/analysis
*CRISPR-Cas Systems/genetics
Humans
*DNA/chemistry/genetics
Nucleic Acid Amplification Techniques/methods
Inverted Repeat Sequences
CRISPR-Associated Proteins/metabolism
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid39965890,
year = {2025},
author = {Chen, H and Cheng, X and Wang, Y and Han, N and Liu, L and Wei, H and Tu, Z and Gu, Z and Song, R and Wang, S and Rong, Z},
title = {Two-Dimensional Nanozyme-Catalyzed Colorimetric CRISPR Assay for the Microfluidic Detection of Monkeypox Virus.},
journal = {Analytical chemistry},
volume = {97},
number = {8},
pages = {4407-4415},
doi = {10.1021/acs.analchem.4c05570},
pmid = {39965890},
issn = {1520-6882},
mesh = {*Colorimetry/methods ; Catalysis ; Graphite/chemistry ; Humans ; Platinum/chemistry ; Nanostructures/chemistry ; Benzidines/chemistry ; CRISPR-Cas Systems/genetics ; Gold/chemistry ; Limit of Detection ; Microfluidic Analytical Techniques ; },
abstract = {The recent monkeypox epidemic outbreaks worldwide highlight the urgent need for fast and precise diagnostic solutions, especially in resource-limited settings. Here, a two-dimensional nanozyme-catalyzed colorimetric CRISPR assay for the microfluidic detection of the monkeypox virus (MPXV) was established. We utilized graphene oxide as a substrate for the adsorption of gold seeds and the deposition of a porous Pt shell to prepare high-performance two-dimensional GO@Pt nanomaterials. The viral nucleic acids released from clinical samples initiated a single-step recombinase polymerase amplification-CRISPR/Cas13a for the trans-cleavage of ssRNA reporters labeled with FAM and biotin. These reporters can be recognized by FAM antibody-conjugated GO@Pt nanozymes and streptavidin-coated magnetic beads. The formed sandwich immunocomplexes can catalyze the oxidation of a colorless 3,3',5,5'-tetramethylbenzidine substrate with a distinct color change. The proposed GO@Pt-catalyzed colorimetric CRISPR assay exhibited a limit of detection of 1 copy/μL of MPXV in 60 min. Forty clinical samples, including rash fluid swabs and oral swabs, were tested with 100% agreement with the real-time PCR. These results indicate the excellent potential of GO@Pt-catalyzed colorimetric CRISPR for the sensitive and accurate testing of MPXV under resource-constrained conditions.},
}

*Colorimetry/methods
Catalysis
Graphite/chemistry
Humans
Platinum/chemistry
Nanostructures/chemistry
Benzidines/chemistry
CRISPR-Cas Systems/genetics
Gold/chemistry
Limit of Detection
Microfluidic Analytical Techniques

@article {pmid39961250,
year = {2025},
author = {Xiang, Q and Guo, F and Li, D and Xu, F and Zhou, W},
title = {Low-background CRISPR/Cas12a sensing system with circular CRISPR RNA for amplified fluorescent detection of antibody in human serum.},
journal = {Talanta},
volume = {288},
number = {},
pages = {127730},
doi = {10.1016/j.talanta.2025.127730},
pmid = {39961250},
issn = {1873-3573},
mesh = {Humans ; *CRISPR-Cas Systems ; Antibodies/chemistry/immunology ; RNA, Circular/blood ; Biosensing Techniques/methods ; CRISPR-Associated Proteins/chemistry ; Fluorescence ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Regular monitoring of serum antibody levels is crucial for preventing interference with therapeutic effectiveness and reducing the risk of toxicity. To address this, a CRISPR/Cas12a sensing system with circular CRISPR RNAs ([C]crRNAs) is described for highly sensitive detection of anti-digoxin (Anti-Dig) antibodies in human serum. In this work, the topology structure of [C]crRNAs effectively suppresses the function of linear crRNAs ([L]crRNAs), making them unable to regulate the cis-/trans-cleavage activity of the Cas12a system. Therefore, a low-background is obtained in the absence of targets. The target Anti-Dig antibodies trigger the assembly of the complete multicomponent nucleic acid enzyme (MNAzyme) with active enzyme activity, which can transform [C]crRNAs into [L]crRNAs. The [L]crRNAs further recover the trans-cleavage activity of the CRISPR/Cas12a system, which can degrade single-stranded reporter DNA to generate a significantly enhanced fluorescent signal. This method enables sensitive detection of Anti-Dig antibodies as low as 15 pM within 60 min and exhibits a linear detection range of 25 pM-50 nM. It also exhibits excellent selectivity against non-target antibodies and has been successfully validated in diluted serum samples, achieving a recovery rate ranging from 96.16 % to 103.08 %. This novel CRISPR/Cas12a sensing system with [C]crRNA represents a powerful and efficient tool for detecting low-abundance biomarkers in complex biological samples.},
}

Humans
*CRISPR-Cas Systems
Antibodies/chemistry/immunology
RNA, Circular/blood
Biosensing Techniques/methods
CRISPR-Associated Proteins/chemistry
Fluorescence
Limit of Detection
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid39954412,
year = {2025},
author = {Liu, ML and Li, Y and Yang, WJ and Yang, WW and Zhuo, Y and He, XJ},
title = {Engineering multi-activator-encoded DNA nanonet to accelerate CRISPR-Cas12a activation for rapid and sensitive electrochemiluminescence bioassay.},
journal = {Talanta},
volume = {288},
number = {},
pages = {127724},
doi = {10.1016/j.talanta.2025.127724},
pmid = {39954412},
issn = {1873-3573},
mesh = {*CRISPR-Cas Systems/genetics ; *DNA/chemistry/genetics ; *Luminescent Measurements/methods ; *Biosensing Techniques/methods ; *DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism/genetics ; *Electrochemical Techniques/methods ; Humans ; Nanostructures/chemistry ; Limit of Detection ; CRISPR-Associated Proteins/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Despite CRISPR-associated (Cas) nucleases have emerged as a versatile and highly programmable tool for biosensing and molecular diagnostics, the efficient manipulation of targeted CRISPR-Cas12a activation requires further improvement. Herein, we engineered a target-response DNA nanodevice called multi-activator-encoded DNA nanonet (MAIDA) which displayed efficient manipulation of CRISPR-Cas12a trans-activity for apurinic/apyrimidinic endonuclease 1 (APE1) activity monitoring. The MAIDA nanodevice was constructed by multi-activator loops (MA loops) encoded with three activator sequences and target-response loops (TR loops) encoded with three abasic sites to generate interlocked DNA nanonet. Notably, the activator sequences on MA loop were pre-hybridized with TR loop, which not only generate AP sites but also inhibit the CRISPR-Cas12a activation in the initial state. When APE1 is present, the AP sites on the MAIDA nanodevice were recognized and cleaved to the release of MA loops, which could activate the trans-cleavage of CRISPR-Cas12a and then output the signal through electrochemiluminescence (ECL) biosensor. Finally, the experimental results demonstrate that the MA loops increase the ECL response of CRISPR-Cas12a by 1.5-fold compared with the conventional single-linear activators, and the limit of detection (LOD) of APE1 by the proposed biosensor is 1.46 × 10[-10] U/μL. The MAIDA nanodevice promoted the efficient manipulation of targeted CRISPR-Cas12a activation with high sensitivity and selectivity, which provided a promising tool for enhancing DNA-based sensing and analytical applications.},
}

*CRISPR-Cas Systems/genetics
*DNA/chemistry/genetics
*Luminescent Measurements/methods
*Biosensing Techniques/methods
*DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism/genetics
*Electrochemical Techniques/methods
Humans
Nanostructures/chemistry
Limit of Detection
CRISPR-Associated Proteins/genetics
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid39949273,
year = {2025},
author = {Li, T and Li, R and Li, Z and Li, Z and Wang, M and He, X and Zhang, G and Zhang, Y and Yang, Y and Li, Y},
title = {Unveiling a novel RNA G-triplex structure: its function and potential in CRISPR-based diagnostics.},
journal = {Chemical communications (Cambridge, England)},
volume = {61},
number = {20},
pages = {4002-4005},
doi = {10.1039/d4cc06581f},
pmid = {39949273},
issn = {1364-548X},
mesh = {*CRISPR-Cas Systems/genetics ; *Nucleic Acid Conformation ; *Hemin/chemistry ; *RNA/chemistry/genetics ; Benzothiazoles/chemistry ; Mesoporphyrins/chemistry ; Biosensing Techniques ; Humans ; Models, Molecular ; },
abstract = {We report the discovery of a novel higher-order RNA structure, RNA G-triplex (rG3), formed by the TERRA sequence. Through CD spectroscopy, NMR analysis, and molecular modeling, we confirmed its stable, parallel conformation. rG3 exhibits strong binding to thioflavin T (ThT), N-methyl mesoporphyrin IX (NMM), and hemin, showcasing its potential as a biosensing element. Additionally, CRISPR-Cas13a trans-cleaves rG3, demonstrating its utility as a sensitive reporter in diagnostic applications. These findings expand the structural diversity of RNA and suggest new avenues for RNA-based biosensors and CRISPR diagnostics.},
}

*CRISPR-Cas Systems/genetics
*Nucleic Acid Conformation
*Hemin/chemistry
*RNA/chemistry/genetics
Benzothiazoles/chemistry
Mesoporphyrins/chemistry
Biosensing Techniques
Humans
Models, Molecular

@article {pmid39923676,
year = {2025},
author = {Li, X and Han, Z and Guo, P and Zhang, X and Hu, Y and Cao, J},
title = {LbCas12a-based DNA POCT facilitates fast genotyping on farm.},
journal = {Talanta},
volume = {287},
number = {},
pages = {127672},
doi = {10.1016/j.talanta.2025.127672},
pmid = {39923676},
issn = {1873-3573},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Genotyping Techniques/methods ; Swine ; Polymorphism, Single Nucleotide ; African Swine Fever Virus/genetics/isolation & purification ; Sheep ; CRISPR-Associated Proteins/genetics ; Farms ; DNA/genetics ; Genotype ; Endodeoxyribonucleases/genetics ; Bacterial Proteins ; },
abstract = {Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 12a (CRISPR/Cas12a) detection system is now widely used for nucleic acid detection and disease diagnosis. However, there are still fewer detections for single nucleotide polymorphisms (SNPs) and limited diversified detection systems for pathogen and SNP sites detection, which greatly limits their applications. Obviously, the development of a more diversified and convenient suite of detection tools is essential to unlock the full potential of CRISPR/Cas12a technology and to expand its applications across a wider range of scenarios. We have successfully developed an integrated CRISPR/Cas12a assay system. This system introduces crRNA during protein expression, reducing the number of steps and reaction time by adding only a fluorescent reporter gene and target DNA during subsequent detection. It enables on-site visualization of the assay in combination with a Recombinase polymerase amplification (RPA) reaction. Combined with the RPA reaction, we are able to rapidly detect African swine fever virus (ASFV) pathogens with high specificity. The system also enables genotyping of the SNP site of the porcine prolificacy-associated estrogen receptor (ESR) gene and the sheep prolificacy-associated Fecundity booroola (FecB) gene. Visualization is possible up to a final concentration of 3 nM, and effective differentiation of low concentrations within the concentration range of the assay. The integrated CRISPR/Cas12a assay system we developed has a robust design that ensures high-fidelity genotyping and pathogen detection are no longer restricted to the lab, allowing for rapid field analysis, which is crucial for timely interventions in agricultural and clinical settings. In addition, it has the advantages of low cost, easy operation and visualization of results.},
}

Animals
*CRISPR-Cas Systems/genetics
*Genotyping Techniques/methods
Swine
Polymorphism, Single Nucleotide
African Swine Fever Virus/genetics/isolation & purification
Sheep
CRISPR-Associated Proteins/genetics
Farms
DNA/genetics
Genotype
Endodeoxyribonucleases/genetics
Bacterial Proteins

@article {pmid39892116,
year = {2025},
author = {Yu, L and Tang, Y and Sun, Y and Wang, H and Yi, H and Zhong, Y and Shao, Z and Zhou, S and He, S and Cao, K and Peng, L and Chen, Z},
title = {DMSO enhanced one-pot HDA-CRISPR/Cas12a biosensor for ultrasensitive detection of Monkeypox virus.},
journal = {Talanta},
volume = {287},
number = {},
pages = {127660},
doi = {10.1016/j.talanta.2025.127660},
pmid = {39892116},
issn = {1873-3573},
mesh = {*Biosensing Techniques ; *CRISPR-Cas Systems ; *Monkeypox virus/isolation & purification ; *Mpox, Monkeypox/diagnosis/virology ; *Nucleic Acid Amplification Techniques/methods ; DNA Helicases ; Dimethyl Sulfoxide ; },
abstract = {We present a dimethyl sulfoxide (DMSO)-enhanced one-pot HDA-CRISPR/Cas12a biosensor for the ultrasensitive detection of the monkeypox virus (MPXV). The MPXV B6R gene was initially amplified using DMSO-enhanced helicase-dependent amplification (HDA) in the bottom of the reaction tubes. DMSO was employed to enhance the amplification efficiency of HDA. CRISPR/Cas12a reagents, pre-added to the caps of the reaction tubes, were subsequently combined with HDA products to generate fluorescence signals. This DMSO-enhanced HDA-CRISPR/Cas12a biosensor enables the detection of synthetic B6R DNA within 1 hour, with a detection limit of 9 aM and a dynamic range of 10 aM to 100 pM. Our work demonstrated that 5% DMSO can enhance the sensitivity of the HDA -CRISPR/Cas12a assay by four orders of magnitude. For clinical applications, this approach can detect as low as 0.4 copies/μL of MPXV pseudovirus. A DMSO-enhanced HDA-CRISPR/Cas12a lateral flow biosensor (LFB) was developed for MPXV point-of-care testing (POCT), achieving a LOD of 10 fM. This method exhibits high specificity in distinguishing the monkeypox virus from closely related orthopoxviruses, including variola, vaccinia, cowpox, ectromelia, and camelpox. The assay is rapid (sample-to-answer times less than 1 h), cost-effective, and compatible with both fluorescence detection and the LFB for visual readouts.},
}

*Biosensing Techniques
*CRISPR-Cas Systems
*Monkeypox virus/isolation & purification
*Mpox, Monkeypox/diagnosis/virology
*Nucleic Acid Amplification Techniques/methods
DNA Helicases
Dimethyl Sulfoxide

@article {pmid39887213,
year = {2025},
author = {Kyawe, PP and Liu, P and Jiang, Z and Bradley, ES and Cicuto, T and Trombly, MI and Silverman, N and Fitzgerald, KA and McDougall, WM and Wang, JP},
title = {CRISPR editing of candidate host factors that impact influenza A virus infection.},
journal = {Microbiology spectrum},
volume = {13},
number = {3},
pages = {e0262724},
pmid = {39887213},
issn = {2165-0497},
support = {N660011924036//U.S. Department of Defense (DOD)/ ; },
mesh = {Humans ; *Influenza A virus/genetics ; A549 Cells ; *Adenosine Deaminase/genetics/metabolism ; *CRISPR-Cas Systems ; *RNA-Binding Proteins/genetics/metabolism ; *Virus Replication/genetics ; *Influenza, Human/virology/genetics/metabolism ; *Gene Editing ; N-Acetylgalactosaminyltransferases/genetics/metabolism ; Animals ; Host-Pathogen Interactions/genetics ; Enterovirus B, Human/genetics ; Chemokine CXCL10/genetics/metabolism ; Dogs ; },
abstract = {Influenza A virus (IAV) is a respiratory pathogen with a segmented negative-sense RNA genome that can cause epidemics and pandemics. The host factors required for the complete IAV infectious cycle have not been fully identified. Here, we examined three host factors for their contributions to IAV infectivity. We performed CRISPR-mediated knockout of cytidine monophosphate N-acetylneuraminic acid synthetase (CMAS) as well as CRISPR-mediated overexpression of beta-1,4 N-acetylgalactosaminyltransferase 2 (B4GALNT2) and adenosine deaminase acting on RNA 1 (ADAR1) in the human bronchial epithelial A549 cell line and evaluated the impact on IAV and other RNA viruses. We confirmed that knockout of CMAS or overexpression of B4GALNT2 restricts IAV infection by diminishing binding to the cell surface but has no effect on vesicular stomatitis virus infection. Although ADAR1 overexpression does not significantly inhibit IAV replication, it has a pro-viral effect with coxsackie B virus (CVB) infection. This pro-viral effect is not likely secondary to reduced type I interferon (IFN) production, as the induction of the IFN-stimulated genes ISG15 and CXCL10 is negligible in both parent and ADAR1-overexpressing A549 cells following CVB challenge. In contrast, ISG15 and CXCL10 production is robust and equal for parent and ADAR1-overexpressing A549 cells challenged with IAV. Taken together, these data provide insights into how host factors can be further explored to understand the dynamics of pro- and anti-viral factors.IMPORTANCEInfluenza A virus (IAV) remains a global threat due to its ability to cause pandemics, making the identification of host factors essential for developing new antiviral strategies. In this study, we utilized CRISPR-based techniques to investigate host factors that impact IAV infectivity. Knockout of CMAS, a key enzyme in sialic acid biosynthesis, significantly reduced IAV binding and infection by disrupting sialic acid production on the cell surface. Overexpression of B4GALNT2 had similar effects, conferring resistance to IAV infection through diminished cell-surface binding. Overexpression of ADAR1, known for its role in RNA editing and immune regulation, impacted IAV replication minimally but enhanced coxsackie B virus replication. Such findings reveal the diverse roles of host factors in viral infection, offering insights for targeted therapeutic development against IAV and other pathogens.},
}

Humans
*Influenza A virus/genetics
A549 Cells
*Adenosine Deaminase/genetics/metabolism
*CRISPR-Cas Systems
*RNA-Binding Proteins/genetics/metabolism
*Virus Replication/genetics
*Influenza, Human/virology/genetics/metabolism
*Gene Editing
N-Acetylgalactosaminyltransferases/genetics/metabolism
Animals
Host-Pathogen Interactions/genetics
Enterovirus B, Human/genetics
Chemokine CXCL10/genetics/metabolism
Dogs

