CRISPR Journal最新文献

{"title":"Safe Speeding on the Highway of Science.","authors":"Rodolphe Barrangou","doi":"10.1177/25731599261473241","DOIUrl":"10.1177/25731599261473241","url":null,"abstract":"","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":"183"},"PeriodicalIF":3.6,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148563716","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":"An Optimized Workflow for <i>In Vitro</i> Transcription of Single Guide RNAs Minimizes Innate Immune Activation.","authors":"Xin Wang, Wim Trypsteen, Jasper Anckaert, Eric de Bony, Pieter Mestdagh","doi":"10.1177/25731599261467974","DOIUrl":"10.1177/25731599261467974","url":null,"abstract":"<p><p>CRISPR interference (CRISPRi) often uses single guide RNAs (sgRNAs) generated by <i>in vitro</i> transcription (IVT); however, IVT-derived RNAs can trigger innate immune responses that confound functional analyses. Here, we evaluate innate immune activation induced by IVT sgRNAs in a CRISPRi setting and show that enzymatic removal of the 5'-triphosphate group alone is insufficient to consistently eliminate this response. We therefore assessed modifications of IVT reaction conditions and found that supplementation with sodium chloride or urea further attenuated immune activation. Based on immune suppression, sgRNA yield, and knockdown efficiency, 0.15 M NaCl was selected for the optimized IVT condition. This condition showed a lower double-stranded RNA (dsRNA) concentration, providing direct support for reduced dsRNA by-products as a contributor to diminished immune activation. By integrating NaCl-supplemented IVT with phosphatase treatment, we establish an optimized and scalable workflow that minimizes innate immune responses while preserving sgRNA-mediated target knockdown efficiency in stable CRISPRi cells.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":"223-232"},"PeriodicalIF":3.6,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148649765","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":"Induction of SpCas9-Directed Immune Responses Using Lipid Nanoparticles and Identification of SpCas9-Derived T Cell Epitopes in C57BL/6 Mice.","authors":"Deja Porenta, Naomi Benne, Alice Sijts, Femke Broere, Enrico Mastrobattista","doi":"10.1177/25731599261456887","DOIUrl":"10.1177/25731599261456887","url":null,"abstract":"<p><p>A major goal of clinically oriented CRISPR-Cas9-based applications is safe and effective <i>in vivo</i> gene editing (knockout or correction) with precise targeting. Substantial efforts have been devoted to the preclinical development of novel drug delivery platforms that enable efficient, targeted delivery. However, the immune responses induced by CRISPR-Cas9 treatment are often overlooked. Preexisting immunity to clinically relevant Cas9 proteins has already been established as a consequence of natural exposure to Cas9-bearing bacteria, which may implicate the safety and efficacy of CRISPR-Cas9-based therapies. Naturally, CRISPR-Cas9 therapies should be nonimmunogenic to avoid amplifying existing Cas9-specific immunity, especially cytotoxic T cell responses. Nonviral delivery systems, such as lipid nanoparticles (LNPs), are widely regarded as less immunogenic than more traditionally used viral vectors, even though LNPs are suitable as a vaccination platforms. In this study, we investigate the induction of SpCas9-directed immunity in C57BL/6 mice upon repeated dosing of LNPs encapsulating Cas9-coding mRNA in two different settings: (1) a vaccination-resembling setting using intramuscularly administered adjuvanted LNPs, and (2) a therapy-resembling setting using intravenously injected, liver-targeting LNPs. In both settings, Cas9-specific T cell responses were detected by evaluating increased total IFN-γ levels upon <i>ex vivo</i> restimulation of isolated splenocytes. However, undetectable Cas9-reactive antibodies induced in the therapeutic setting emphasize the discrepancy between humoral and cellular responses. To improve future monitoring of Cas9-specific T cell responses, we report six Cas9-derived epitopes recognized by CD8⁺ T cells, as well as a CD4⁺ T cell polypeptide carrying one of the CD8⁺ T cell epitopes that induced strong IFN-γ production <i>ex vivo</i>. This work is intended to facilitate the preclinical monitoring of Cas9-specific T cell responses in C57BL/6 mice and support the development of safe CRISPR-Cas9-based therapies.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":"151-166"},"PeriodicalIF":3.6,"publicationDate":"2026-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148260546","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":"Are We Making Genetically Modified Humans?","authors":"Rodolphe Barrangou","doi":"10.1177/25731599261456158","DOIUrl":"10.1177/25731599261456158","url":null,"abstract":"","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":"119"},"PeriodicalIF":3.6,"publicationDate":"2026-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148177286","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>Corrigendum to:</i> \"Subtypes of Type I-E CRISPR-Cas Systems Distribution in Human <i>Escherichia coli</i> Isolates from China\".","authors":"","doi":"10.1177/25731599261457400","DOIUrl":"10.1177/25731599261457400","url":null,"abstract":"","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":"177"},"PeriodicalIF":3.6,"publicationDate":"2026-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148213307","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 Potent CRISPR-Cas12l