CRISPR Journal最新文献

{"title":"Under Pressure: Can CRISPR Deliver Despite Contextual Headwinds?","authors":"Rodolphe Barrangou","doi":"10.1177/25731599261442503","DOIUrl":"https://doi.org/10.1177/25731599261442503","url":null,"abstract":"","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":"9 2","pages":"55"},"PeriodicalIF":4.0,"publicationDate":"2026-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147730725","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Subtypes of Type I-E CRISPR-Cas Systems Distribution in Human <i>Escherichia coli</i> Isolates from China.","authors":"Yingyi Guo, Jiahui Li, Anwarul Haque, Jiakang Chen, Xu Yang, Yijing Wang, Likang Yao, Chuyue Zhuo, Jiong Wang, Nanhao He, Yexin Lin, Shunian Xiao, Baomo Liu, Chao Zhuo","doi":"10.1177/25731599261430828","DOIUrl":"10.1177/25731599261430828","url":null,"abstract":"<p><p>The correlation between CRISPR-Cas systems and plasmid-mediated bacterial antibiotic resistance is increasingly growing attention. However, currently no reports exist on the relationship between the CRISPR-Cas systems and the carriage of <i>bla</i>_NDM or plasmids in <i>E. coli</i>. Here, molecular characterization and phylogenetic analysis of 639 <i>E. coli</i> isolated from humans in China were carried out. Depending on similarity in sequence, the type I-E CRISPR-Cas systems in <i>E. coli</i> can be grouped into two distinct clades, which we refer to for descriptive purposes within this study as the type I-E-S1 and I-E-S2, whereas the type I-E-S2 CRISPR-Cas system is further divided into I-E-S2a and I-E-S2b systems based on the presence of <i>cas8e</i> and <i>cas11</i>. ST167 (phylogroup A) and ST410 (phylogroup C) <i>E. coli</i> were observed bearing the type I-E-S1 and I-E-S2b systems, respectively. Compared with strains carrying the I-E-S1 type CRISPR-Cas system, the <i>bla</i>_NDM carrying rate, the positive rate of IncX3 plasmid, and the positive rate of IncF plasmid of strains with the I-E-S2a type CRISPR-Cas system were evidently lower (<i>p</i> < 0.05); the <i>bla</i>_NDM carrying rate and the positive rate of IncF plasmid of strains with the I-E-S2b type CRISPR-Cas system were evidently higher (<i>p</i> < 0.05). The <i>bla</i>_NDM positive rate and IncF plasmid positive rate of strains carrying the I-E-S2a type CRISPR-Cas system were significantly lower than those of strains carrying the I-E-S2b type CRISPR-Cas system (<i>p</i> < 0.001). It proves that the I-E-S1, I-E-S2a, and I-E-S2b type CRISPR-Cas systems are beneficial for spreading <i>bla</i>_NDM and IncX3 plasmids. We found significant differences in the <i>cas</i> gene sequences of the I-E-S1 and I-E-S2 type CRISPR loci. The type I-E CRISPR-Cas systems in <i>E. coli</i> isolated from Chinese sources are classified further for the first time, revealing their high correlation with <i>bla</i>_NDM, phylogenetic groups, and multilocus sequence typing. This work paves the way for a deeper understanding of the role that CRISPR-Cas systems play in the rise of resistant <i>E. coli</i> ST167 and ST410.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":"89-102"},"PeriodicalIF":4.0,"publicationDate":"2026-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147482564","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Toward Next-Gen Cell Therapy for Pediatric Patients: Neonatal Hepatocytes Tolerate Electroporation-Mediated Gene Editing and Engraft in the Liver.","authors":"Justin R Gibson, Abishek Dhungana, Menam Pokhrel, Benjamin Bessah Arthur, Olumide Adebayo, Daniel Hossack, Renee N Cottle","doi":"10.1177/25731599261430830","DOIUrl":"10.1177/25731599261430830","url":null,"abstract":"<p><p>Hepatocyte transplantation (HTx) offers a safer, less invasive alternative to orthotopic liver transplantation for inherited metabolic liver diseases, especially in high-risk pediatric patients. Combining HTx with <i>ex vivo</i> gene editing is a promising autologous therapeutic strategy using the patient's cells. We investigated the feasibility of this approach by applying CRISPR-Cas9 gene knock-out to neonatal mouse hepatocytes and comparing their engraftment potential with that of mature adult cells in the <i>Fah^-/-</i> mouse model of hereditary tyrosinemia type I (HT1). Electroporation-mediated gene editing did not significantly impair the ability of neonatal hepatocytes to engraft <i>in vivo</i>. Quantitative histological analysis revealed comparable liver repopulation levels between recipients of gene-edited neonatal cells and adult cells after hepatoxicity-mediated selection, providing a benchmark for electroporation-mediated gene editing in neonatal hepatocytes, and supporting the development of genetically corrected neonatal hepatocyte products as a crucial long-term or bridge-to-transplant therapeutic strategy for pediatric liver disease.