@article {pmid39879802,
year = {2025},
author = {Yuan, G and Cheng, D and Huang, J and Wang, M and Xia, X and An, H and Xie, F and Li, X and Chen, J and Tang, Y and Peng, C},
title = {An integrated and multifunctional homemade cell sensor platform based on Si-d-CQDs and CRISPR-Cas12a for CD31 detection during endothelial-to-mesenchymal transition.},
journal = {Talanta},
volume = {287},
number = {},
pages = {127612},
doi = {10.1016/j.talanta.2025.127612},
pmid = {39879802},
issn = {1873-3573},
mesh = {Humans ; *CRISPR-Cas Systems ; *Platelet Endothelial Cell Adhesion Molecule-1/metabolism/analysis ; Electrochemical Techniques/methods ; Biosensing Techniques/methods ; Electrodes ; Epithelial-Mesenchymal Transition ; Human Umbilical Vein Endothelial Cells ; Tin Compounds ; },
abstract = {Endothelial-to-mesenchymal transition (EndMT) plays a crucial role in the initiation and progression of atherosclerosis and various disease processes. Cluster of differentiation 31 (CD31) is a significant marker in EndMT. Detecting CD31 is essential for early-stage monitoring of EndMT and diagnosing atherosclerosis. Herein, we propose a homemade electrochemical array sensor comprising four electrodes, applied for cell cultivation, electrical stimulation, and simultaneous electrochemical detection, offering a three-in-one approach for CD31 detection during EndMT. To enhance the analytical performance of the cell sensor, indium tin oxide/chitosan-MXene/polyaniline (ITO/CS-MXene/PANI) composites were synthesized. The synthesis process involved the polymerization of PANI on the surface of the CS-MXene-modified ITO electrode, resulting in the creation of highly biocompatible active sites for cell immobilization. Si-d-CQDs@acDNA-AptCD31-Fc, with exceptional photophysical and chemical properties, was integrated into the array sensor setup, which enabled the dual-mode detection of fluorescent and electrochemical signals in cultured cells. A CRISPR-Cas12a system was employed to cleave Si-d-CQDs@acDNA-AptCD31-Fc. Subsequently, the fragmented Fc molecules were enriched via electrochemistry to further amplify the electrochemical signals. Through the unique combination of programmable Si-d-CQDs@acDNA-AptCD31-Fc, the CRISPR-Cas12a system, and voltage enrichment, a novel "signal-on-off-super on" signal amplification strategy was developed. The cell sensor exhibited a wide linear range from 1 × 10[1] particles mL[-1] to 1 × 10[6] particles mL[-1] (R[2] = 0.9912) and a detection limit of 4 particles mL[-1]. The proposed strategy presents a promising approach for developing CRISPR-Cas12a-based tools for detecting various stages of EndMT and opens a new window for dual-mode analysis applications.},
}

Humans
*CRISPR-Cas Systems
*Platelet Endothelial Cell Adhesion Molecule-1/metabolism/analysis
Electrochemical Techniques/methods
Biosensing Techniques/methods
Electrodes
Epithelial-Mesenchymal Transition
Human Umbilical Vein Endothelial Cells
Tin Compounds

@article {pmid39707619,
year = {2025},
author = {Liao, W and Guo, R and Li, J and Liu, N and Jiang, L and Whelan, J and Shou, H},
title = {CRISPR/Cas9-mediated mutagenesis of SEED FATTY ACID REDUCER genes significantly increased seed oil content in soybean.},
journal = {Plant & cell physiology},
volume = {66},
number = {2},
pages = {273-284},
doi = {10.1093/pcp/pcae148},
pmid = {39707619},
issn = {1471-9053},
support = {2021C02057//Key Research Projects of Zhejiang Province/ ; 2021YFF1001204//Research and Development Program of China/ ; B14027//111 Project of Ministry of Education/ ; },
mesh = {*Glycine max/genetics/metabolism ; *Seeds/genetics/metabolism ; *CRISPR-Cas Systems ; *Fatty Acids/metabolism ; *Plant Oils/metabolism ; *Mutagenesis ; Gene Editing/methods ; Plant Proteins/genetics/metabolism ; Plants, Genetically Modified ; Genes, Plant ; Gene Expression Regulation, Plant ; Germination/genetics ; },
abstract = {Increasing seed oil content (SOC) is an important breeding goal for soybean breeding. While significant efforts have been made to improve SOC through metabolic pathway engineering, research to increase soybean SOC by reducing lipid degradation and fatty acid (FA) decomposition during seed maturation process is limited. Seed fatty acid reducers (SFARs) are members of the GDSL enzyme family and play a crucial role in lipid metabolism. Among them, a pair of the GmSFAR4 genes is highly expressed in soybean seeds during seed desiccation and germination. In the study, GmSFAR4a/b double mutants were generated using CRISPR/Cas9-mediated gene editing technique. The seed FA content of GmSFAR4a/b double mutants was significantly increased by ∼8% compared to wild type when grown in greenhouse, and ∼17% when grown in the field, without any adverse effects on seed vitality and plant growth. Our work enriches the understanding of soybean seed oil metabolism and provides a new approach to increase soybean SOC.},
}

*Glycine max/genetics/metabolism
*Seeds/genetics/metabolism
*CRISPR-Cas Systems
*Fatty Acids/metabolism
*Plant Oils/metabolism
*Mutagenesis
Gene Editing/methods
Plant Proteins/genetics/metabolism
Plants, Genetically Modified
Genes, Plant
Gene Expression Regulation, Plant
Germination/genetics

@article {pmid39692461,
year = {2025},
author = {Li, X and Hu, H and Wang, H and Liu, J and Jiang, W and Zhou, F and Zhang, J},
title = {DNA nanotechnology-based strategies for minimising hybridisation-dependent off-target effects in oligonucleotide therapies.},
journal = {Materials horizons},
volume = {12},
number = {5},
pages = {1388-1412},
doi = {10.1039/d4mh01158a},
pmid = {39692461},
issn = {2051-6355},
mesh = {Humans ; *Nanotechnology/methods ; *Oligonucleotides/therapeutic use/pharmacology/administration & dosage ; Oligonucleotides, Antisense/therapeutic use/pharmacology/administration & dosage ; DNA/genetics ; Genetic Therapy/methods ; Nucleic Acid Hybridization ; Animals ; RNA, Small Interfering/administration & dosage/therapeutic use ; CRISPR-Cas Systems ; },
abstract = {Targeted therapy has emerged as a transformative breakthrough in modern medicine. Oligonucleotide drugs, such as antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs), have made significant advancements in targeted therapy. Other oligonucleotide-based therapeutics like clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems are also leading a revolution in targeted gene therapy. However, hybridisation-dependent off-target effects, arising from imperfect base pairing, remain a significant and growing concern for the clinical translation of oligonucleotide-based therapeutics. These mismatches in base pairing can lead to unintended steric blocking or cleavage events in non-pathological genes, affecting the efficacy and safety of the oligonucleotide drugs. In this review, we examine recent developments in oligonucleotide-based targeted therapeutics, explore the factors influencing sequence-dependent targeting specificity, and discuss the current approaches employed to reduce the off-target side effects. The existing strategies, such as chemical modifications and oligonucleotide length optimisation, often require a trade-off between specificity and binding affinity. To further address the challenge of hybridisation-dependent off-target effects, we discuss DNA nanotechnology-based strategies that leverage the collaborative effects of nucleic acid assembly in the design of oligonucleotide-based therapies. In DNA nanotechnology, collaborative effects refer to the cooperative interactions between individual strands or nanostructures, where multiple bindings result in more stable and specific hybridisation behaviour. By requiring multiple complementary interactions to occur simultaneously, the likelihood of unintended partially complementary binding events in nucleic acid hybridisation should be reduced. And thus, with the aid of collaborative effects, DNA nanotechnology has great promise in achieving both high binding affinity and high specificity to minimise the hybridisation-dependent off-target effects of oligonucleotide-based therapeutics.},
}

Humans
*Nanotechnology/methods
*Oligonucleotides/therapeutic use/pharmacology/administration & dosage
Oligonucleotides, Antisense/therapeutic use/pharmacology/administration & dosage
DNA/genetics
Genetic Therapy/methods
Nucleic Acid Hybridization
Animals
RNA, Small Interfering/administration & dosage/therapeutic use
CRISPR-Cas Systems

@article {pmid39514364,
year = {2025},
author = {Doctor, Y and Sanghvi, M and Mali, P},
title = {A Manual for Genome and Transcriptome Engineering.},
journal = {IEEE reviews in biomedical engineering},
volume = {18},
number = {},
pages = {250-267},
doi = {10.1109/RBME.2024.3494715},
pmid = {39514364},
issn = {1941-1189},
support = {OT2 OD032742/OD/NIH HHS/United States ; R01 HG012351/HG/NHGRI NIH HHS/United States ; },
mesh = {Humans ; *Transcriptome/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Animals ; Genetic Engineering ; Genome/genetics ; },
abstract = {Genome and transcriptome engineering have emerged as powerful tools in modern biotechnology, driving advancements in precision medicine and novel therapeutics. In this review, we provide a comprehensive overview of the current methodologies, applications, and future directions in genome and transcriptome engineering. Through this, we aim to provide a guide for tool selection, critically analyzing the strengths, weaknesses, and best use cases of these tools to provide context on their suitability for various applications. We explore standard and recent developments in genome engineering, such as base editors and prime editing, and provide insight into tool selection for change of function (knockout, deletion, insertion, substitution) and change of expression (repression, activation) contexts. Advancements in transcriptome engineering are also explored, focusing on established technologies like antisense oligonucleotides (ASOs) and RNA interference (RNAi), as well as recent developments such as CRISPR-Cas13 and adenosine deaminases acting on RNA (ADAR). This review offers a comparison of different approaches to achieve similar biological goals, and consideration of high-throughput applications that enable the probing of a variety of targets. This review elucidates the transformative impact of genome and transcriptome engineering on biological research and clinical applications that will pave the way for future innovations in the field.},
}

Humans
*Transcriptome/genetics
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Animals
Genetic Engineering
Genome/genetics

@article {pmid39513325,
year = {2025},
author = {Lin, J and Xiao, D and Wu, M and Chen, X and Xu, Q and Wang, S and Zang, L},
title = {Pleiotropic effects of Ebony on pigmentation and development in the Asian multi-coloured ladybird beetle, Harmonia axyridis (Coleoptera: Coccinellidae).},
journal = {Insect molecular biology},
volume = {34},
number = {2},
pages = {263-277},
doi = {10.1111/imb.12968},
pmid = {39513325},
issn = {1365-2583},
support = {KJCX20230115//Technical Innovation Program of Beijing Academy of Agriculture and Forestry Sciences/ ; 2023YFD1400600//National Key Research and Development Program of China/ ; },
mesh = {Animals ; *Coleoptera/genetics/growth & development/physiology ; *Pigmentation/genetics ; Insect Proteins/genetics/metabolism ; Larva/growth & development/genetics/physiology ; Genetic Pleiotropy ; Female ; CRISPR-Cas Systems ; Pupa/growth & development/genetics ; Male ; Predatory Behavior ; Melanins/metabolism ; },
abstract = {Melanin plays a pivotal role in insect body pigmentation, significantly contributing to their adaptation to diverse biotic and abiotic environmental challenges. Several genes involved in insect melanin synthesis showed pleiotropic effects on insect development and reproduction. Among these, the N-β-alanyl dopamine synthetase gene (Ebony) is integral to the pigmentation process. However, the full spectrum of its pleiotropic impacts is not yet thoroughly understood. In this study, we identified and characterised the HaEbony gene in the Asian multi-coloured ladybird beetle (Harmonia axyridis) and found that HaEbony gene is a conserved gene within the Coleoptera order. We aimed to further explore the multiple roles of HaEbony in the physiology and behaviour in H. axyridis. The CRISPR/Cas9 system was applied to generate multiple HaEbony knockout allele (HaEbony[+/-]), showing nucleotide deletion in the G0 and G1 generations. Remarkably, the resultant HaEbony[+/-] mutants consistently displayed darker pigmentation than their wild-type counterparts across larval, pupal and adult stages. Furthermore, these HaEbony[+/-] individuals (G0) demonstrated an enhanced predatory efficiency, evidenced by a higher number of aphids consumed compared to the wild type. A significant finding was the reduced egg hatchability in both G0 and G1 generations of the HaEbony[+/-] group, highlighting a potential reproductive fitness cost associated with HaEbony deficiency. In conclusion, our study not only sheds light on the multifaceted roles of HaEbony in H. axyridis but also highlights the potential of employing CRISPR/Cas9-targeted modifications of the Ebony gene. Such genetic interventions could enhance the environmental adaptability and predatory efficacy of ladybirds, presenting a novel strategy in biological control application.},
}

Animals
*Coleoptera/genetics/growth & development/physiology
*Pigmentation/genetics
Insect Proteins/genetics/metabolism
Larva/growth & development/genetics/physiology
Genetic Pleiotropy
Female
CRISPR-Cas Systems
Pupa/growth & development/genetics
Male
Predatory Behavior
Melanins/metabolism

@article {pmid40029441,
year = {2025},
author = {Pei, Y and Cao, W and Kong, X and Wang, S and Sun, Z and Zuo, Y and Hu, Z},
title = {CRISPR/Cas9-mediated efficient PlCYP81Q38 mutagenesis in Phryma leptostachya.},
journal = {Planta},
volume = {261},
number = {4},
pages = {73},
pmid = {40029441},
issn = {1432-2048},
support = {2023-JC-QN-0213//Natural Science Foundation of Shaanxi Province/ ; 2023YFD1700700//National Key Research and Development Program of China/ ; 2452020221//Chinese Universities Scientific Fund from Science/ ; },
mesh = {*CRISPR-Cas Systems ; *Lignans/metabolism ; *Gene Editing/methods ; *Mutagenesis ; Plant Proteins/genetics/metabolism ; Cytochrome P-450 Enzyme System/genetics/metabolism ; Dioxoles ; Plant Roots/genetics/metabolism ; },
abstract = {Combined with hairy root transformation, the CRISPR/Cas9 system was established to initiate targeted mutagenesis of PlCYP81Q38, which influenced lignan accumulation in Phryma leptostachya. Phryma leptostachya is a traditional Chinese medicinal herb renowned for its applications in both conventional medicine and natural botanical insecticides, with lignans as the main active ingredients. During the biosynthesis of lignans, PlCYP81Q38, a P450 protein, is assumed to play a crucial role and is accountable for the production of sesamin from (+)-pinoresinol. As a cutting-edge genome editing tool, the CRISPR/Cas9 system is widely employed across diverse species for gene functional research but yet to be harnessed in P. leptostachya. This study utilized the CRISPR/Cas9 system in conjunction with hairy root transformation to initiate targeted mutagenesis in PlCYP81Q38 gene. Employing binary vectors, pYLCRISPR/Cas9Pubi-H, complemented by dual single-stranded guided RNAs (sgRNAs), enabled precise editing at two gene sites and the deletion of large fragments. This editing system resulted in mutagenesis rates surpassing 79%, achieving a notable rate of 61.9% fragment deletion mutants. Liquid chromatography/tandem mass spectrometry confirmed the impact on lignan biosynthesis by PlCYP81Q38-targeted mutagenesis, resulting in the accumulation of pinoresinol and disrupted production of sesamin, 6-demethoxy-leptostachyol acetate, and leptostachyol acetate. Furthermore, the knockout of PlCYP81Q38 up-regulated its upstream pathway genes, such as dirigent gene, cinnamoyl-CoA reductase genes, cinnamyl-alcohol dehydrogenase genes, and p-coumarate 3-hydroxylase genes, identified through gene co-expression analysis. Collectively, mediated by the CRISPR/Cas9 platform, the new biotechnology for targeted genome editing within P. leptostachya, our findings affirm the significant roles of PlCYP81Q38 in the lignan biosynthesis pathway and highlight the potential of CRISPR/Cas9 in exploring the functional genome and secondary metabolite biosynthesis of this plant species.},
}

*CRISPR-Cas Systems
*Lignans/metabolism
*Gene Editing/methods
*Mutagenesis
Plant Proteins/genetics/metabolism
Cytochrome P-450 Enzyme System/genetics/metabolism
Dioxoles
Plant Roots/genetics/metabolism

@article {pmid40029034,
year = {2025},
author = {Cheng, X and Zhao, W and Chen, D and Ren, D and Qian, T and Xia, X and Wang, X and Li, Q and Yang, J and Gu, Y and Zhang, P and Yin, K and Yu, P and Dong, W},
title = {Ultrasensitive Detection of FEN1 Activity for Cancer Diagnosis Using a CRISPR/Cas13a-Based Triple Cascade Amplification System.},
journal = {Advanced healthcare materials},
volume = {14},
number = {6},
pages = {e2404411},
doi = {10.1002/adhm.202404411},
pmid = {40029034},
issn = {2192-2659},
support = {22104090//National Natural Science Foundation of China/ ; 82102184//National Natural Science Foundation of China/ ; 82204349//National Natural Science Foundation of China/ ; 82170526//National Natural Science Foundation of China/ ; 62305207//National Natural Science Foundation of China/ ; PT21011//Shanghai High-Level Local University construction project/ ; 23ZR1436800//Shanghai 2023 "Science and Technology Innovation Action Plan" Natural Science Foundation Project/ ; ZYYZDXK-2023070//NATCM's Project of High-level Construction of Key TCM Disciplines/ ; 2023AH010073//Program for Excellent Sci-tech Innovation Teams of Universities in Anhui Province/ ; LCZX2201//Clinical Research Foundation of Huadong Hospital/ ; //the Junior Thousand Talents Program of China/ ; },
mesh = {Humans ; *Flap Endonucleases/metabolism ; *CRISPR-Cas Systems/genetics ; *Neoplasms/diagnosis/metabolism/genetics ; Biosensing Techniques/methods ; Cell Line, Tumor ; },
abstract = {Flap endonuclease 1 (FEN1) is closely associated with tumor progression and proliferation, making it a promising biomarker for cancer diagnosis. However, developing a sensitive, reliable, and user-friendly method for quantitative FEN1 detection remains technically challenging. In this study, an ultrasensitive FEN1 biosensor is established using a target-induced cleavage-ligation-transcription-activation cascade strategy (LTACas13a) to enhance the cleavage ability of CRISPR/Cas13a. The LTACas13a method has shown excellent performance in screening FEN1 inhibitors and detecting endogenous FEN1 activity in living cells, as well as in clinical biological samples such as human serum and tissue samples. Additionally, using a universal dumbbell probe derived from FEN1, a multiplex LTACas13a strategy is developed for detecting various DNA glycosylases, including formamidopyrimidine DNA glycosylase, uracil DNA glycosylase, and human alkyl adenine DNA glycosylase. This straightforward approach provides a reliable and effective diagnostic tool for early-stage cancer detection and offers significant opportunities for FEN1 biosensing and related drug discovery.},
}