Double-Strand Break Gene Editor.","authors":"Tomas Urbaitis, Laima Trinkuniene, Ieva Lenkaite, Monika Petrauskyte, Renatas Krasauskas, Migle Stitilyte, Modestas Sabaliauskas, Giedrius Sasnauskas, Giedre Tamulaitiene, Joshua K Young, Virginijus Siksnys, Giedrius Gasiunas","doi":"10.1177/25731599261448428","DOIUrl":"10.1177/25731599261448428","url":null,"abstract":"<p><p>Recently, a new family of CRISPR-Cas12 endonucleases from an unexplored phylum of bacteria, <i>Armatimonadota</i>, was discovered. Named Cas12l, they are compact (800-900 aa), recognize a 5' C-rich protospacer adjacent motif, and present an N-terminal domain that stretches from the beginning to the end of the ribonucleoprotein-bound DNA target site, effectively locking it in place. Here, structure-guided rational design supplemented with AI-based large protein language model predictions was used to improve rates of DNA target cleavage of a family member, Asp2Cas12l. Compared to the wild-type, engineered variants exhibited an approximately 10-fold increase in double-strand break (DSB) editing efficiency in human cells with less target-to-target variation. Moreover, frequencies of editing were comparable to those of SpCas9 at overlapping target sites, and their DSBs efficiently corrected by homology-directed repair (39-56% of editing outcomes). Altogether, this study extends our understanding of CRISPR-Cas12 protein engineering and offers a potent new alternative for DSB-mediated genome editing in human cells.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":"126-140"},"PeriodicalIF":3.6,"publicationDate":"2026-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147984323","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":"Advice from the CRISPR Whisperer.","authors":"Dorothy Semenow","doi":"10.1177/25731599261428458","DOIUrl":"10.1177/25731599261428458","url":null,"abstract":"","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":"123-125"},"PeriodicalIF":3.6,"publicationDate":"2026-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148260477","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":"Increasing the Effective Gene Drive Homing Rate by Targeting the Haploinsufficient Spermatogenesis Gene <i>Klhl10</i>.","authors":"Ceili L Peng, W Sebastian Kamau, Julien Freeman, Zachary J Hill, Kevin M Esvelt","doi":"10.1177/25731599261452112","DOIUrl":"10.1177/25731599261452112","url":null,"abstract":"<p><p>CRISPR-based gene drives represent a powerful new technology for limiting disease transmission and controlling invasive populations. These systems rely on homology-directed repair (HDR) to \"drive\" a genetic element through a population. However, mammals tend to favor non-homologous end joining (NHEJ), which generates mutations that halt further drive propagation. Here, we describe the experimental characterization of a putative target locus for a gene drive system targeting the haploinsufficient spermatogenesis gene <i>Klhl10</i> in the laboratory mouse. Using a newly designed \"coding sequence cassette,\" we introduce downstream guide RNAs within the gene, ensuring that sperm undergoing NHEJ are selectively removed from the population. As a proof of principle, we demonstrate that targeting <i>Klhl10</i> with constitutively expressed LbCas12a results in strong selection against frameshift-containing sperm, validating the core purification mechanism required for this drive strategy. Unexpectedly, we also observed that female offspring lacked most frameshift mutations, suggesting a previously unrecognized role for <i>Klhl10</i> in oogenesis or early embryonic development.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":"167-176"},"PeriodicalIF":3.6,"publicationDate":"2026-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148220794","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":"Optimization of Genome-Wide CRISPR Screens Using Dual-Guide RNA Infection with Cas9 Electroporation (DICE).","authors":"Caitlin Purman, Charles Lu, Apexa Modi, Vinny Vijaykumar, Michael J Flister, Anneke I den Hollander, Sabah Kadri, Joshua D Stender","doi":"10.1177/25731599261448141","DOIUrl":"10.1177/25731599261448141","url":null,"abstract":"<p><p>Single-guide RNA (sgRNA) lentiviral infection with Cas9 protein electroporation (SLICE) enables CRISPR screening in primary cell types that require transient Cas9 expression, yet is limited by scalability and robustness. Here, we introduce dual guide RNA infection with Cas9 electroporation (DICE), which expresses two guides from the same lentiviral construct that target the same gene. In genome-wide screens, DICE outperformed SLICE in defining essential genes and modulators of PD-L1 expression in Interferon-gamma-activated THP1 cells. Collectively, these data demonstrate that DICE can be utilized for reduced-scale CRISPR screens in cell types with transient Cas9 protein expression without sacrificing screening quality.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":"141-150"},"PeriodicalIF":3.6,"publicationDate":"2026-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147943781","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 Learns to Read the Epigenome.","authors":"Joshua B Black, Charles A Gersbach","doi":"10.1177/25731599261460335","DOIUrl":"10.1177/25731599261460335","url":null,"abstract":"","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":"120-122"},"PeriodicalIF":3.6,"publicationDate":"2026-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148240584","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}