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":"103-114"},"PeriodicalIF":4.0,"publicationDate":"2026-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147610705","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Viability-Based Assessment Reveals True Efficiency of CRISPR-Cas9 Transfection Methods in Rabbit Spermatozoa.","authors":"Sarah K Topfer, Madi Rutherford, Frances Zewe, Tanja Strive, Kevin P Oh","doi":"10.1177/25731599261424870","DOIUrl":"10.1177/25731599261424870","url":null,"abstract":"<p><p>Modern genome editing tools such as CRISPR-Cas9 have revolutionized mammalian genome engineering, yet translation to <i>in vivo</i> applications remains limited by low efficiency and frequent occurrence of mosaicism. Sperm-mediated delivery of editing reagents is one proposed alternative that may mitigate these issues. This method depends on efficient transfection of genome editing materials into viable spermatozoa, a critical yet frequently overlooked parameter. Using FACS, we compared electroporation (Neon NxT) and lipofection (CRISPRMAX) for introducing CRISPR-Cas9 ribonucleoproteins into viable rabbit spermatozoa. Electroporation, shown to enable Cas9 and plasmid transfection in spermatozoa from other species, performed poorly once dead spermatozoa were excluded. In contrast, Lipofectamine CRISPRMAX improved transfection efficiency with minimal effects on spermatozoa viability and motility. These findings emphasize the importance of distinguishing true transfection (transfection of viable spermatozoa) from total transfection and highlight lipofection as a promising alternative to electroporation for sperm-based genome editing, with potential applications in rabbit genome engineering.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":"59-70"},"PeriodicalIF":4.0,"publicationDate":"2026-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147610618","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Conditional Expression of Cas9 and dCas9 in <i>Lucilia cuprina</i> Reveals dCas9-Associated Lethality.","authors":"Alexis Kriete, Tatiana Basika, Rossina Novas, Olivia L Mathieson, Esther J Belikoff, Manuel Kleiner, Maxwell J Scott","doi":"10.1177/25731599261436373","DOIUrl":"10.1177/25731599261436373","url":null,"abstract":"<p><p>Conditional sex transformation systems could improve genetic control strategies against insect pests. Here, we developed and tested CRISPR-based, tetracycline-repressible sex transformation strains in the Australian sheep blowfly, <i>Lucilia cuprina</i>. Using Tet-Off-regulated expression of Cas9 and dCas9, we targeted the sex-determining gene <i>transformer</i> with the goal of converting females into males. Conditional Cas9 expression enabled knockout of a visual marker gene, confirming inducible genome editing. However, strains expressing <i>transformer</i>-targeting sgRNA arrays did not undergo sex transformation. Embryonic microinjection of <i>transformer</i>-targeting sgRNAs into Cas9-expressing embryos produced intersex individuals, indicating that sgRNA expression from the integrated arrays was insufficient to disrupt the sex determination pathway. In contrast, high-level dCas9 expression was associated with developmental delays, reduced body weight, and lethality. These findings establish the first conditional CRISPR expression system in <i>L. cuprina</i> and demonstrate that Cas9 is compatible with inducible gene editing, whereas dCas9 is poorly tolerated at high expression levels.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":"71-88"},"PeriodicalIF":4.0,"publicationDate":"2026-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147624690","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"CRISPR Medicine Needs Infrastructure, Not Just Innovation.","authors":"Jens-Ole Bock","doi":"10.1177/25731599261429007","DOIUrl":"10.1177/25731599261429007","url":null,"abstract":"","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":"56-58"},"PeriodicalIF":4.0,"publicationDate":"2026-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147610689","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"<i>Streptococcus uberis</i> Cas9-A Compact Type II-A Nuclease Recognizing a Unique PAM and Functional in Human Cells.","authors":"Aleksandra Vasileva, Marina Abramova, Polina Selkova, Anatolii Arseniev, Olga Musharova, Polina Malysheva, Alina Demkina, Mikhail Khodorkovskii, Konstantin Severinov","doi":"10.1177/25731599251404417","DOIUrl":"10.1177/25731599251404417","url":null,"abstract":"<p><p>Several type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 RNA-guided nucleases are commonly used for genome engineering. Their relatively large size and requirements for specific protospacer adjacent motif (PAM) sequences flanking their targets prompt continuous searches for additional more compact Cas9 enzymes with new PAM specificities. Here, we present SuCas9, a compact nuclease from <i>Streptococcus uberis</i>, a bacterium inhabiting the mammary glands of dairy cattle. SuCas9 recognizes a novel 5'-NNAAA-3' PAM, efficiently cleaves DNA <i>in vitro</i>, and is active in human cells. SuCas9 thus expands the available genome editing toolset and may find biotechnological and medicinal applications in the future.