Humans
*Flap Endonucleases/metabolism
*CRISPR-Cas Systems/genetics
*Neoplasms/diagnosis/metabolism/genetics
Biosensing Techniques/methods
Cell Line, Tumor

@article {pmid40027883,
year = {2025},
author = {Zhang, X and Tao, C and Li, M and Zhang, S and Liang, P and Huang, Y and Liu, H and Wang, Y},
title = {Engineering of SauriCas9 with enhanced specificity.},
journal = {Molecular therapy. Nucleic acids},
volume = {36},
number = {1},
pages = {102455},
pmid = {40027883},
issn = {2162-2531},
abstract = {SauriCas9 is a compact Cas9 nuclease showing promise for in vivo therapeutic applications. However, concerns about off-target effects necessitated improvements in specificity. We addressed this by introducing mutations to eliminate polar contacts between Cas9 and the target DNA, resulting in the SauriCas9-R253A variant with enhanced specificity. To validate its efficacy, we employed SauriCas9-R253A to disrupt three genes (B2M, TRAC, and PDCD1), a strategy integral to the development of allogeneic chimeric antigen receptor T cell (CAR-T) therapies. Our results demonstrated that the most efficient single-guide RNAs for SauriCas9-R253A exhibited comparable activity to SpCas9 and showed no detectable off-target effects in the disruption of these genes, highlighting its therapeutic potential.},
}

@article {pmid40026698,
year = {2025},
author = {Álvarez-Rodríguez, A and Li, Z and Jin, BK and Stijlemans, B and Geldhof, P and Magez, S},
title = {A CRISPR-Cas-based recombinase polymerase amplification assay for ultra-sensitive detection of active Trypanosoma brucei evansi infections.},
journal = {Frontiers in molecular biosciences},
volume = {12},
number = {},
pages = {1512970},
pmid = {40026698},
issn = {2296-889X},
abstract = {INTRODUCTION: Control of Trypanosoma brucei evansi (T. b. evansi) infections remains a significant challenge in managing Surra, a widespread veterinary disease affecting both wild and domestic animals. In the absence of an effective vaccine, accurate diagnosis followed by treatment is crucial for successful disease management. However, existing diagnostic methods often fail to detect active infections, particularly in field conditions. Recent advancements in CRISPR-Cas technology, combined with state-of-the-art isothermal amplification assays, offer a promising solution. This approach has led us to the development of a TevRPA-CRISPR assay, a highly sensitive and specific T. b. evansi diagnostic tool suitable for both laboratory and field settings.

METHODS: First, the TevCRISPR-Cas12b cleavage assay was developed and optimized, and its analytical sensitivity was evaluated. Next, this technology was integrated with the TevRPA to create the TevRPA-CRISPR test, with the reaction conditions being optimized and its analytical sensitivity and specificity assessed. Finally, the test's accuracy in detecting both active and cured T. b. evansi infections was evaluated.

RESULTS: The optimized TevCRISPR-Cas12b cleavage assay demonstrated the ability to detect T. b. evansi target DNA at picomolar concentrations. Integrating TevCRISPR-Cas12b with RPA in Two-Pot and One-Pot TevRPA-CRISPR tests achieved up to a 100-fold increase in analytical sensitivity over RPA alone, detecting attomolar concentrations of T. b. evansi target DNA, while maintaining analytical specificity for T. b. evansi. Both assays exhibited performance comparable to the gold standard TevPCR in experimental mouse infections, validating their effectiveness for detecting active infections and assessing treatment efficacy.

DISCUSSION: The TevRPA-CRISPR tests prove highly effective for diagnosing active infections and assessing treatment efficacy, while being adaptable for both laboratory and field use. Thus, the TevRPA-CRISPR assays emerge as a promising addition to current diagnostic tools, offering efficient and reliable detection of active T. b. evansi infections.},
}

@article {pmid40025688,
year = {2025},
author = {He, F and Liu, X and Wang, H and Li, X and Wu, Y and Zhang, D and Liang, S},
title = {The Transcriptional Regulator DhyR Positively Modulates Daptomycin Biosynthesis in Streptomyces roseosporus.},
journal = {Microbial biotechnology},
volume = {18},
number = {3},
pages = {e70110},
doi = {10.1111/1751-7915.70110},
pmid = {40025688},
issn = {1751-7915},
support = {2023SCUH0068//Basic Research Funding for Colleges and Universities- 'From 0 to 1' Innovative Research Program of Sichuan University/ ; 2024ZYD0107//Sichuan Province Science and Technology Support Program/ ; },
mesh = {*Streptomyces/genetics/metabolism ; *Daptomycin/biosynthesis ; *Gene Expression Regulation, Bacterial ; *Multigene Family ; Biosynthetic Pathways/genetics ; Transcription Factors/genetics/metabolism ; Gene Expression Profiling ; Anti-Bacterial Agents/biosynthesis/metabolism ; Gene Deletion ; Bacterial Proteins/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Daptomycin (DAP) is a cyclic lipopeptide antibiotic produced by Streptomyces roseosporus, and its biosynthesis is precisely regulated by a complex regulatory network. Although the biosynthetic pathway of DAP has been elucidated, the regulatory mechanism governing its biosynthesis at the transcriptional level is not yet fully understood. In the present study, a new transcriptional regulator, DhyR, was identified. A deletion mutant of dhyR was constructed using the CRISPR-Cas9 tool to elucidate the biological role of DhyR thanks to functional and transcriptomic analyses. The results demonstrated that DhyR positively regulates DAP biosynthesis in S. roseosporus. The in-frame deletion of the dhyR gene resulted in a significant downregulation of the transcription levels of all structural genes within the DAP biosynthetic gene cluster and a significant decrease in DAP yield. In contrast, overexpression of dhyR enhanced the transcription levels of the DAP biosynthetic gene cluster, leading to a 23% increase in DAP yield. Deletion of dhyR caused significant changes in the expression of multiple genes involved in carbohydrate metabolism, energy metabolism and amino acid metabolic pathways through transcriptome analysis. Especially, deletion of dhyR led to a significant downregulation of transcription levels of three DAP biosynthesis-associated genes, including atrA, depR1 and ssig-05090. In summary, DhyR positively regulates DAP biosynthesis in S. roseosporus by influencing the expression of the DAP gene cluster and modulating precursor flux. It functions as a pleiotropic regulator of primary and secondary metabolism in S. roseosporus.},
}

*Streptomyces/genetics/metabolism
*Daptomycin/biosynthesis
*Gene Expression Regulation, Bacterial
*Multigene Family
Biosynthetic Pathways/genetics
Transcription Factors/genetics/metabolism
Gene Expression Profiling
Anti-Bacterial Agents/biosynthesis/metabolism
Gene Deletion
Bacterial Proteins/genetics/metabolism
CRISPR-Cas Systems

@article {pmid40024074,
year = {2025},
author = {Mukherjee, AG and R, K and Gopalakrishnan, AV},
title = {Recent advances in CRISPR technology in the milieu of oral squamous cell carcinoma.},
journal = {Pathology, research and practice},
volume = {269},
number = {},
pages = {155860},
doi = {10.1016/j.prp.2025.155860},
pmid = {40024074},
issn = {1618-0631},
abstract = {Oral squamous cell carcinoma (OSCC) is a widely recognized cancer that constitutes over 90 % of all oral cancer cases [1]. Thanks to advancements in cancer treatment, such as radiation, chemotherapy, and molecular target therapy, the 5-year relative survival rate for OSCC has more than doubled in the past 26 years. However, the exact cause and molecular process of OSCC have not been fully understood, and further investigation is necessary. CRISPR/Cas technology is seen as a groundbreaking tool in molecular biology, providing unparalleled accuracy in altering genes. Its use in conditions like OSCC shows excellent potential for advancing research and treatment development. OSCC, characterized by the growth of cancer cells in the lining of the mouth, presents challenges in its treatment and control. Traditional therapies like surgery, radiation, and chemotherapy often show limited success and may lead to severe side effects. The promise of CRISPR/Cas technology in tackling critical aspects of OSCC is apparent. In this correspondence, we discuss the recent advances in CRISPR technology in the OSCC milieu.},
}

@article {pmid40022609,
year = {2025},
author = {Lee, WH and Bates, EA and Kipp, ZA and Pauss, SN and Martinez, GJ and Blair, CA and Hinds, TD},
title = {Insulin receptor responsiveness governs TGFβ-induced hepatic stellate cell activation: Insulin resistance instigates liver fibrosis.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {39},
number = {5},
pages = {e70427},
pmid = {40022609},
issn = {1530-6860},
support = {R01DK121797//HHS | NIH | NIDDK | Division of Diabetes, Endocrinology, and Metabolic Diseases (DEM)/ ; R01DA058933//HHS | NIH | National Institute on Drug Abuse (NIDA)/ ; F31HL170972//HHS | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; F31HL175979//HHS | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; 25PRE1374495//American Heart Association (AHA)/ ; },
mesh = {Cell Line ; Humans ; *Insulin Resistance ; *Receptor, Insulin/metabolism ; *Hepatic Stellate Cells/metabolism ; Transforming Growth Factor beta/metabolism ; CRISPR-Cas Systems ; *Liver Cirrhosis/metabolism ; Cell Proliferation ; Cell Movement ; Signal Transduction ; Gene Regulatory Networks ; },
abstract = {The insulin receptor (INSR) has been shown to be hyperactive in hepatic stellate cells (HSCs) in humans and rodents with liver fibrosis. To explore HSC cellular mechanisms that INSR regulates during pro-fibrotic stimulation, we used CRISPR-Cas9 technology. We knocked out a portion of the INSR gene in human LX2 HSC cells (INSR[e5-8] KO) that regulates insulin responsiveness but not the insulin-like growth factor (IGF) or transforming growth factor-β (TGFβ) signaling. The INSR[e5-8] KO HSCs had significantly higher cell growth, BrdU incorporation, and lower TP53 expression that suppresses growth, and they also exhibited increased migration compared to the Scramble control. We treated the scramble control and INSR[e5-8] KO HSCs with insulin or TGFβ and profiled hundreds of kinase activities using the PamGene PamStation kinome technology. Our analysis showed that serine/threonine kinase (STK) activities were reduced, and most of the protein-tyrosine kinase (PTK) activities were increased in the INSR[e5-8] KO compared to the Scramble control HSCs. To study gene transcripts altered in activated Scramble control and INSR[e5-8] KO HSCs, we treated them with TGFβ for 24 h. We isolated RNA for sequencing and found that the INSR[e5-8] KO cells, compared to control HSCs, had altered transcriptional responsiveness to TGFβ stimulation, collagen-activated signaling, smooth muscle cell differentiation pathways, SMAD protein signaling, collagen metabolic process, integrin-mediated cell adhesion, and notch signaling. This study demonstrates that reduced INSR responsiveness enhances HSC growth and selectively mediates TGFβ-induced HSC activation. These findings provide new insights into the development of more effective treatments for liver fibrosis.},
}

Cell Line
Humans
*Insulin Resistance
*Receptor, Insulin/metabolism
*Hepatic Stellate Cells/metabolism
Transforming Growth Factor beta/metabolism
CRISPR-Cas Systems
*Liver Cirrhosis/metabolism
Cell Proliferation
Cell Movement
Signal Transduction
Gene Regulatory Networks

@article {pmid40021632,
year = {2025},
author = {Gao, S and Weng, B and Wich, D and Power, L and Chen, M and Guan, H and Ye, Z and Xu, C and Xu, Q},
title = {Improving adenine base editing precision by enlarging the recognition domain of CRISPR-Cas9.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {2081},
pmid = {40021632},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *CRISPR-Associated Protein 9/metabolism/genetics/chemistry ; *Adenine/metabolism/chemistry ; Humans ; Streptococcus pyogenes/genetics/enzymology ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Protein Domains ; HEK293 Cells ; Bacterial Proteins/genetics/metabolism/chemistry ; },
abstract = {Domain expansion contributes to diversification of RNA-guided-endonucleases including Cas9. However, it remains unclear how REC domain expansion could benefit Cas9. In this study, we identify an insertion spot that is compatible with large REC insertion and succeeds in enlarging the non-catalytic REC domain of Streptococcus pyogenes Cas9. The natural-evolution-like giant SpCas9 (GS-Cas9) is created and shows substantially improved editing precision. We further discover that enlarging the REC domain could enable regulation of the N-terminal adenine deaminase TadA8e tethered to the Cas9 scaffold, which contributes to substantially reducing unexpected editing and improving the precision of the adenine base editor ABE8e. We provide proof of concept for evolution-inspired expansion of Cas9 and offer an alternative solution for optimizing gene editors. Our study also indicates a vast potential for engineering the topological malleability of RNA-guided endonucleases and base editors.},
}

*CRISPR-Cas Systems
*Gene Editing/methods
*CRISPR-Associated Protein 9/metabolism/genetics/chemistry
*Adenine/metabolism/chemistry
Humans
Streptococcus pyogenes/genetics/enzymology
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
Protein Domains
HEK293 Cells
Bacterial Proteins/genetics/metabolism/chemistry

@article {pmid40021494,
year = {2025},
author = {Sajjad, MW and Imran, I and Muzamil, F and Naqvi, RZ and Amin, I},
title = {AZD7648 (DNA-PKcs inhibitor): a two-edged sword for editing genomes.},
journal = {Functional & integrative genomics},
volume = {25},
number = {1},
pages = {49},
pmid = {40021494},
issn = {1438-7948},
mesh = {*Gene Editing ; *DNA-Activated Protein Kinase/genetics/antagonists & inhibitors ; Humans ; CRISPR-Cas Systems ; DNA End-Joining Repair/drug effects ; Recombinational DNA Repair ; Animals ; Pyrimidines ; Schiff Bases ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR-Cas9) has been the most practical technique in genome editing for the last decade. Its molecular mechanism includes steps that occur in a sequence, starting from a break in a double strand to repair. After a double-strand break in the DNA strand, the repairing of DNA done via Homology-Directed Repair (HDR) is considered important in different organisms as it is ideal for precise genome editing and the reduction of unintended mutations. Still, it is mostly dominated by the Non-Homologous End Joining (NHEJ) pathway. A recent study by Cullot et al. published in Nature Biotechnology showed interesting features of AZD7648 (a DNA-PKcs inhibitor) that increase the probability of HDR event while DNA repairing (Cullot et al. 2024).},
}

*Gene Editing
*DNA-Activated Protein Kinase/genetics/antagonists & inhibitors
Humans
CRISPR-Cas Systems
DNA End-Joining Repair/drug effects
Recombinational DNA Repair
Animals
Pyrimidines
Schiff Bases

@article {pmid40020637,
year = {2025},
author = {Deng, A and Mao, Z and Jin, X and Lv, W and Huang, L and Zhong, H and Wang, S and Shi, Y and Zhou, T and Zhao, J and Huang, Q and Luo, X and Ma, L and Zou, H and Fu, R and Huang, G},
title = {ID-CRISPR: A CRISPR/Cas12a platform for label-free and sensitive detection of rare mutant alleles using self-interference DNA hydrogel reporter.},
journal = {Biosensors & bioelectronics},
volume = {278},
number = {},
pages = {117309},
doi = {10.1016/j.bios.2025.117309},
pmid = {40020637},
issn = {1873-4235},
abstract = {Accurate and sensitive detection of single nucleotide variants (SNVs) is paramount for cancer diagnosis and treatment. The CRISPR/Cas12a system shows promise for SNV detection due to its high sensitivity and single-base specificity. However, most CRISPR/Cas12a-based methods rely on F/Q-labeled single-stranded DNA (ssDNA) reporters, which are susceptible to fluorescence fluctuations, thereby reducing accuracy. To address these limitations, researchers have proposed using DNA hydrogels as signal transducers in CRISPR/Cas12a systems. Yet, the encapsulation of indicators into DNA hydrogels introduces additional instability, which could compromise both detection sensitivity and linearity. In this study, we integrated hyperspectral interferometry into a DNA hydrogel-based CRISPR/Cas12a detection platform (ID-CRISPR) to achieve sensitive label-free SNV detection. Using EGFR L858R SNV as a model target, we demonstrated that ID-CRISPR can detect mutant allele frequencies (MAFs) as low as 0.1% with a limit of detection (LOD) of 5 aM, while also showing its potential for quantifying SNV abundance. Its clinical utility was confirmed through analysis of lung tumor samples, with results consistent with sequencing data. Therefore, ID-CRISPR provides a sensitive, label-free, and user-friendly platform for SNV detection, offering new insights into combining optical sensing with DNA hydrogel technology in CRISPR/Cas assays.},
}

@article {pmid39970722,
year = {2025},
author = {Lai, Y and Wang, J and Xie, N and Liu, G and Lacap-Bugler, DC},
title = {Identification of a novel forkhead transcription factor MtFKH1 for cellulase and xylanase gene expression in Myceliophthora thermophila (ATCC 42464).},
journal = {Microbiological research},
volume = {294},
number = {},
pages = {128097},
doi = {10.1016/j.micres.2025.128097},
pmid = {39970722},
issn = {1618-0623},
mesh = {*Gene Expression Regulation, Fungal ; *Cellulase/genetics/metabolism ; *Fungal Proteins/genetics/metabolism ; *Sordariales/genetics/enzymology ; *Forkhead Transcription Factors/genetics/metabolism ; *Promoter Regions, Genetic ; Endo-1,4-beta Xylanases/genetics/metabolism ; Cellulose/metabolism ; Gene Expression Profiling ; CRISPR-Cas Systems ; Spores, Fungal/genetics ; },
abstract = {Myceliophthora thermophila is a thermophilic fungus, known to produce industrially important enzymes in biorefineries. The mechanism underlying cellulase and xylanase expression in filamentous fungi is a complex regulatory network controlled by numerous transcription factors (TFs). These TFs in M. thermophila remain unclear. Here, we identified and characterised a novel cellulase and xylanase regulator MtFKH1 in M. thermophila through comparative transcriptomic and genetic analyses. Five of the eight potential TFs, which showed differential expression levels when grown on Avicel and glucose, were successfully deleted using the newly designed CRISPR/Cas9 system. This system identified the forkhead TF MtFKH1. The disruption of Mtfkh1 elevated the cellulolytic and xylanolytic enzyme activities, whereas the overexpression of Mtfkh1 led to considerable decrease in cellulase and xylanase production in M. thermophila cultivated on Avicel. The loss of Mtfkh1 also exhibited an impairment in sporulation in M. thermophila. Real-time quantitative reverse transcription PCR (RT-qPCR) and the electrophoretic mobility shift assays (EMSAs) demonstrated that MtFKH1 regulates the gene expression and specifically bind to the promoter regions of genes encoding β-glucosidase (bgl1/MYCTH_66804), cellobiohydrolase (cbh1/MYCTH_109566), and xylanase (xyn1/MYCTH_112050), respectively. Furthermore, DNase I footprinting analysis identified binding motif of MtFKH1 in the upstream region of Mtbgl1, with strongest binding affinity. Finally, transcriptomic profiling and Gene Ontology (GO) enrichment analyses of Mtfkh1 deletion mutant revealed that the regulon of MtFKH1 were significantly prevalent in hydrolase activity (acting on glycosyl bonds), polysaccharide binding, and carbohydrate metabolic process functional categories. These findings expand our knowledge on how forkhead transcription factor regulates lignocellulose degradation and provide a novel target for engineering of fungal cell factories with the hyperproduction of cellulase and xylanase.},
}

*Gene Expression Regulation, Fungal
*Cellulase/genetics/metabolism
*Fungal Proteins/genetics/metabolism
*Sordariales/genetics/enzymology
*Forkhead Transcription Factors/genetics/metabolism
*Promoter Regions, Genetic
Endo-1,4-beta Xylanases/genetics/metabolism
Cellulose/metabolism
Gene Expression Profiling
CRISPR-Cas Systems
Spores, Fungal/genetics

@article {pmid39956401,
year = {2025},
author = {Puri, B and Majumder, S and Gaikwad, AB},
title = {CRISPR/Cas9 based knockout of lncRNA MALAT1 attenuates TGF-β1 induced Smad 2/3 mediated fibrosis during AKI-to-CKD transition.},
journal = {European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences},
volume = {207},
number = {},
pages = {107044},
doi = {10.1016/j.ejps.2025.107044},
pmid = {39956401},
issn = {1879-0720},
mesh = {*RNA, Long Noncoding/genetics ; Animals ; *Acute Kidney Injury/genetics/metabolism ; *Fibrosis/genetics ; *Smad3 Protein/genetics/metabolism ; *CRISPR-Cas Systems ; Rats ; *Transforming Growth Factor beta1/genetics/metabolism ; Cell Line ; *Renal Insufficiency, Chronic/genetics/metabolism ; *Smad2 Protein/genetics/metabolism ; Gene Knockout Techniques ; },
abstract = {Acute kidney injury (AKI) is a significant clinical issue with potential long-term consequences, as even a single episode can progress to chronic kidney disease (CKD). The AKI-to-CKD transition involves complex pathophysiology, including persistent inflammation, apoptosis, and fibrosis. Long noncoding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has been recognized as a potential therapeutic target for various kidney diseases, including AKI and CKD. In our previous study, we conducted the transcriptomic analysis of lncRNAs in-vitro and animal models of AKI-to-CKD transition and found several dysregulated lncRNAs such as MALAT1, MEG3, NEAT1, MIAT, and H19 in this transition. Among these, we have selected lncRNA MALAT1 to further validate its role in AKI-to-CKD transition as a therapeutic target via a cluster regularly intercept short palindromic protein (CRISPR) associated protein 9 (Cas9)-mediated knockout approach in NRK52E cells. Guide RNAs (gRNAs) were designed to target MALAT1, and the PX459 turbo green fluorescence protein (GFP) plasmid containing MALAT1 gRNA1&2 was transfected into NRK52E cells using CRISPRMAX. Results demonstrated that MALAT1 knockout significantly reduced MALAT1 expression and attenuated Smad2/3-mediated fibrosis by decreasing pSmad2, pSmad2/3, Smad4, vimentin, fibronectin, collagen-I, and α-SMA expression levels, while increasing Smad7, Smurf2, and E-cadherin levels. These findings suggest that targeting the MALAT1/Smad2/3 pathway could be a potential therapeutic target for mitigating fibrosis to prevent AKI-to-CKD transition.},
}

*RNA, Long Noncoding/genetics
Animals
*Acute Kidney Injury/genetics/metabolism
*Fibrosis/genetics
*Smad3 Protein/genetics/metabolism
*CRISPR-Cas Systems
Rats
*Transforming Growth Factor beta1/genetics/metabolism
Cell Line
*Renal Insufficiency, Chronic/genetics/metabolism
*Smad2 Protein/genetics/metabolism
Gene Knockout Techniques

@article {pmid39904299,
year = {2025},
author = {Zhang, Y and Gao, L and Shi, Z and Wu, Q and Miao, X},
title = {Paper-based electrochemiluminescence telomerase activity detection using hybridization chain reaction and CRISPR/Cas12a dual signal amplification.},
journal = {Bioelectrochemistry (Amsterdam, Netherlands)},
volume = {164},
number = {},
pages = {108916},
doi = {10.1016/j.bioelechem.2025.108916},
pmid = {39904299},
issn = {1878-562X},
mesh = {*Telomerase/genetics ; Humans ; *Luminescent Measurements/methods ; *Nucleic Acid Hybridization ; *CRISPR-Cas Systems ; *Electrochemical Techniques/methods ; Paper ; Biosensing Techniques/methods ; Limit of Detection ; Metal Nanoparticles/chemistry ; Gold/chemistry ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {Sensitive telomerase activity detection becomes particularly significance since the important value of it in early cancer diagnosis as a potential biomarker. Herein, we developed a paper-based analytical devices (PADs) for telomerase activity detection, using positively charged Au@luminol nanoparticles ((+)Au@luminol NPs) as electrochemiluminescence (ECL) signal probe coupling with hybridization chain reaction (HCR) and CRISPR/Cas12a dual signal amplification. Firstly, the initial strong ECL signal was obtained based on the electrostatic adsorption of (+)Au@luminol NPs onto the surface of HCR double-stranded hybrid aggregates. In the presence of telomerase, the primer was efficiently elongated with telomeric repeats of (TTAGGG)n to release activator DNA and trigger the CRISPR/Cas12a, which can prevent the happen of HCR and the adsorption of (+)Au@luminol NPs through cleaving the capture probe on the electrode surface, such results directly inducing the decrease of the ECL signal that was proportional to telomerase concentration, due to the efficient signal amplification of HCR and CRISPR/Cas12a, a low detection limit of 2.3 cells/mL for telomerase could be detected. Moreover, the sensor realized the effective application for telomerase extracts analysis in human serum samples, making it possess potential application value for telomerase activity assays in cancer diagnostics.},
}

*Telomerase/genetics
Humans
*Luminescent Measurements/methods
*Nucleic Acid Hybridization
*CRISPR-Cas Systems
*Electrochemical Techniques/methods
Paper
Biosensing Techniques/methods
Limit of Detection
Metal Nanoparticles/chemistry
Gold/chemistry
Nucleic Acid Amplification Techniques/methods

@article {pmid39880143,
year = {2025},
author = {Li, L and Luo, M and Zhou, L and Wang, Y and Jiao, Y and Wang, C and Gong, C and Cen, X and Yao, S},
title = {Glucocorticoid pre-administration improves LNP-mRNA mediated protein replacement and genome editing therapies.},
journal = {International journal of pharmaceutics},
volume = {672},
number = {},
pages = {125282},
doi = {10.1016/j.ijpharm.2025.125282},
pmid = {39880143},
issn = {1873-3476},
mesh = {Animals ; *Gene Editing/methods ; *RNA, Messenger/administration & dosage ; *Glucocorticoids/administration & dosage ; *Nanoparticles/administration & dosage ; Mice ; Lipids/chemistry ; Male ; Mice, Inbred C57BL ; Genetic Therapy/methods ; Hepatocytes/metabolism ; Phagocytosis/drug effects ; CRISPR-Cas Systems ; Humans ; Liver/metabolism ; Macrophages/metabolism/drug effects ; Gene Transfer Techniques ; Liposomes ; },
abstract = {Lipid nanoparticles (LNPs) are among the most promising non-viral mRNA delivery systems for gene therapeutic applications. However, the in vivo delivery of LNP-mRNA remains challenging due to multiple intrinsic barriers that hinder LNPs from reaching their target cells. In this study, we sought to enhance LNP delivery by manipulating intrinsic regulatory mechanisms involved in their metabolism. We demonstrated that activation of the glucocorticoid pathway significantly increased the systemic delivery of LNP-mRNA in both mice and monkeys, achieving up to a fourfold improvement. This enhancement was primarily attributed to the glucocorticoid-mediated inhibition of macrophage phagocytosis in circulation and the liver, which resulted in higher LNP accumulation in hepatocytes. Consequently, glucocorticoid activation improved the therapeutic efficacy of LNP-based protein replacement and CRISPR/Cas9 genome editing therapies. Together, these findings establish a practical strategy to enhance the systemic delivery of RNA-based protein replacement and genome editing therapeutics, highlighting the potential of manipulating endogenous mechanisms to optimize exogenous gene delivery.},
}

Animals
*Gene Editing/methods
*RNA, Messenger/administration & dosage
*Glucocorticoids/administration & dosage
*Nanoparticles/administration & dosage
Mice
Lipids/chemistry
Male
Mice, Inbred C57BL
Genetic Therapy/methods
Hepatocytes/metabolism
Phagocytosis/drug effects
CRISPR-Cas Systems
Humans
Liver/metabolism
Macrophages/metabolism/drug effects
Gene Transfer Techniques
Liposomes

@article {pmid39739998,
year = {2025},
author = {Liang, H and Mu, X and Huang, Y and Zhao, S and Tian, J},
title = {Magnetic Assisted DNA Logic Gate Nanomachine Based on CRISPR/Cas12a for Recognition of Dual miRNAs.},
journal = {Chemistry, an Asian journal},
volume = {20},
number = {5},
pages = {e202401209},
doi = {10.1002/asia.202401209},
pmid = {39739998},
issn = {1861-471X},
support = {22064003//National Natural Science Foundation of China/ ; 21465007//National Natural Science Foundation of China/ ; 2015GXNSFGA139003//Guangxi Natural Science Foundation of China/ ; //Bagui Scholar of Guangxi Province/ ; //Duxiu Scholar of Guangxi Normal University/ ; CMEMR2014-A08//State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry and Pharmaceutical Sciences of Guangxi Normal University/ ; },
mesh = {*MicroRNAs/analysis/genetics ; *CRISPR-Cas Systems/genetics ; Humans ; *DNA/chemistry ; DNA, Catalytic/chemistry/metabolism ; Biosensing Techniques ; Logic ; Computers, Molecular ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {The anomalous expression of microRNA poses a serious threat to human life and health safety, and serves as an important biomarker for cancer detection. In this study, a novel magnetic-assisted DNA logic gate nanomachine triggered by miRNA-21 and miRNA-155 was designed based on the trans-cleavage activity of CRISPR/Cas12a activated by a split DNA activator, using only a single crRNA and signal probe, which simplified the detection procedure and complex nucleic acid amplification. The presence of target molecules, miRNA-21 and miRNA-155, can stimulate the DNA walker machine assembled on magnetic beads, which releases activator under the action of DNAzyme. Then the trans-cleavage activity of CRISPR/Cas12a is initiated and the system signal significantly increases. Based on this, an AND logic gate nanomachine was constructed for simultaneous analysis of miRNA-21 and miRNA-155. The detection limits of miRNA-21 and miRNA-15 were 9.00 pM and 42.00 pM, respectively, and this method was successfully applied to miRNA analysis in cell samples. This nanomachine combined the DNA walker with DNA logic circuit and CRISPR/Cas12a system, providing a new approach for simultaneous detection of multiple targets and further expanding the application of gene editing in the analysis and sensing of multiple target substances.},
}

*MicroRNAs/analysis/genetics
*CRISPR-Cas Systems/genetics
Humans
*DNA/chemistry
DNA, Catalytic/chemistry/metabolism
Biosensing Techniques
Logic
Computers, Molecular
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid38631265,
year = {2024},
author = {Wang, H and Hang, X and Wang, H and Peng, J and Yu, H and Wang, L},
title = {Label/immobilization-free Cas12a-based electrochemiluminescence biosensor for sensitive DNA detection.},
journal = {Talanta},
volume = {275},
number = {},
pages = {126114},
doi = {10.1016/j.talanta.2024.126114},
pmid = {38631265},
issn = {1873-3573},
mesh = {*Luminescent Measurements/methods ; CRISPR-Associated Proteins/genetics/metabolism ; Endodeoxyribonucleases/genetics/metabolism ; CRISPR-Cas Systems ; Bacterial Proteins/genetics/metabolism ; Feasibility Studies ; Humans ; *DNA, Viral/analysis/genetics ; *Human papillomavirus 16/genetics/isolation & purification ; Hydrogels ; Reproducibility of Results ; *Electrochemical Techniques/methods ; },
abstract = {Electrochemiluminescence (ECL) is one of the most sensitive techniques in the field of diagnostics. However, they typically require luminescent labeling and electrode surface biological modification, which is a time-consuming and laborious process involving multiple steps and may also lead to low reaction efficiency. Fabricating label/modification-free biosensors has become one of the most attractive parts for simplifying the ECL assays. In this work, the ECL luminophores carbon dots (CDs) were encapsulated in DNA hydrogel in situ by a simple rolling circle amplification (RCA) reaction. Upon binding of the target DNA, active Cas12a induces a collateral cleavage of the hydrogel's ssDNA backbone, resulting in a programmable degradation of the hydrogel and the release of CDs. By directly measuring the released CDs ECL, a simple and rapid label/modification-free detection of the target HPV-16 was realized. It is noted that this method allowed for 0.63 pM HPV-16 DNA detection without any amplification step, and it could take only ∼60 min for a fast test of a human serum sample. These results showed that our label/modification-free ECL biosensor has great potential for use in simple, rapid, and sensitive point-of-care (POC) detection.},
}

*Luminescent Measurements/methods
CRISPR-Associated Proteins/genetics/metabolism
Endodeoxyribonucleases/genetics/metabolism
CRISPR-Cas Systems
Bacterial Proteins/genetics/metabolism
Feasibility Studies
Humans
*DNA, Viral/analysis/genetics
*Human papillomavirus 16/genetics/isolation & purification
Hydrogels
Reproducibility of Results
*Electrochemical Techniques/methods

@article {pmid40019808,
year = {2025},
author = {Cook, AL and Moyer, AL and Boxer, L and Norris, AL},
title = {Re: Novel Off-Targeting Events Identified after Genome-Wide Analysis of CRISPR-Cas Edited Pigs.},
journal = {The CRISPR journal},
volume = {},
number = {},
pages = {},
doi = {10.1089/crispr.2024.0091},
pmid = {40019808},
issn = {2573-1602},
}

@article {pmid40019770,
year = {2025},
author = {Mao, X and Lu, Y and Gao, Z and Zhong, J and Xiao, A and Lin, J and Hu, J and Shu, B and Zhang, C},
title = {Modular Microfluidic Sensor Integrating Nucleic Acid Extraction, CRISPR/Cas13a, and Electrochemiluminescence for Multichannel RNA Detection.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.4c06197},
pmid = {40019770},
issn = {1520-6882},
abstract = {Rapid and accurate screening of pathogens is crucial for disease detection. Here, a modular microfluidic sensor has been constructed for RNA detection, with integrated nucleic acid extraction, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a reaction, and electrochemiluminescence (ECL) detection. The sensor consists of nucleic acid processing and detection modules. The nucleic acid processing module is used for nucleic acid extraction, RNA distribution, and the CRISPR/Cas reaction. Specifically, immiscible filtration assisted by surface tension is employed for nucleic acid extraction, significantly reducing the extraction time. Magnetic force is utilized for RNA distribution and transportation, minimizing the need for microstructures, such as microvalves and micropumps. Multichannel CRISPR/Cas13a reactions enable biological recognition, signal amplification, and multiplex detection. The fiber material-based detection module controls fluid flow and performs dry chemistry-based ECL detection. A novel multichannel closed bipolar electrode-based ECL (MCBPE-ECL) system is employed, with simplified experimental operations and enhanced sensitivity. Together, the multichannel CRISPR/Cas13a reactions and MCBPE-ECL enable the sensor's multiplexed detection. Under optimized conditions, the sensor can complete RNA extraction and detection in 30 min, with a detection limit of 0.372 fM for Escherichia coli 16S rRNA. Furthermore, in human blood samples, the detection limit for E. coli is 63.8 cfu/mL. Notably, the sensor can simultaneously determine the growth curves of single colonies of E. coli and Staphylococcus aureus strains in the same culture medium, demonstrating its multiplexed detection capability.},
}

@article {pmid40017424,
year = {2025},
author = {Song, Y and Park, H and Thirumalaraju, P and Kovilakath, N and Hardie, JM and Bigdeli, A and Bai, Y and Chang, S and Yoo, J and Kanakasabapathy, MK and Kim, S and Chun, J and Chen, H and Li, JZ and Tsibris, AM and Kuritzkes, DR and Shafiee, H},
title = {Deactivated Cas9-Engineered Magnetic Micromotors toward a Point-of-Care Digital Viral RNA Assay.},
journal = {ACS nano},
volume = {},
number = {},
pages = {},
doi = {10.1021/acsnano.4c14913},
pmid = {40017424},
issn = {1936-086X},
abstract = {Digital nucleic acid assays, known for their high sensitivity and specificity, typically rely on fluorescent readouts and expensive and complex nanowell manufacturing, which constrain their broader use in point-of-care (POC) application. Here, we introduce an alternative digital molecular diagnostics, termed dCRISTOR, by seamlessly integrating deactivated Cas9 (dCas9)-engineered micromotors, extraction-free loop-mediated isothermal amplification (LAMP), low-cost bright field microscopy, and deep learning-enabled image processing. The micromotor, composed of a polystyrene sphere attached to a magnetic bead, incorporates a dCas9 ribonucleoprotein complex. The presence of human immunodeficiency virus-1 (HIV-1) RNA in a sample results in the formation of large-sized amplicons that can be specifically captured by the micromotors, reducing their velocity induced by an external magnetic field. The micromotor is propelled by an external magnetic field, which eliminates the need for chemical fuels, reducing system complexity, and allowing for precise control over micromotor movement, enhancing accuracy and reliability. A convolutional neural network classification-based multiobject tracking algorithm, CNN-MOT, accurately measures the change in micromotor motion, facilitating the binary digital assay format ("1" or "0") for simplified result interpretation without user bias. Incorporating an extraction-free LAMP assay streamlines the dCRISTOR workflow, enabling qualitative HIV-1 detection in spiked plasma (n = 21) that demonstrates 100% sensitivity and specificity and achieves a limit of detection (LOD) of 0.96 copies/μL. The assay also achieved 100% correlation with reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in clinical patient samples (n = 9). The dCRISTOR assay, a label-free digital nucleic acid testing system that eliminates the need for fluorescence readouts, absorbance measurements, or expensive manufacturing processes, represents a substantial advancement in digital viral RNA diagnostics.},
}