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":"21-35"},"PeriodicalIF":4.0,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145858981","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"CRISPR Friendship: A Decade of CRISPR Innovation and Collaboration in Aquaculture Genetics.","authors":"Karim M Khalil, Ahmed H Elaswad","doi":"10.1177/25731599251401530","DOIUrl":"10.1177/25731599251401530","url":null,"abstract":"","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":"7-8"},"PeriodicalIF":4.0,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146031600","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A Streamlined CRISPRa Architecture with Dual Pol II/Pol III Promoter and Optimized scRNA Enables Robust and Tunable Gene Activation.","authors":"Dylan Gallo, Martine Bes, Thibault Mounier, Caroline Calatayud, Anne-Cécile Meunier, Christophe Périn","doi":"10.1177/25731599251408333","DOIUrl":"10.1177/25731599251408333","url":null,"abstract":"<p><p>CRISPR activation (CRISPRa) offers a powerful approach to upregulate endogenous genes; yet, existing systems in plants can be complex or difficult to integrate with CRISPR interference (CRISPRi). Here, we present a streamlined and flexible CRISPRa platform that enables robust gene activation. Using a dual-luciferase reporter, we benchmarked a range of guide RNA scaffolds, effector proteins, and promoters. We developed a novel single-guide RNA (sgRNA) architecture, harboring two MS2 aptamers inserted into the tetraloop and driven by a composite Pol II/Pol III promoter, as the most efficient configuration. This scaffold outperformed gR2.0- and SunTag-based constructs, reaching up to 100-fold activation of a minimal 35S promoter and up to 215-fold induction of three endogenous rice genes in protoplast assays. In contrast, scaffold RNAs (scRNAs) with aptamers at the 3' end or in excessive copy numbers were ineffective. Exploratory AlphaFold modeling supports a possible role for aptamer positioning and MCP-VP64 dimerization, although this remains a working hypothesis. This modular design enables tunable gene regulation in rice protoplasts and provides a practical platform for high-throughput screening and synthetic gene circuit prototyping in plants. Given that scRNA geometry and promoter architecture are universal features of CRISPR-based transcriptional modulation, the system is expected to be broadly portable across species. While the architecture is intended to be compatible with CRISPRi, future studies will be needed to establish its practical use in combined CRISPRa/i settings.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":"36-48"},"PeriodicalIF":4.0,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146127584","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"BAR-CAT: Targeted Recovery of Synthetic Genes via Barcode-Directed CRISPR-dCas9 Enrichment.","authors":"Natanya K Villegas, Mindy H Tran, Abigail Keller, Calin Plesa","doi":"10.1177/25731599251401526","DOIUrl":"10.1177/25731599251401526","url":null,"abstract":"<p><p>Modern gene synthesis platforms enable investigations of protein function and genome biology at an unprecedented scale. Yet, the proportion of error-free constructs in diverse gene libraries decreases with length due to the propagation of oligo synthesis errors. To rescue these error-free constructs, we developed Barcode-Assisted Retrieval CRISPR-Activated Targeting (BAR-CAT), an <i>in vitro</i> method that uses multiplexed dCas9-single-guide RNA (sgRNA) complexes to extract barcodes corresponding to error-free constructs. After a 15-min incubation and wash regimen, three low-bundance targets in a 300,000-member test library were enriched 600-fold, greatly reducing downstream requirements. When applied to a 384-gene DropSynth gene library, BAR-CAT enriched 12 targets up to 122-fold and revealed practical limits imposed by sgRNA competition and library complexity, which now guide ongoing protocol scaling. By eliminating laborious clone-by-clone validation and working directly on plasmid libraries, BAR-CAT provides a platform for recovering perfect synthetic genes, subsetting large libraries, and ultimately lowering the cost of functional genomics at scale.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":"9-20"},"PeriodicalIF":4.0,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145670634","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}