@article {pmid40017406,
year = {2025},
author = {Liu, G},
title = {Advancing CRISPR/Cas Biosensing with Integrated Devices.},
journal = {ACS sensors},
volume = {10},
number = {2},
pages = {575-576},
doi = {10.1021/acssensors.5c00330},
pmid = {40017406},
issn = {2379-3694},
}

@article {pmid39765088,
year = {2025},
author = {Yang, R and Zhao, L and Fang, M and Kong, W and Luan, Y},
title = {CRISPR-Cas12a-driven aptasensor for sensitive detection of alternariol by using a personal glucose meter.},
journal = {Talanta},
volume = {286},
number = {},
pages = {127496},
doi = {10.1016/j.talanta.2024.127496},
pmid = {39765088},
issn = {1873-3573},
mesh = {*Aptamers, Nucleotide/chemistry ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; *Lactones/chemistry ; Limit of Detection ; CRISPR-Associated Proteins/chemistry/genetics ; Humans ; Bacterial Proteins/genetics/chemistry ; Blood Glucose Self-Monitoring ; beta-Fructofuranosidase/chemistry ; Endodeoxyribonucleases ; },
abstract = {Alternariol (AOH) has attracted much attention as an emerging toxin in edible herbs that can pose potential carcinogenic risks to human. However, the rapid detection of AOH to ensure food safety remains a challenge. Here, a CRISPR-Cas12a-mediated aptamer-based sensor (aptasensor) was proposed for the sensitive quantification of AOH by using a personal glucose meter. First, fluorescent probes were used to validate crRNA sequences that could activate Cas12a trans-cleavage activity. Owing to the high affinity of aptamer, different concentrations of AOH were able to release different levels of Cas12a cleavage activity to cut magnetic bead-modified invertase probes. The free invertase was collected to catalyze hydrolysis of sucrose to glucose. After optimizing a series of key parameters, the constructed aptasensor with dual signal amplification of Cas12a and invertase allowed for highly-sensitive AOH detection in a linear range of 0.45-3000 ng/mL with a detection limit of 0.048 ng/mL. In addition, this aptasensor was successfully applied in edible herb samples with satisfactory recovery rates of 92.19-102.40 %. This CRISPR-Cas12a-mediated aptasensing platform provides a simple and powerful alternative for rapid quantitative detection of AOH, which is of great significance for food safety.},
}

*Aptamers, Nucleotide/chemistry
*CRISPR-Cas Systems/genetics
*Biosensing Techniques/methods
*Lactones/chemistry
Limit of Detection
CRISPR-Associated Proteins/chemistry/genetics
Humans
Bacterial Proteins/genetics/chemistry
Blood Glucose Self-Monitoring
beta-Fructofuranosidase/chemistry
Endodeoxyribonucleases

@article {pmid39724854,
year = {2025},
author = {Li, X and Dong, J and Deng, L and Huo, D and Yang, M and Hou, C},
title = {CRISPR/Cas12a regulated preassembled bulb-shaped G-quadruplex signal unit for FL/CM dual-mode ultrasensitive detection of miRNA-155.},
journal = {Talanta},
volume = {286},
number = {},
pages = {127413},
doi = {10.1016/j.talanta.2024.127413},
pmid = {39724854},
issn = {1873-3573},
mesh = {*G-Quadruplexes ; *MicroRNAs/analysis/blood ; Humans ; *CRISPR-Cas Systems/genetics ; Biosensing Techniques/methods ; Limit of Detection ; Colorimetry/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {High sensitivity and specificity in microRNA detection are of great significance for early cancer screening. This study employed a pre-assembled bulb-shaped G-quadruplex signal unit (G4MB) as a novel and efficient label-free probe. The products amplified by the miRNA-155-targeted exponential amplification reaction (EXPAR) activated the trans-cleavage activity of CRISPR/Cas12a, disrupting the G4MB structure to achieve dual-channel fluorescence/colorimetric (FL/CM) inverse signal output. Due to the strong signal amplification of EXPAR, the highly efficient cleavage by CRISPR/Cas12a, and the ultra-high response signal of the structurally stable G4MB probe, the FL mode achieved a high signal-to-noise ratio (S/N) of approximately 12.5. The CM mode, combined with smart devices for RGB curve adjustment, successfully corrected the background and provided precise and objective image data support while allowing results to be observed with the naked eye. Additionally, the sensor system exhibited high accuracy in complex human serum environments and RNA extracted from three different types of cells. Moreover, the G4MB probe required no complicated labeling, demonstrated structural stability, and had a rapid response. Most importantly, this study analyzed the advantages of the G4MB and applied it to miRNA detection for the first time, providing practical insights for biosensor construction, molecular diagnostics, and clinical applications.},
}

*G-Quadruplexes
*MicroRNAs/analysis/blood
Humans
*CRISPR-Cas Systems/genetics
Biosensing Techniques/methods
Limit of Detection
Colorimetry/methods
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid38145395,
year = {2025},
author = {Mutale-Joan, C and El Arroussi, H},
title = {Biotechnological strategies overcoming limitations to H. pluvialis-derived astaxanthin production and Morocco's potential.},
journal = {Critical reviews in food science and nutrition},
volume = {65},
number = {8},
pages = {1404-1419},
doi = {10.1080/10408398.2023.2294163},
pmid = {38145395},
issn = {1549-7852},
mesh = {*Xanthophylls/metabolism ; *Biotechnology/methods ; Morocco ; *Microalgae/metabolism ; *Biomass ; CRISPR-Cas Systems ; Chlorophyceae/metabolism ; },
abstract = {Haematococcus pluvialis is the richest source of natural astaxanthin, but the production of H. pluvialis-derived astaxanthin is usually limited by its slow cell proliferation and astaxanthin accumulation. Efforts to enhance biomass productivity, astaxanthin accumulation, and extraction are ongoing. This review highlights different approaches that have previously been studied in microalgal species for enhanced biomass productivity, as well as optimized methods for astaxanthin accumulation and extraction, and how these methods could be combined to bypass the challenges limiting natural astaxanthin production, particularly in H. pluvialis, at all stages (biomass production, and astaxanthin accumulation and extraction). Biotechnological approaches, such as overexpressing low CO2 inducible genes, utilizing complementary carbon sources, CRISPR-Cas9 bioengineering, and the use of active compounds, for biomass productivity are outlined. Direct astaxanthin extraction from H. pluvialis zoospores and Morocco's potential for microalgal-based astaxanthin production are equally discussed. This review emphasizes the need to engineer an optimized H. pluvialis-derived astaxanthin production system combining two or more of these strategies for increased growth, and astaxanthin productivity, to compete in the larger, lower-priced market in aquaculture and nutraceutical sectors.},
}

*Xanthophylls/metabolism
*Biotechnology/methods
Morocco
*Microalgae/metabolism
*Biomass
CRISPR-Cas Systems
Chlorophyceae/metabolism

@article {pmid40015840,
year = {2025},
author = {Chen, S and Ruan, Y and Li, Z and Zhou, C and Chen, Q and Zhou, X and Zhang, Y and Yang, C and Pan, H},
title = {CRISPR/Cas9-mediated editing of the melanization gene ebony in the 28-spotted ladybeetle, Henosepilachna vigintioctopunctata.},
journal = {Pesticide biochemistry and physiology},
volume = {208},
number = {},
pages = {106231},
doi = {10.1016/j.pestbp.2024.106231},
pmid = {40015840},
issn = {1095-9939},
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Melanins/metabolism ; Insect Proteins/genetics/metabolism ; Lepidoptera/genetics ; RNA Interference ; Pigmentation/genetics ; },
abstract = {The melanization process, which is essential for the proper functioning of the cuticle, has been extensively investigated for its enzymatic roles and physiological effects. Henosepilachna vigintioctopunctata, a significant pest species, presents considerable economic threats. However, due to the variable efficiency of RNA interference for genetic manipulation, establishing a CRISPR/Cas9 system is crucial for providing a more precise and reliable method for functional genomics in this non-model insect. In this study, we first utilized RNAi to investigate Hvebony, which encodes N-β-alanyldopamine, a critical compound in cuticle melanization. Subsequently, we introduced CRISPR/Cas9 for the first time in H. vigintioctopunctata. RNAi experiments revealed that knockdown of Hvebony resulted in abnormal melanin accumulation and low mortality rates, indicating its involvement in cuticle tanning. A novel CRISPR/Cas9 workflow was established, successfully resulting in the knocking out of Hvebony and the creation of a stable mutant strain characterized by dark pigmentation and low fitness costs. This study establishes Hvebony as a promising molecular marker for genetic studies in H. vigintioctopunctata. Moreover, it can be utilized in the development of genome editing control strategies and for analyses of gene function in H. vigintioctopunctata.},
}

Animals
*CRISPR-Cas Systems
*Gene Editing/methods
Melanins/metabolism
Insect Proteins/genetics/metabolism
Lepidoptera/genetics
RNA Interference
Pigmentation/genetics

@article {pmid40015249,
year = {2025},
author = {Dederer, HG},
title = {Human health and genetic technology.},
journal = {Trends in biotechnology},
volume = {43},
number = {3},
pages = {522-532},
doi = {10.1016/j.tibtech.2024.12.006},
pmid = {40015249},
issn = {1879-3096},
mesh = {Humans ; *Genetic Therapy/methods ; Organisms, Genetically Modified/genetics ; Animals ; DNA, Recombinant/genetics ; },
abstract = {The 1975 Asilomar conference contributed to the misperception that recombinant DNA (rDNA) technology is inherently risky to human health and the environment. It thus paved the way toward process-based regulation of genetically modified organisms (GMOs), such as in the EU. Initially, this regulatory approach obstructed technological uses of rDNA related to human health. However, regulators gradually softened the rules applicable to laboratories or industrial facilities. This encouraged R&D and production of pharmaceuticals derived from GMOs. Nevertheless, administering pharmaceuticals containing GMOs to patients may still be confronted with burdensome process-based GMO law on the deliberate release of GMOs into the environment. On the other hand, pharmaceutical law may need to be updated regarding, for example, novel gene therapies or xenotransplantation.},
}

Humans
*Genetic Therapy/methods
Organisms, Genetically Modified/genetics
Animals
DNA, Recombinant/genetics

@article {pmid40014250,
year = {2025},
author = {Bolideei, M and Barzigar, R and Gahrouei, RB and Mohebbi, E and Haider, KH and Paul, S and Paul, MK and Mehran, MJ},
title = {Applications of Gene Editing and Nanotechnology in Stem Cell-Based Therapies for Human Diseases.},
journal = {Stem cell reviews and reports},
volume = {},
number = {},
pages = {},
pmid = {40014250},
issn = {2629-3277},
abstract = {Stem cell research is a dynamic and fast-advancing discipline with great promise for the treatment of diverse human disorders. The incorporation of gene editing technologies, including ZFNs, TALENs, and the CRISPR/Cas system, in conjunction with progress in nanotechnology, is fundamentally transforming stem cell therapy and research. These innovations not only provide a glimmer of optimism for patients and healthcare practitioners but also possess the capacity to radically reshape medical treatment paradigms. Gene editing and nanotechnology synergistically enhance stem cell-based therapies' precision, efficiency, and applicability, offering transformative potential for treating complex diseases and advancing regenerative medicine. Nevertheless, it is important to acknowledge that these technologies also give rise to ethical considerations and possible hazards, such as inadvertent genetic modifications and the development of genetically modified organisms, therefore creating a new age of designer infants. This review emphasizes the crucial significance of gene editing technologies and nanotechnology in the progress of stem cell treatments, particularly for degenerative pathologies and injuries. It emphasizes their capacity to restructure and comprehensively revolutionize medical treatment paradigms, providing fresh hope and optimism for patients and healthcare practitioners.},
}

@article {pmid40014102,
year = {2025},
author = {Jiang, Y and Xue, R and Chang, Y and Cao, D and Liu, B and Li, Y},
title = {The knockout of Gγ subunit HvGS3 by CRISPR/Cas9 gene editing improves the lodging resistance of barley through dwarfing and stem strengthening.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {138},
number = {3},
pages = {61},
pmid = {40014102},
issn = {1432-2242},
support = {2023-NK-A3//Geomatics Technology and Application Key Laboratory of Qinghai Province/ ; 077GJHZ2023028GC//Global Common Challenges Project of International Partnership Program of Chinese Academy of Sciences/ ; 2023-1-1//Qinghai Science and Technology Department/ ; },
mesh = {*Hordeum/genetics/growth & development ; *CRISPR-Cas Systems ; *Gene Editing ; *Gibberellins/metabolism ; *Plant Proteins/genetics/metabolism ; *Plant Stems/genetics/growth & development ; *Lignin/metabolism ; Gene Expression Regulation, Plant ; Plants, Genetically Modified/genetics ; Phenotype ; Gene Knockout Techniques ; },
abstract = {Gγ subunits participate in multiple biological processes, but their biological function in barley is unknown. Here, CRISPR/Cas9 gene editing was used to knockout HvGS3 in barley. The height of hvgs3 plants were reduced by 37.8 ~ 43.1% compared to wild type, and the culm lodging resistance index (CLRI) of the second internode of stems was increased by 76.6%. The decrease in cell length of the second internode was similar to its node length. The shorter cells may be the main reason for the declines in the internode length and plant height. The number and area of vascular bundles, the epidermal thickness, and the mechanical tissue thickness were significantly higher in hvgs3 due to the higher lignin content. Transcriptome analysis showed higher expression of structural genes related to lignin biosynthesis. Gibberellin (GA) biosynthesis was suppressed through the down-regulation of the GA3ox gene, and the application of gibberellin restored the plant height of hvgs3, indicating that plant height was altered by hindering gibberellin biosynthesis. These results shed new light on the functions of the Gγ subunit GS3 and provide a resource for breeding new lodging-resistant barley cultivars.},
}

*Hordeum/genetics/growth & development
*CRISPR-Cas Systems
*Gene Editing
*Gibberellins/metabolism
*Plant Proteins/genetics/metabolism
*Plant Stems/genetics/growth & development
*Lignin/metabolism
Gene Expression Regulation, Plant
Plants, Genetically Modified/genetics
Phenotype
Gene Knockout Techniques

@article {pmid39924908,
year = {2025},
author = {Gao, Y and Ang, YS and Yung, LL},
title = {CRISPR-Cas12a-Assisted DNA Circuit for Nonmicroscopic Detection of Cell Surface Receptor Clustering.},
journal = {ACS sensors},
volume = {10},
number = {2},
pages = {977-985},
doi = {10.1021/acssensors.4c02770},
pmid = {39924908},
issn = {2379-3694},
mesh = {Humans ; *CRISPR-Cas Systems ; DNA/chemistry ; Cell Line, Tumor ; Receptor, ErbB-3/metabolism ; Receptor, ErbB-2/metabolism ; CRISPR-Associated Proteins/metabolism/chemistry ; Bacterial Proteins/chemistry/metabolism/genetics ; Endodeoxyribonucleases/chemistry/metabolism ; ErbB Receptors/metabolism/genetics ; },
abstract = {Protein-protein interactions (PPIs) on the cell surface have been of great interest due to their high clinical relevance and significance; however, the methods for detecting PPIs heavily rely on microscopic instruments. In this work, we designed a Cas12a-assisted DNA circuit for detecting cell surface receptor clustering events without a dependence on microscopy. This nonmicroscopic approach is based on the proximity principle, where localized protein-protein interactions such as receptor clustering are converted into DNA barcodes. These barcodes can then be identified by Cas12a for signal generation in the bulk. The compatibility of the circuit with Cas12a was first experimentally verified. Several leak reactions were identified and minimized. Lastly, we implemented this design in human breast cancer cell line models to distinguish the different levels of human epidermal growth factor receptor 2 (HER2) homodimers and heterodimers with HER1 and HER3 semiquantitatively without the use of a microscope. Overall, our proposed Cas12a-assisted DNA circuit for detecting cell surface receptor clustering shows the potential for fast screening in diagnostic applications and drug discovery, demonstrating the promising use of enzymatic DNA circuits in biological applications.},
}

Humans
*CRISPR-Cas Systems
DNA/chemistry
Cell Line, Tumor
Receptor, ErbB-3/metabolism
Receptor, ErbB-2/metabolism
CRISPR-Associated Proteins/metabolism/chemistry
Bacterial Proteins/chemistry/metabolism/genetics
Endodeoxyribonucleases/chemistry/metabolism
ErbB Receptors/metabolism/genetics

@article {pmid39910784,
year = {2025},
author = {Pascual-Garrigos, A and Lozano-Torres, B and Das, A and Molloy, JC},
title = {Colorimetric CRISPR Biosensor: A Case Study with Salmonella Typhi.},
journal = {ACS sensors},
volume = {10},
number = {2},
pages = {717-724},
doi = {10.1021/acssensors.4c02029},
pmid = {39910784},
issn = {2379-3694},
mesh = {*Colorimetry/methods ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; *Salmonella typhi/genetics/isolation & purification ; beta-Galactosidase/genetics/metabolism ; Humans ; },
abstract = {There is a critical need to implement a sensitive and specific point-of-care (POC) biosensor that addresses the instrument limitations and manufacturing challenges faced in resource-constrained contexts. In this paper we focus on enteric fever which is a highly contagious and prevalent infection in low- and middle-income countries. Although easily treatable, its ambiguous symptoms paired with a lack of fast, accurate and affordable diagnostics lead to incorrect treatments which exacerbate the disease burden, including increasing antibiotic resistance. In this study, we develop a readout module for CRISPR-Cas12a that produces a colorimetric output that is visible to the naked eye and can act as a cascade signal amplifier in any CRISPR assay based on trans-cleavage. We achieve this by immobilizing an oligo covalently linked to a β-galactosidase (LacZ) enzyme, which is cleaved in the presence of DNA target-activated CRISPR-Cas12a. Upon cleavage, the colorimetric enzyme is released, and the supernatant transferred to an environment containing X-Gal producing an intense blue color. This method is capable of detecting amplified bacterial genomic DNA and has a lower limit of detection (LoD) to standard fluorescent assays while removing the requirement for costly equipment. Furthermore, it remained active 4 weeks after lyophilization, allowing for the possibility of shipment without cold chain, significantly reducing deployment costs.},
}

*Colorimetry/methods
*Biosensing Techniques/methods
*CRISPR-Cas Systems/genetics
*Salmonella typhi/genetics/isolation & purification
beta-Galactosidase/genetics/metabolism
Humans

@article {pmid39886746,
year = {2025},
author = {Yuan, X and Geng, W and Ji, J and Yan, Z and Wei, J and Wu, Y and Yang, R},
title = {CRISPR/Cas13a-Programmed Cu NCs and Z-Scheme T-COF/Ag2S for Photoelectrochemical Biosensing of circRNA.},
journal = {ACS sensors},
volume = {10},
number = {2},
pages = {1270-1279},
doi = {10.1021/acssensors.4c03180},
pmid = {39886746},
issn = {2379-3694},
mesh = {*Biosensing Techniques/methods ; *Copper/chemistry ; *Electrochemical Techniques/methods ; Humans ; *RNA, Circular/blood/genetics ; *Silver Compounds/chemistry ; *CRISPR-Cas Systems ; Metal-Organic Frameworks/chemistry ; Lung Neoplasms/blood ; Metal Nanoparticles/chemistry ; Limit of Detection ; Sulfides/chemistry ; Photochemical Processes ; },
abstract = {Circular RNAs (circRNAs), as a class of noncoding RNA molecules with a circular structure exhibit high stability and spatiotemporal-specific expression, making them ideal cancer biomarkers for liquid biopsy. Herein, a new photoelectrochemical (PEC) biosensor for a highly sensitive circRNA assay in the whole blood of lung cancer patients was designed based on CRISPR/Cas13a-programmed Cu nanoclusters (Cu NCs) and a Z-scheme covalent organic framework/silver sulfide (T-COF/Ag2S) composite. This Z-scheme T-COF/Ag2S composite accelerates electron transfer and produces an excellent initial photocurrent. When CRISPR/Cas13a precisely targets circRNA, it nonspecifically cleaves the triple-helix molecular structure to release DNA fragments (C'/C"). After the C'/C" opens the DNA hairpin probe (HP) modified on the electrode, hybridization chain reactions are performed to produce abundant AT-rich double-stranded DNA with the addition of H1 and H2 probes. Upon the incubation of Cu[2+], Cu NCs are in situ formed via the A-Cu[2+]-T bonds and can effectively quench the photocurrent of the Z-scheme T-COF/Ag2S due to the energy transfer process. This developed PEC biosensor for the circRNA assay shows a low limit of detection of 0.5 fM, and the reusability of DNA-modified magnetic beads (MB-DNA) reduces the detection cost. Moreover, the PEC biosensor can accurately quantify the circRNA level and distinguish the circRNA expression in whole blood from healthy controls and lung cancer patients, offering strong potential in clinical diagnosis.},
}

*Biosensing Techniques/methods
*Copper/chemistry
*Electrochemical Techniques/methods
Humans
*RNA, Circular/blood/genetics
*Silver Compounds/chemistry
*CRISPR-Cas Systems
Metal-Organic Frameworks/chemistry
Lung Neoplasms/blood
Metal Nanoparticles/chemistry
Limit of Detection
Sulfides/chemistry
Photochemical Processes

@article {pmid39862216,
year = {2025},
author = {Fu, R and Wang, Y and Qiao, S and Xu, P and Xianyu, Y and Zhang, J},
title = {CRISPR-Cas12a-Mediated Growth of Gold Nanoparticles for DNA Detection in Agarose Gel.},
journal = {ACS sensors},
volume = {10},
number = {2},
pages = {1429-1439},
doi = {10.1021/acssensors.4c03461},
pmid = {39862216},
issn = {2379-3694},
mesh = {*Gold/chemistry ; *Metal Nanoparticles/chemistry ; *Sepharose/chemistry ; *CRISPR-Cas Systems/genetics ; Humans ; DNA/chemistry/genetics ; Biosensing Techniques/methods ; BRCA1 Protein/genetics ; Limit of Detection ; CRISPR-Associated Proteins ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {The rapid, simple, and sensitive detection of nucleic acid biomarkers plays a significant role in clinical diagnosis. Herein, we develop a label-free and point-of-care approach for isothermal DNA detection through the trans-cleavage activity of CRISPR-Cas12 and the growth of gold nanomaterials in agarose gel. The presence of the target can activate CRISPR-Cas12a to cleave single-stranded DNA, thus modulating the length and number of DNA sequences that mediate the growth of gold nanoparticles (AuNPs) or gold nanorods (AuNRs). Due to the extraordinary plasmonic property of gold nanomaterials, they present characteristic absorption/color after the growth with unique shapes. The sensing strategy is applied to detect BRCA-1, a biomarker related to breast cancer, with limits of detection of 1.72 pM (AuNP-based) and 2.07 pM (AuNR-based). AuNPs/AuNRs can be immobilized in agarose gels that display different colors in the presence of target DNA sequences. The agarose gel-based test allows for a readout by the naked eye or the RGB value with a smartphone. The approach is isothermal and label-free without any surface modification of nanomaterials, which holds great potential for the detection of nucleic acids in clinical applications.},
}

*Gold/chemistry
*Metal Nanoparticles/chemistry
*Sepharose/chemistry
*CRISPR-Cas Systems/genetics
Humans
DNA/chemistry/genetics
Biosensing Techniques/methods
BRCA1 Protein/genetics
Limit of Detection
CRISPR-Associated Proteins
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid39715471,
year = {2025},
author = {Nguyen, NHK and Rafiee, R and Parcha, PK and Tagmount, A and Rubnitz, J and Ribeiro, R and Cao, X and Pounds, SB and Vulpe, CD and Lamba, JK},
title = {Genome-wide CRISPR/Cas9 screen identifies AraC-daunorubicin-etoposide response modulators associated with outcomes in pediatric AML.},
journal = {Blood advances},
volume = {9},
number = {5},
pages = {1078-1091},
doi = {10.1182/bloodadvances.2024014157},
pmid = {39715471},
issn = {2473-9537},
support = {T32 HG008958/HG/NHGRI NIH HHS/United States ; },
mesh = {Humans ; *Leukemia, Myeloid, Acute/genetics/drug therapy/mortality ; *CRISPR-Cas Systems ; *Etoposide/pharmacology/therapeutic use ; Child ; *Daunorubicin/therapeutic use/pharmacology ; *Cytarabine/therapeutic use/pharmacology ; Drug Resistance, Neoplasm/genetics ; Antineoplastic Combined Chemotherapy Protocols/therapeutic use ; Prognosis ; Cell Line, Tumor ; Biomarkers, Tumor ; Treatment Outcome ; },
abstract = {Cytarabine, daunorubicin, and etoposide (ADE) have been the standard backbone of induction chemotherapy regimen for patients with pediatric acute myeloid leukemia (pAML) for >5 decades. However, chemoresistance is still a major concern, and a significant proportion of pAML becomes resistant to ADE treatment and relapse, leading to poor survival. Therefore, there is a considerable need to identify mechanisms mediating drug resistance for overcoming chemoresistance. Herein, we performed synthetic lethal CRISPR/Cas9 screens using the ADE components to identify response markers. We further integrated significant markers in 3 independent pAML clinical cohorts treated with only an ADE regimen to identify drug response biomarkers with prognostic significance. We were able to identify several mediators that represent clinically and biologically significant marker genes for ADE treatment, such as BCL2, CLIP2, and VAV3, which are resistant markers to ADE, with high expression associated with poor outcomes in pAML treated with ADE, and GRPEL1, HCFC1, and TAF10, which are sensitive markers to ADE, with high expression showing beneficial outcomes. Notably, BCL2, CLIP2, and VAV3 knockdowns in their expression in AML cell lines sensitized the cells more to the ADE components, suggesting that these modulators should be further studied as potential therapeutic targets to overcome chemoresistance.},
}

Humans
*Leukemia, Myeloid, Acute/genetics/drug therapy/mortality
*CRISPR-Cas Systems
*Etoposide/pharmacology/therapeutic use
Child
*Daunorubicin/therapeutic use/pharmacology
*Cytarabine/therapeutic use/pharmacology
Drug Resistance, Neoplasm/genetics
Antineoplastic Combined Chemotherapy Protocols/therapeutic use
Prognosis
Cell Line, Tumor
Biomarkers, Tumor
Treatment Outcome

@article {pmid39690508,
year = {2025},
author = {Wang, W and Yan, L and Li, J and Zhang, C and He, Y and Li, S and Xia, L},
title = {Engineering a robust Cas12i3 variant-mediated wheat genome editing system.},
journal = {Plant biotechnology journal},
volume = {23},
number = {3},
pages = {860-873},
doi = {10.1111/pbi.14544},
pmid = {39690508},
issn = {1467-7652},
support = {2021YFF1000204//National Key Research and Development Program of China/ ; 32188102//National Natural Science Foundation of China/ ; 2023ZD04074//STI 2030-Major Projects/ ; B23CJ0208//Hainan Seed Industry Lab/ ; //National Engineering Research Centre of Crop Molecular Breeding/ ; },
mesh = {*Triticum/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Genome, Plant/genetics ; CRISPR-Associated Proteins/genetics/metabolism ; Humans ; HEK293 Cells ; Plants, Genetically Modified/genetics ; },
abstract = {Wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD) is one of the most important food crops in the world. CRISPR/Cas12i3, which belongs to the type V-I Cas system, has attracted extensive attention recently due to its smaller protein size and its less-restricted canonical 'TTN' protospacer adjacent motif (PAM). However, due to its relatively lower editing efficacy in plants and the hexaploidy complex nature of wheat, Cas12i3/Cas12i3-5M-mediated genome editing in wheat has not been documented yet. Here, we report the engineering of a robust Cas12i3-5M-mediated genome editing system in wheat through the fusion of T5 exonuclease (T5E) in combination with an optimised crRNA expression strategy (Opt). We first showed that fusion of T5E, rather than ExoI, to Cas12i3-5M increased the gene editing efficiencies by up to 1.34-fold and 3.87-fold, compared to Cas12i3-5M and Cas12i3 in HEK293T cells, respectively. However, its editing efficiency remains low in wheat. We then optimised the crRNA expression strategy and demonstrated that Opt-T5E-Cas12i3-5M could enhance the editing efficiency by 1.20- to 1.33-fold and 4.05- to 7.95-fold in wheat stable lines compared to Opt-Cas12i3-5M and Opt-Cas12i3, respectively, due to progressive 5'-end resection of the DNA strand at the cleavage site with increased deletion size. The Opt-T5E-Cas12i3-5M enabled an editing efficiency ranging from 60.71% to 90.00% across four endogenous target genes in stable lines of three elite Chinese wheat varieties. Together, the developed robust Opt-T5E-Cas12i3-5M system enriches wheat genome editing toolkits for either biological research or genetic improvement and may be extended to other important polyploidy crop species.},
}

*Triticum/genetics
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Genome, Plant/genetics
CRISPR-Associated Proteins/genetics/metabolism
Humans
HEK293 Cells
Plants, Genetically Modified/genetics

@article {pmid39652457,
year = {2025},
author = {Zhao, A and Li, Q and Meng, P and Liu, P and Wu, S and Lang, Z and Song, Y and Macho, AP},
title = {Reduced content of gamma-aminobutyric acid enhances resistance to bacterial wilt disease in tomato.},
journal = {Plant biotechnology journal},
volume = {23},
number = {3},
pages = {792-806},
doi = {10.1111/pbi.14539},
pmid = {39652457},
issn = {1467-7652},
support = {XDB27040204//Chinese Academy of Sciences/ ; //Chinese 1000 talents program/ ; 2022YFD2100100//National Key Research and Development Program of China/ ; },
mesh = {*Solanum lycopersicum/microbiology/genetics/metabolism ; *gamma-Aminobutyric Acid/metabolism ; *Plant Diseases/microbiology/immunology ; *Disease Resistance/genetics ; *Ralstonia solanacearum/physiology/pathogenicity ; CRISPR-Cas Systems ; Gene Editing ; Plant Roots/microbiology/genetics/metabolism ; Glutamate Decarboxylase/metabolism/genetics ; Mutation ; },
abstract = {Bacteria within the Ralstonia solanacearum species complex cause devastating diseases in numerous crops, causing important losses in food production and industrial supply. Despite extensive efforts to enhance plant tolerance to disease caused by Ralstonia, efficient and sustainable approaches are still missing. Before, we found that Ralstonia promotes the production of gamma-aminobutyric acid (GABA) in plant cells; GABA can be used as a nutrient by Ralstonia to sustain the massive bacterial replication during plant colonization. In this work, we used CRISPR-Cas9-mediated genome editing to mutate SlGAD2, which encodes the major glutamate decarboxylase responsible for GABA production in tomato, a major crop affected by Ralstonia. The resulting Slgad2 mutant plants show reduced GABA content, and enhanced tolerance to bacterial wilt disease upon Ralstonia inoculation. Slgad2 mutant plants did not show altered susceptibility to other tested biotic and abiotic stresses, including drought and heat. Interestingly, Slgad2 mutant plants showed altered microbiome composition in roots and soil. We reveal a strategy to enhance plant resistance to Ralstonia by the manipulation of plant metabolism leading to an impairment of bacterial fitness. This approach could be particularly efficient in combination with other strategies based on the manipulation of the plant immune system, paving the way to a sustainable solution to Ralstonia in agricultural systems.},
}

*Solanum lycopersicum/microbiology/genetics/metabolism
*gamma-Aminobutyric Acid/metabolism
*Plant Diseases/microbiology/immunology
*Disease Resistance/genetics
*Ralstonia solanacearum/physiology/pathogenicity
CRISPR-Cas Systems
Gene Editing
Plant Roots/microbiology/genetics/metabolism
Glutamate Decarboxylase/metabolism/genetics
Mutation

@article {pmid39630060,
year = {2025},
author = {Zhou, Y and Shi, L and Li, X and Wei, S and Ye, X and Gao, Y and Zhou, Y and Cheng, L and Cheng, L and Duan, F and Li, M and Zhang, H and Qian, Q and Zhou, W},
title = {Genetic engineering of RuBisCO by multiplex CRISPR editing small subunits in rice.},
journal = {Plant biotechnology journal},
volume = {23},
number = {3},
pages = {731-749},
doi = {10.1111/pbi.14535},
pmid = {39630060},
issn = {1467-7652},
support = {2023ZD0407203//Biological Breeding-National Science and Technology Major Project/ ; 32330079//Program of National Natural Science Foundation of China/ ; 32472040//General Program of National Natural Science Foundation of China/ ; },
mesh = {*Oryza/genetics/enzymology ; *Ribulose-Bisphosphate Carboxylase/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Photosynthesis/genetics ; Plants, Genetically Modified/genetics ; Mutation/genetics ; Plant Proteins/genetics/metabolism ; Genetic Engineering/methods ; },
abstract = {Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is required for photosynthetic carbon assimilation, as it catalyses the conversion of inorganic carbon into organic carbon. Despite its importance, RuBisCO is inefficient; it has a low catalytic rate and poor substrate specificity. Improving the catalytic performance of RuBisCO is one of the key routes for enhancing plant photosynthesis. As the basic subunit of RuBisCO, RbcS affects the catalytic properties and plays a key role in stabilizing the structure of holoenzyme. Yet, the understanding of functions of RbcS in crops is still largely unknown. Toward this end, we employed CRISPR-Cas9 technology to randomly edit five rbcS genes in rice (OsrbcS1-5), generating a series of knockout mutants. The mutations of predominant rbcS genes in rice photosynthetic tissues, OsrbcS2-5, conferred inhibited growth, delayed heading and reduced yield in the field conditions, accompanying with lower RuBisCO contents and activities and significantly reduced photosynthetic efficiency. The retarded phenotypes were severer caused by multiple mutations. In addition, we revealed that these mutants had fewer chloroplasts and starch grains and a lower sugar content in the shoot base, resulting in fewer rice tillers. Further structural analysis of the mutated RuBisCO enzyme in one rbcs2,3,5 mutant line uncovered no significant differences from the wild-type protein, indicating that the mutations of rbcS did not compromise the protein assembly or the structure. Our findings generated a mutant pool with genetic diversities, which offers a valuable resource and novel insights into unravelling the mechanisms of RuBisCO in rice. The multiplex genetic engineering approach of this study provides an effective and feasible strategy for RuBisCO modification in crops, further facilitate the photosynthesis improvement and sustainable crop production.},
}

*Oryza/genetics/enzymology
*Ribulose-Bisphosphate Carboxylase/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Photosynthesis/genetics
Plants, Genetically Modified/genetics
Mutation/genetics
Plant Proteins/genetics/metabolism
Genetic Engineering/methods

@article {pmid39342963,
year = {2025},
author = {Jiang, F and Ding, X and Wang, X and Fu, K and Jia, Z and Liang, L and Guo, W},
title = {Rapid and Sensitive On-Site Nucleic Acid Detection of Three Main Fusarium Pathogens of Maize Stalk Rot Based on RPA-CRISPR/Cas12a.},
journal = {Plant disease},
volume = {109},
number = {2},
pages = {289-296},
doi = {10.1094/PDIS-08-24-1678-SR},
pmid = {39342963},
issn = {0191-2917},
mesh = {*Fusarium/genetics/isolation & purification ; *Zea mays/microbiology ; *Plant Diseases/microbiology ; *CRISPR-Cas Systems ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {Maize stalk rot is a soilborne disease that poses a serious threat to maize production worldwide, with the most significant cause being fungal stalk rot. The development of a visual and rapid detection method for the maize stalk rot pathogen is significant for its prompt and accurate identification, enhancing agricultural production efficiency, and implementing timely preventive measures. These measures will help safeguard the maize yield and quality, ultimately reducing agricultural losses. In this study, we aimed to develop an efficient method to detect maize stalk rot pathogens. We focused on three pathogenic fungi commonly found in maize-producing regions worldwide: Fusarium verticillioides, F. proliferatum, and F. graminearum. Based on translation elongation factor 1-α, we developed a rapid detection technique using recombinase polymerase amplification-CRISPR/Cas12a, combined with test strips to develop an on-site rapid visual detection test for these pathogens. The method showed detection sensitivity for F. verticillioides, F. proliferatum, and F. graminearum within 20 min at concentrations of 7.8 pg/μl, 0.11 ng/μl, and 0.13 ng/μl, respectively. The sensitivity increased with increasing reaction time. Testing of field disease samples indicated that the method is effective in detecting nucleic acids obtained through crude extraction methods. In conclusion, we developed a visually rapid detection technology that does not rely on complex instruments and equipment for the on-site early detection of F. verticillioides, F. proliferatum, and F. graminearum in the field to implement effective control measures, ensuring stable and high maize yields.},
}

*Fusarium/genetics/isolation & purification
*Zea mays/microbiology
*Plant Diseases/microbiology
*CRISPR-Cas Systems
Nucleic Acid Amplification Techniques/methods

@article {pmid40013512,
year = {2025},
author = {Gondalia, N and Quiroz, LF and Lai, L and Singh, AK and Khan, M and Brychkova, G and McKeown, PC and Chatterjee, M and Spillane, C},
title = {Harnessing promoter elements to enhance gene editing in plants: perspectives and advances.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/pbi.14533},
pmid = {40013512},
issn = {1467-7652},
abstract = {Genome-edited plants, endowed with climate-smart traits, have been promoted as tools for strengthening resilience against climate change. Successful plant gene editing (GE) requires precise regulation of the GE machinery, a process controlled by the promoters, which drives its transcription through interactions with transcription factors (TFs) and RNA polymerase. While constitutive promoters are extensively used in GE constructs, their limitations highlight the need for alternative approaches. This review emphasizes the promise of tissue/organ specific as well as inducible promoters, which enable targeted GE in a spatiotemporal manner with no effects on other tissues. Advances in synthetic biology have paved the way for the creation of synthetic promoters, offering refined control over gene expression and augmenting the potential of plant GE. The integration of these novel promoters with synthetic systems presents significant opportunities for precise and conditional genome editing. Moreover, the advent of bioinformatic tools and artificial intelligence is revolutionizing the characterization of regulatory elements, enhancing our understanding of their roles in plants. Thus, this review provides novel insights into the strategic use of promoters and promoter editing to enhance the precision, efficiency and specificity of plant GE, setting the stage for innovative crop improvement strategies.},
}

@article {pmid40011959,
year = {2025},
author = {Schmidt, H and Zhang, M and Chakarov, D and Bansal, V and Mourelatos, H and Sánchez-Rivera, FJ and Lowe, SW and Ventura, A and Leslie, CS and Pritykin, Y},
title = {Genome-wide CRISPR guide RNA design and specificity analysis with GuideScan2.},
journal = {Genome biology},
volume = {26},
number = {1},
pages = {41},
pmid = {40011959},
issn = {1474-760X},
support = {5T32CA160001//MSKCC TROT program/ ; P01 CA129243/NH/NIH HHS/United States ; U01 HG009395/NH/NIH HHS/United States ; U01 HG009395/NH/NIH HHS/United States ; DP2 AI171161/NH/NIH HHS/United States ; CAREER 2238831//National Science Foundation/ ; },
mesh = {Animals ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Mice ; *CRISPR-Cas Systems ; Genome ; Gene Editing/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {We present GuideScan2 for memory-efficient, parallelizable construction of high-specificity CRISPR guide RNA (gRNA) databases and user-friendly design and analysis of individual gRNAs and gRNA libraries for targeting coding and non-coding regions in custom genomes. GuideScan2 analysis identifies widespread confounding effects of low-specificity gRNAs in published CRISPR screens and enables construction of a gRNA library that reduces off-target effects in a gene essentiality screen. GuideScan2 also enables the design and experimental validation of allele-specific gRNAs in a hybrid mouse genome. GuideScan2 will facilitate CRISPR experiments across a wide range of applications.},
}

Animals
*RNA, Guide, CRISPR-Cas Systems/genetics
Mice
*CRISPR-Cas Systems
Genome
Gene Editing/methods
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40011853,
year = {2025},
author = {Sheng, T and Su, H and Yao, L and Qu, Z and Liu, H and Shao, W and Zhang, X},
title = {RhoB regulates prostate cancer cell proliferation and docetaxel sensitivity via the PI3K-AKT signaling pathway.},
journal = {BMC cancer},
volume = {25},
number = {1},
pages = {354},
pmid = {40011853},
issn = {1471-2407},
mesh = {Humans ; *rhoB GTP-Binding Protein/metabolism/genetics ; *Docetaxel/pharmacology ; Male ; *Proto-Oncogene Proteins c-akt/metabolism ; *Cell Proliferation/drug effects ; *Phosphatidylinositol 3-Kinases/metabolism ; *Signal Transduction/drug effects ; *Prostatic Neoplasms/pathology/drug therapy/genetics/metabolism ; *Epithelial-Mesenchymal Transition/drug effects ; *Cell Movement/drug effects ; Cell Line, Tumor ; Antineoplastic Agents/pharmacology/therapeutic use ; CRISPR-Cas Systems ; Drug Resistance, Neoplasm/genetics ; Gene Knockout Techniques ; Gene Expression Regulation, Neoplastic/drug effects ; },
abstract = {Docetaxel is a widely used first-line treatment for castration-resistant prostate cancer (CRPC). RhoB, a member of the Rho GTPase family, plays a major role in prostate cancer metastasis by modulating the PI3K-AKT signaling pathway. It is crucial in regulating cytoskeletal reassembly, cell migration, focal adhesion (FA) dynamics. To investigate RhoB's function in prostate cancer, CRISPR/Cas9 gene editing technique was utilized to knock out the RhoB gene in prostate cancer cells. Successful gene editing was confirmed by using T7 endonuclease I (T7EI) assays and Sanger sequencing. Knocking out RhoB enhanced epithelial-mesenchymal transition (EMT) and decreased the IC50 value of docetaxel in RhoB-knockout PC-3 cells. This suggests increased sensitivity to docetaxel. Furthermore, RhoB knockout prompted the migration and invasion of prostate cancer cells, effects that were reversed upon RhoB overexpression. Interestingly, RhoB status did not significantly influence the cell cycle of prostate cancer cells. RNA sequencing of PC-3 cells with either overexpressed or knock-out RhoB revealed that RhoB regulates pathways involved in FA, ECM receptor interaction, and PI3K-AKT signaling. These pathways directly influence the EMT process, cell migration, and invasion in prostate cancer cells. Notably, RhoB overexpression activated PI3K-AKT signaling when PC-3 cells were treated with low concentration of DTXL (50 nM, 72 h). This activation reduced DTXL's cytotoxicity, suggesting may confer chemoresistance via PI3K-AKT pathway activation.},
}

Humans
*rhoB GTP-Binding Protein/metabolism/genetics
*Docetaxel/pharmacology
Male
*Proto-Oncogene Proteins c-akt/metabolism
*Cell Proliferation/drug effects
*Phosphatidylinositol 3-Kinases/metabolism
*Signal Transduction/drug effects
*Prostatic Neoplasms/pathology/drug therapy/genetics/metabolism
*Epithelial-Mesenchymal Transition/drug effects
*Cell Movement/drug effects
Cell Line, Tumor
Antineoplastic Agents/pharmacology/therapeutic use
CRISPR-Cas Systems
Drug Resistance, Neoplasm/genetics
Gene Knockout Techniques
Gene Expression Regulation, Neoplastic/drug effects

@article {pmid40011813,
year = {2025},
author = {Lukasiak, S and Kalinka, A and Gupta, N and Papadopoulos, A and Saeed, K and McDermott, U and Hannon, GJ and Ross-Thriepland, D and Walter, D},
title = {A benchmark comparison of CRISPRn guide-RNA design algorithms and generation of small single and dual-targeting libraries to boost screening efficiency.},
journal = {BMC genomics},
volume = {26},
number = {1},
pages = {198},
pmid = {40011813},
issn = {1471-2164},
mesh = {Humans ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Algorithms ; *Benchmarking ; Gene Library ; Genome, Human ; },
abstract = {Genome-wide CRISPR sgRNA libraries have emerged as transformative tools to systematically probe gene function. While these libraries have been iterated over time to be more efficient, their large size limits their use in some applications. Here, we benchmarked publicly available genome-wide single-targeting sgRNA libraries and evaluated dual targeting as a strategy for pooled CRISPR loss-of-function screens. We leveraged this data to design two minimal genome-wide human CRISPR-Cas9 libraries that are 50% smaller than other libraries and that preserve specificity and sensitivity, thus enabling broader deployment at scale.},
}

Humans
*CRISPR-Cas Systems
*RNA, Guide, CRISPR-Cas Systems/genetics
*Algorithms
*Benchmarking
Gene Library
Genome, Human

@article {pmid40011676,
year = {2025},
author = {Cheng, ECK and Lam, JKC and Kwon, SC},
title = {Cytosolic CRISPR RNAs for efficient application of RNA-targeting CRISPR-Cas systems.},
journal = {EMBO reports},
volume = {},
number = {},
pages = {},
pmid = {40011676},
issn = {1469-3178},
support = {17110223//Research Grants Council, University Grants Committee ()/ ; Start-up//University of Hong Kong (HKU)/ ; },
abstract = {Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) technologies have evolved rapidly over the past decade with the continuous discovery of new Cas systems. In particular, RNA-targeting CRISPR-Cas13 proteins are promising single-effector systems to regulate target mRNAs without altering genomic DNA, yet the current Cas13 systems are restrained by suboptimal efficiencies. Here, we show that U1 promoter-driven CRISPR RNAs (crRNAs) increase the efficiency of various applications, including RNA knockdown and editing, without modifying the Cas13 protein effector. We confirm that U1-driven crRNAs are exported into the cytoplasm, while conventional U6 promoter-driven crRNAs are mostly confined to the nucleus. Furthermore, we reveal that the end positions of crRNAs expressed by the U1 promoter are consistent regardless of guide sequences and lengths. We also demonstrate that U1-driven crRNAs, but not U6-driven crRNAs, can efficiently repress the translation of target genes in combination with catalytically inactive Cas13 proteins. Finally, we show that U1-driven crRNAs can counteract the inhibitory effect of miRNAs. Our simple and effective engineering enables unprecedented cytosolic RNA-targeting applications.},
}

@article {pmid40010333,
year = {2025},
author = {Keith, NC and Snyder, RA and Euler, CW and Modell, JW},
title = {Bacteria exploit viral dormancy to establish CRISPR-Cas immunity.},
journal = {Cell host & microbe},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.chom.2025.02.001},
pmid = {40010333},
issn = {1934-6069},
abstract = {CRISPR-Cas systems provide prokaryotes with adaptive immunity against foreign genetic elements, including bacteriophages, by recording DNA-based immunological memories of infection called "spacers." How cells without preexisting immunity survive a rapid lytic infection long enough to acquire a new spacer and utilize it for defense remains a mystery. Here, we show that bacteria exploit the alternative dormant or "lysogenic" life cycle of temperate phages to establish CRISPR-Cas immunity. During a phage infection, immunization rates are significantly enhanced in cells entering lysogeny compared to those undergoing lysis. Furthermore, in the absence of a foreign threat, bacteria can acquire spacers targeting prophages residing within the chromosome. In this case, self-targeting by Cas9 promotes curing of the prophage, allowing immunized cells to avoid autoimmunity. The preferred acquisition of spacers during the establishment and maintenance of lysogeny may explain why most spacers in natural bacterial isolates target temperate phages.},
}

@article {pmid40008014,
year = {2025},
author = {Huang, Y and Chen, Z and Huang, H and Ding, S and Zhang, M},
title = {Important applications of DNA nanotechnology combined with CRISPR/Cas systems in biotechnology.},
journal = {RSC advances},
volume = {15},
number = {8},
pages = {6208-6230},
pmid = {40008014},
issn = {2046-2069},
abstract = {DNA nanotechnology leverages the specificity of Watson-Crick base pairing and the inherent attributes of DNA, enabling the exploitation of molecular characteristics, notably self-assembly, in nucleic acids to fabricate novel, controllable nanoscale structures and mechanisms. In the emerging field of DNA nanotechnology, DNA is not only a genetic material, but also a versatile multifunctional polymer, comprising deoxyribonucleotides, and facilitating the construction of precisely dimensioned and precise shaped two-dimensional (2D) and three-dimensional (3D) nanostructures. DNA molecules act as carriers of biological information, with notable advancements in bioimaging, biosensing, showing the profound impact. Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated systems (Cas) constitute self-defense mechanisms employed by bacteria and archaea to defend against viral invasion. With the discovery and modification of various functional Cas proteins, coupled with the identification of increasingly designable and programmable CRISPR RNAs (crRNAs), the potential of the CRISPR/Cas system in the field of molecular diagnostics is steadily being realized. Structural DNA nanotechnology provides a customizable and modular platform for accurate positioning of nanoscopic materials, for e.g., biomedical uses. This addressability has just recently been applied in conjunction with the newly developed gene engineering tools to enable impactful, programmable nanotechnological applications. As of yet, self-assembled DNA nanostructures have been mainly employed to enhance and direct the delivery of CRISPR/Cas, but lately the groundwork has also been laid out for other intriguing and complex functions. These recent advances will be described in this perspective. This review explores biosensing detection methods that combine DNA nanotechnology with CRISPR/Cas systems. These techniques are used in biosensors to detect small molecules such as DNA, RNA, and etc. The combination of 2D and 3D DNA nanostructures with the CRISPR/Cas system holds significant value and great development prospects in the detection of important biomarkers, gene editing, and other biological applications in fields like biosensing.},
}

@article {pmid40007105,
year = {2025},
author = {Ebrecht, AC and Luelf, UJ and Govender, K and Opperman, DJ and Urlacher, VB and Smit, MS},
title = {Use of Whole Cells and Cell-Free Extracts of Catalase-Deficient E. coli for Peroxygenase-Catalyzed Reactions.},
journal = {Biotechnology and bioengineering},
volume = {},
number = {},
pages = {},
doi = {10.1002/bit.28959},
pmid = {40007105},
issn = {1097-0290},
support = {//This work was supported by the South African Council for Scientific and Industrial Research-Industrial Biocatalysis Hub (CSIR-IBH) initiative funded by the South African Department of Science and Innovation (DSI) and the Technology Innovation Agency (TIA)./ ; },
abstract = {Unspecific peroxygenases (UPOs) and cytochrome P450 monooxygenases (CYPs) with peroxygenase activity are becoming the preferred biocatalysts for oxyfunctionalization reactions. While whole cells (WCs) or cell-free extracts (CFEs) of Escherichia coli are often preferred for cofactor-dependent monooxygenase reactions, hydrogen peroxide (H2O2) driven peroxygenase reactions are generally performed with purified enzymes, because the catalases produced by E. coli are expected to quickly degrade H2O2. We used the CRISPR/Cas system to delete the catalase encoding chromosomal genes, katG, and katE, from E. coli BL21-Gold(DE3) to obtain a catalase-deficient strain. A short UPO, DcaUPO, and two CYP peroxygenases, SscaCYP_E284A and CYP102A1_21B3, were used to compare the strains for peroxygenase expression and subsequent sulfoxidation, epoxidation, and benzylic hydroxylation activity. While 10 mM H2O2 was depleted within 10 min after addition to WCs and CFEs of the wild-type strain, at least 60% remained after 24 h in WCs and CFEs of the catalase-deficient strain. CYP peroxygenase reactions, with generally lower turnover frequencies, benefited the most from the use of the catalase-deficient strain. Comparison of purified peroxygenases in buffer versus CFEs of the catalase-deficient strain revealed that the peroxygenases in CFEs generally performed as well as the purified proteins. We also used WCs from catalase-deficient E. coli to screen three CYP peroxygenases, wild-type SscaCYP, SscaCYP_E284A, and SscaCYP_E284I for activity against 10 substrates comparing H2O2 consumption with substrate consumption and product formation. Finally, the enzyme-substrate pair with highest activity, SscaCYP_E284I, and trans-β-methylstyrene, were used in a preparative scale reaction with catalase-deficient WCs. Use of WCs or CFEs from catalase-deficient E. coli instead of purified enzymes can greatly benefit the high-throughput screening of enzyme or substrate libraries for peroxygenase activity, while they can also be used for preparative scale reactions.},
}

@article {pmid40005257,
year = {2025},
author = {Yuan, Z},
title = {From Origin to the Present: Establishment, Mechanism, Evolutions and Biomedical Applications of the CRISPR/Cas-Based Macromolecular System in Brief.},
journal = {Molecules (Basel, Switzerland)},
volume = {30},
number = {4},
pages = {},
pmid = {40005257},
issn = {1420-3049},
mesh = {*CRISPR-Cas Systems ; Humans ; *Gene Editing/methods ; *Genetic Therapy/methods ; Animals ; Neoplasms/genetics/therapy ; },
abstract = {Advancements in biological and medical science are intricately linked to the biological central dogma. In recent years, gene editing techniques, especially CRISPR/Cas systems, have emerged as powerful tools for modifying genetic information, supplementing the central dogma and holding significant promise for disease diagnosis and treatment. Extensive research has been conducted on the continuously evolving CRISPR/Cas systems, particularly in relation to challenging diseases, such as cancer and HIV infection. Consequently, the integration of CRISPR/Cas-based techniques with contemporary medical approaches and therapies is anticipated to greatly enhance healthcare outcomes for humans. This review begins with a brief overview of the discovery of the CRISPR/Cas system. Subsequently, using CRISPR/Cas9 as an example, a clear description of the classical molecular mechanism underlying the CRISPR/Cas system was given. Additionally, the development of the CRISPR/Cas system and its applications in gene therapy and high-sensitivity disease diagnosis were discussed. Furthermore, we address the prospects for clinical applications of CRISPR/Cas-based gene therapy, highlighting the ethical considerations associated with altering genetic information. This brief review aims to enhance understanding of the CRISPR/Cas macromolecular system and provide insight into the potential of genetic macromolecular drugs for therapeutic purposes.},
}

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

@article {pmid40004557,
year = {2025},
author = {Wang, J and Wang, H and Zhai, J and Zhu, F and Ren, Y and Zhou, J and Zhang, Z and Luo, L and Xu, W},
title = {Identification of Ziziphus jujuba cv. Dongzao DNA Demethylase ZjROS1 Gene Family and Construction of CRISPR/Cas9-Mediated Gene-Editing Vector.},
journal = {Genes},
volume = {16},
number = {2},
pages = {},
pmid = {40004557},
issn = {2073-4425},
support = {YCX24138//Graduate Innovation Program of North Minzu University/ ; 2022QCXTD04//Innovation Team for Genetic Improvement of Economic Forests/ ; 32060669//National Natural Science Foundation of China/ ; },
mesh = {*Ziziphus/genetics/growth & development ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Gene Expression Regulation, Plant ; Plant Proteins/genetics/metabolism ; DNA Methylation/genetics ; Fruit/genetics/growth & development ; Multigene Family ; },
abstract = {DNA methylation is one of the earliest and most extensively studied epigenetic regulatory mechanisms. The ROS1 (Repressor of Silencing 1) gene was first discovered in Arabidopsis thaliana, and it is a DNA demethylase that can remove 5-methylcytosine from DNA, thereby affecting DNA methylation levels and gene expression. Objectives: The objective of this study was to investigate the role of ROS1 in the development and maturation of Ziziphus jujuba cv. "Dongzao" fruit. Methods: We cloned the ROS1 gene and conducted bioinformatics and expression characteristics analyses on it. Results: Three ROS1 genes, named ZjROS1-1~3, was identified, and each member protein was localized in the nucleus, cytoskeleton, chloroplast, and vacuole. The promoter contained cis-elements such as light response, plant hormone signal transduction, and stress response cis-elements, and it interacted with many proteins such as CMT, MET, and ZDP. The results of the real-time fluorescence quantitative PCR show that ZjROS1 has specific expression patterns in different tissues of Z. jujuba cv. Dongzao, and the expression of ZjROS1-2 in flowers and fruits is high. At the same time, CRISPR/Cas9 technology was used to construct a gene-editing vector for ZjROS1, which provided a basis for the subsequent genetic transformation. Conclusions: In this study, the biological function of ZjROS1 was clarified and a gene-editing vector was constructed, which provided a theoretical basis for the regulation mechanism of demethylase ZjROS1 in the fruit ripening and development of Z. jujuba cv. Dongzao.},
}

*Ziziphus/genetics/growth & development
*Gene Editing/methods
*CRISPR-Cas Systems
*Gene Expression Regulation, Plant
Plant Proteins/genetics/metabolism
DNA Methylation/genetics
Fruit/genetics/growth & development
Multigene Family

@article {pmid39954255,
year = {2025},
author = {Xiang, RR and Lee, SA and Tyndall, CF and Bhatia, AR and Yin, J and Singler, C and Hauk, BJ and Kipp, MP and Takeda, DY},
title = {CRISPR screening identifies regulators of enhancer-mediated androgen receptor transcription in advanced prostate cancer.},
journal = {Cell reports},
volume = {44},
number = {2},
pages = {115312},
doi = {10.1016/j.celrep.2025.115312},
pmid = {39954255},
issn = {2211-1247},
mesh = {Male ; *Receptors, Androgen/metabolism/genetics ; Humans ; *Homeodomain Proteins/metabolism/genetics ; *Enhancer Elements, Genetic/genetics ; *Hepatocyte Nuclear Factor 3-alpha/metabolism/genetics ; *GATA2 Transcription Factor/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; Animals ; Cell Line, Tumor ; Prostatic Neoplasms, Castration-Resistant/genetics/pathology/metabolism ; Gene Expression Regulation, Neoplastic ; Transcription Factor AP-2/metabolism/genetics ; Prostatic Neoplasms/genetics/pathology/metabolism ; Transcription, Genetic ; Mice ; Protein Binding ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Amplification of the androgen receptor (AR) locus is the most frequent alteration in metastatic castration-resistant prostate cancer (CRPC). Recently, it was discovered that an enhancer of the AR is co-amplified with the AR gene body and contributes to increased AR transcription and resistance to androgen deprivation therapy. However, the mechanism of enhancer activation in advanced disease is unknown. Here, we used CRISPR-Cas9 screening to identify transcription factors that bind to the AR enhancer and modulate enhancer-mediated AR transcription. We demonstrate that HOXB13, GATA2, and TFAP2C bind the AR enhancer in patient-derived xenografts and directly impact features associated with an active chromatin state. Interestingly, the AR enhancer belongs to a set of regulatory elements that require HOXB13 to maintain FOXA1 binding, further delineating the role of HOXB13 in CRPC. This work provides a framework to functionally identify trans-acting factors required for the activation of disease-related noncoding regulatory elements.},
}

Male
*Receptors, Androgen/metabolism/genetics
Humans
*Homeodomain Proteins/metabolism/genetics
*Enhancer Elements, Genetic/genetics
*Hepatocyte Nuclear Factor 3-alpha/metabolism/genetics
*GATA2 Transcription Factor/metabolism/genetics
*CRISPR-Cas Systems/genetics
Animals
Cell Line, Tumor
Prostatic Neoplasms, Castration-Resistant/genetics/pathology/metabolism
Gene Expression Regulation, Neoplastic
Transcription Factor AP-2/metabolism/genetics
Prostatic Neoplasms/genetics/pathology/metabolism
Transcription, Genetic
Mice
Protein Binding
Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid39936934,
year = {2025},
author = {Lu, C and Chen, J and Zhang, S and Tian, Y and Ying, X and Zhang, D and Luo, Z and Si, X and Li, M},
title = {Development of a vitrified CRISPR/Cas12b-based assay for rapid genotyping of SLCO1B1 SNPs without DNA amplification.},
journal = {Analytical methods : advancing methods and applications},
volume = {17},
number = {9},
pages = {2083-2093},
doi = {10.1039/d4ay02015d},
pmid = {39936934},
issn = {1759-9679},
mesh = {*Polymorphism, Single Nucleotide/genetics ; Humans ; *Liver-Specific Organic Anion Transporter 1/genetics ; *CRISPR-Cas Systems/genetics ; *Genotyping Techniques/methods ; Genotype ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {Two single nucleotide polymorphisms (SNPs) in the human SLCO1B1 gene, c.388A>G (rs2306283) and c.521T>C (rs4149056), are independent determinants of the efficacy and side effects of statin drugs. Multinational clinical guidelines recommend testing for SLCO1B1 genotypes before the initial use of statins. Current SLCO1B1 SNP identification methods, primarily based on quantitative fluorescence PCR, rely on expensive equipment, are time-consuming, and require cold-chain storage for reagents, making them unsuitable for use in resource-limited healthcare settings. In this study, we developed a CRISPR/Cas12b-based amplification-free genotyping technique for SLCO1B1 SNPs. Within 30 minutes of the isothermal reaction, genotyping of the c.388A>G and c.521T>C SNPs in the SLCO1B1 gene can be observed by the naked eyes under blue light. Additionally, maltodextrin was identified as an effective vitrification stabilizer for the CRISPR/Cas12b premix. A low-cost vitrification process was optimized to prepare a glass like solid reagent via room-temperature vacuum drying. The vitrified CRISPR/Cas12b reagent retained approximately 88% of its activity after 30 days of storage at 37 °C, eliminating the need for cold-chain storage and allowing for long-term preservation at room temperature. This vitrified CRISPR/Cas12b based rapid SNP detection technique is especially suitable for genotyping drug metabolism genes in primary healthcare settings, providing effective guidance for precision medicine in clinical practice.},
}

*Polymorphism, Single Nucleotide/genetics
Humans
*Liver-Specific Organic Anion Transporter 1/genetics
*CRISPR-Cas Systems/genetics
*Genotyping Techniques/methods
Genotype
Nucleic Acid Amplification Techniques/methods

@article {pmid39930995,
year = {2025},
author = {Nafian, F and Esfahani, KS and Hobabi Aghmiuni, M and Khoushab, S and Illeslamllo, T and Nafian, S and Mohamadiyan, N and Aleyasin, NS and Kamali Doust Azad, B},
title = {Emerging microfluidic technologies for CRISPR-based diagnostics: an overview.},
journal = {Analytical methods : advancing methods and applications},
volume = {17},
number = {9},
pages = {1962-1976},
doi = {10.1039/d5ay00063g},
pmid = {39930995},
issn = {1759-9679},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Microfluidic Analytical Techniques/methods/instrumentation ; Point-of-Care Testing ; Lab-On-A-Chip Devices ; },
abstract = {In recent years, CRISPR (clustered regularly interspaced short palindromic repeats) has emerged as a detection technique with high specificity and sensitivity. However, it still needs improvements in terms of reducing cost, complexity, cross-contamination, technical requirements, and lack of quantification platforms. Microfluidic strategies can advance CRISPR-based technology and be modified to a higher level in the future. This review provides an overview of CRISPR-based detection systems (CRISPR-Dx) and their mechanism. Then, it explains how they have been optimized for fast and accurate point-of-care testing (POCT) using microfluidic devices such as SHINE, CARMEN, DNAiTECH, Dμchip, MAPnavi, FAST, and ITP. We discuss their innovations, primarily focusing on how they develop CRISPR-Dx in detection throughput, quantification, simple operation, visualization, sensitivity, specificity, and anti-contamination.},
}

Humans
*CRISPR-Cas Systems/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Microfluidic Analytical Techniques/methods/instrumentation
Point-of-Care Testing
Lab-On-A-Chip Devices

@article {pmid39838098,
year = {2025},
author = {Whalen, JM and Earley, J and Wisniewski, C and Mercurio, AM and Cantor, SB},
title = {Targeting BRCA1-deficient PARP inhibitor-resistant cells with nickases reveals nick resection as a cancer vulnerability.},
journal = {Nature cancer},
volume = {6},
number = {2},
pages = {278-291},
pmid = {39838098},
issn = {2662-1347},
support = {R01 CA254037/CA/NCI NIH HHS/United States ; 1F32CA268524-01A1//U.S. Department of Health & Human Services | NIH | NCI | Division of Cancer Epidemiology and Genetics, National Cancer Institute (National Cancer Institute Division of Cancer Epidemiology and Genetics)/ ; R01CA285607//U.S. Department of Health & Human Services | NIH | NCI | Division of Cancer Epidemiology and Genetics, National Cancer Institute (National Cancer Institute Division of Cancer Epidemiology and Genetics)/ ; R01 CA254037/CA/NCI NIH HHS/United States ; R01 CA254037/CA/NCI NIH HHS/United States ; },
mesh = {*Poly(ADP-ribose) Polymerase Inhibitors/pharmacology/therapeutic use ; *BRCA1 Protein/genetics/deficiency ; Animals ; *Tumor Suppressor p53-Binding Protein 1/genetics/metabolism ; Humans ; Mice ; *Drug Resistance, Neoplasm/genetics ; Female ; Cell Line, Tumor ; Poly (ADP-Ribose) Polymerase-1/antagonists & inhibitors/genetics/metabolism ; Homologous Recombination ; CRISPR-Cas Systems ; DNA-Binding Proteins/genetics ; Breast Neoplasms/genetics/drug therapy ; },
abstract = {Tumors lacking the BRCA1 and BRCA2 (BRCA) hereditary breast cancer genes display heightened sensitivity to anti-cancer treatments, such as inhibitors of poly (ADP-ribose) polymerase 1 (PARP1). However, when resistance develops, treatments are lacking. Using CRISPR technology, we discovered that enhancing homologous recombination through increased DNA end resection in BRCA1-deficient cells by loss of the 53BP1-Shieldin complex-which is associated with resistance to PARP inhibitors-also heightens sensitivity to DNA nicks. The sensitivity is caused by hyper-resection of nicks into extensive single-stranded regions that trigger cell death. Based on these findings and that nicks limit tumor formation in mice, we propose nickases as a tool for personalized medicine. Moreover, our findings indicate that restricting nick expansion is a critical function of the 53BP1-Shieldin complex.},
}

*Poly(ADP-ribose) Polymerase Inhibitors/pharmacology/therapeutic use
*BRCA1 Protein/genetics/deficiency
Animals
*Tumor Suppressor p53-Binding Protein 1/genetics/metabolism
Humans
Mice
*Drug Resistance, Neoplasm/genetics
Female
Cell Line, Tumor
Poly (ADP-Ribose) Polymerase-1/antagonists & inhibitors/genetics/metabolism
Homologous Recombination
CRISPR-Cas Systems
DNA-Binding Proteins/genetics
Breast Neoplasms/genetics/drug therapy

@article {pmid39716488,
year = {2025},
author = {He, Y and Zalenski, N and Stephenson, AA and Raper, AT and Ghimire, C and Suo, Z},
title = {Conformational transitions of Streptococcus pyogenes Cas9 induced by salt and single-guide RNA binding.},
journal = {The Journal of biological chemistry},
volume = {301},
number = {2},
pages = {108120},
pmid = {39716488},
issn = {1083-351X},
support = {R01 GM122093/GM/NIGMS NIH HHS/United States ; },
mesh = {*Streptococcus pyogenes/enzymology/metabolism ; *RNA, Guide, CRISPR-Cas Systems/metabolism ; *CRISPR-Associated Protein 9/metabolism/chemistry ; *Potassium Chloride/pharmacology/metabolism/chemistry ; Protein Conformation ; Bacterial Proteins/metabolism/chemistry/genetics ; Fluorescence Resonance Energy Transfer ; },
abstract = {Streptococcus pyogenes (Sp) Cas9 has been widely utilized to edit genomes across diverse species. To achieve high efficiency and specificity as a gene-editing enzyme, Sp Cas9 undergoes a series of sequential conformational changes during substrate binding and catalysis. Here, we employed single-molecule FRET techniques to investigate the effect of different KCl concentrations on conformational dynamics of Sp Cas9 in the presence or the absence of a single-guide RNA (sgRNA). In the absence of sgRNA and at low KCl concentrations (75 mM), apo Cas9 surprisingly exhibited two distinct conformations: a primary autoinhibited open conformation (apo Cas9 conformation [Cas9[apo]]) and a secondary sgRNA-bound-like conformation (Cas9[X]). Interestingly, increase in buffer KCl concentration led to a linear increase in the Cas9[X] population and a corresponding decrease in the Cas9[apo] population. In contrast, changes in KCl concentration exerted the opposite effects on the Cas9[X] and Cas9[apo] populations in the presence of sgRNA. Collectively, our findings by using KCl concentration as the probe suggest that Cas9 might employ a conformational sampling mechanism, in addition to the more common induced-fit mechanism established by us previously, for sgRNA binding.},
}

*Streptococcus pyogenes/enzymology/metabolism
*RNA, Guide, CRISPR-Cas Systems/metabolism
*CRISPR-Associated Protein 9/metabolism/chemistry
*Potassium Chloride/pharmacology/metabolism/chemistry
Protein Conformation
Bacterial Proteins/metabolism/chemistry/genetics
Fluorescence Resonance Energy Transfer

@article {pmid39266721,
year = {2025},
author = {Caliendo, F and Vitu, E and Wang, J and Kuo, SH and Sandt, H and Enghuus, CN and Tordoff, J and Estrada, N and Collins, JJ and Weiss, R},
title = {Customizable gene sensing and response without altering endogenous coding sequences.},
journal = {Nature chemical biology},
volume = {21},
number = {3},
pages = {348-359},
pmid = {39266721},
issn = {1552-4469},
support = {W911NF19C0008//United States Department of Defense | Defense Advanced Research Projects Agency (DARPA)/ ; HR0011-20-2-0005//United States Department of Defense | Defense Advanced Research Projects Agency (DARPA)/ ; 5-R01-EB030946-04//U.S. Department of Health & Human Services | NIH | National Institute of Biomedical Imaging and Bioengineering (NIBIB)/ ; 5-R01-EB025256-04//U.S. Department of Health & Human Services | NIH | National Institute of Biomedical Imaging and Bioengineering (NIBIB)/ ; 5R01HD105947-03//U.S. Department of Health & Human Services | NIH | Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)/ ; CNS-1446474//National Science Foundation (NSF)/ ; W911NF-17-3-003//United States Department of Defense | United States Army | U.S. Army Research, Development and Engineering Command | Army Research Office (ARO)/ ; 5RC2DK120535//U.S. Department of Health & Human Services | NIH | National Institute of Diabetes and Digestive and Kidney Diseases (National Institute of Diabetes & Digestive & Kidney Diseases)/ ; },
mesh = {Animals ; CHO Cells ; *Cricetulus ; *Biosensing Techniques/methods ; CRISPR-Cas Systems ; Proto-Oncogene Proteins c-bcl-2/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Expression Regulation ; Synthetic Biology/methods ; Cell Survival ; },
abstract = {Synthetic biology aims to modify cellular behaviors by implementing genetic circuits that respond to changes in cell state. Integrating genetic biosensors into endogenous gene coding sequences using clustered regularly interspaced short palindromic repeats and Cas9 enables interrogation of gene expression dynamics in the appropriate chromosomal context. However, embedding a biosensor into a gene coding sequence may unpredictably alter endogenous gene regulation. To address this challenge, we developed an approach to integrate genetic biosensors into endogenous genes without modifying their coding sequence by inserting into their terminator region single-guide RNAs that activate downstream circuits. Sensor dosage responses can be fine-tuned and predicted through a mathematical model. We engineered a cell stress sensor and actuator in CHO-K1 cells that conditionally activates antiapoptotic protein BCL-2 through a downstream circuit, thereby increasing cell survival under stress conditions. Our gene sensor and actuator platform has potential use for a wide range of applications that include biomanufacturing, cell fate control and cell-based therapeutics.},
}

Animals
CHO Cells
*Cricetulus
*Biosensing Techniques/methods
CRISPR-Cas Systems
Proto-Oncogene Proteins c-bcl-2/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics
Gene Expression Regulation
Synthetic Biology/methods
Cell Survival