CRISPR JournalPub Date : 2024-12-01Epub Date: 2024-09-23DOI: 10.1089/crispr.2024.0053
Benjamin G Gowen, Prachi Khekare, Shannon R McCawley, Kory Melton, Craig Soares, Jean Chan, Vihasi Jani, Pierre Boivin, Ashil Bans, Weng-In Leong, Aaron J Cantor, Jack Walleshauser, Peter B Otoupal, Rina J Mepani, Adam P Silverman, Mary Haak-Frendscho, Spencer C Wei
{"title":"Identification of a Guide RNA Targeting an Ultraconserved Element for Evaluation of Cas9 Genome Editors Across Mammalian Species.","authors":"Benjamin G Gowen, Prachi Khekare, Shannon R McCawley, Kory Melton, Craig Soares, Jean Chan, Vihasi Jani, Pierre Boivin, Ashil Bans, Weng-In Leong, Aaron J Cantor, Jack Walleshauser, Peter B Otoupal, Rina J Mepani, Adam P Silverman, Mary Haak-Frendscho, Spencer C Wei","doi":"10.1089/crispr.2024.0053","DOIUrl":"10.1089/crispr.2024.0053","url":null,"abstract":"","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":"306-309"},"PeriodicalIF":3.7,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142300796","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}
CRISPR JournalPub Date : 2024-12-01Epub Date: 2024-10-22DOI: 10.1089/crispr.2024.0052
Christopher J Cotter, Cong T Trinh
{"title":"CRISPR-GRIT: Guide RNAs with Integrated Repair Templates Enable Precise Multiplexed Genome Editing in the Diploid Fungal Pathogen <i>Candida albicans</i>.","authors":"Christopher J Cotter, Cong T Trinh","doi":"10.1089/crispr.2024.0052","DOIUrl":"10.1089/crispr.2024.0052","url":null,"abstract":"<p><p><i>Candida albicans,</i> an opportunistic fungal pathogen, causes severe infections in immunocompromised individuals. Limited classes and overuse of current antifungals have led to the rapid emergence of antifungal resistance. Thus, there is an urgent need to understand fungal pathogen genetics to develop new antifungal strategies. Genetic manipulation of <i>C. albicans</i> is encumbered by its diploid chromosomes requiring editing both alleles to elucidate gene function. Although the recent development of CRISPR-Cas systems has facilitated genome editing in <i>C. albicans</i>, large-scale and multiplexed functional genomic studies are still hindered by the necessity of cotransforming repair templates for homozygous knockouts. Here, we present CRISPR-GRIT (<u>G</u>uide <u>R</u>NAs with <u>I</u>ntegrated Repair <u>T</u>emplates), a repair template-integrated guide RNA design for expedited gene knockouts and multiplexed gene editing in <i>C. albicans</i>. We envision that this method can be used for high-throughput library screens and identification of synthetic lethal pairs in both <i>C. albicans</i> and other diploid organisms with strong homologous recombination machinery.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":"385-394"},"PeriodicalIF":3.7,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142512851","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}
CRISPR JournalPub Date : 2024-12-01Epub Date: 2024-11-08DOI: 10.1089/crispr.2024.0001
Aysegul Ates, Cihan Tastan, Safak Ermertcan
{"title":"CRISPR-Cas9-Mediated Targeting of Multidrug Resistance Genes in Methicillin-Resistant <i>Staphylococcus aureus</i>.","authors":"Aysegul Ates, Cihan Tastan, Safak Ermertcan","doi":"10.1089/crispr.2024.0001","DOIUrl":"10.1089/crispr.2024.0001","url":null,"abstract":"<p><p>Antibiotic resistance poses a global health crisis limiting the efficacy of available therapeutic agents. We explored CRISPR-Cas-based antimicrobials to combat multidrug resistance in methicillin-resistant <i>Staphylococcus aureus</i> (MRSA), targeting methicillin (<i>mec</i>A), gentamicin (<i>aac</i>A), and ciprofloxacin (<i>grl</i>A, <i>grl</i>B) resistance genes. Engineered CRISPR plasmids with specific single-guide RNAs were electroporated into MRSA strains. Real-time polymerase chain reaction assessed gene expression changes, while antibiotic susceptibility tests (ASTs) evaluated resistance status. Results showed a 1.5-fold decrease in <i>mec</i>A, a 5.5-fold decrease in <i>grl</i>A, a 6-fold decrease in <i>grl</i>B, and a 4-fold decrease in <i>aac</i>A expression. ASTs demonstrated the reversal of resistance to beta-lactam, quinolone, and aminoglycoside antibiotics. Western blot analysis revealed a 70% decrease in penicillin-binding protein 2a expression. Sanger sequencing confirmed point mutations in the <i>grl</i>B and <i>aac</i>A genes. Our findings highlight the potential of CRISPR-Cas9 technology to restore antibiotic efficacy against multidrug-resistant pathogens.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":"374-384"},"PeriodicalIF":3.7,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142607408","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}
CRISPR JournalPub Date : 2024-12-01Epub Date: 2024-11-18DOI: 10.1089/crispr.2024.0018
Siyuan Liu, Yingdi Zhao, Qiqin Mo, Yadong Sun, Hanhui Ma
{"title":"Engineering CjCas9 for Efficient Base Editing and Prime Editing.","authors":"Siyuan Liu, Yingdi Zhao, Qiqin Mo, Yadong Sun, Hanhui Ma","doi":"10.1089/crispr.2024.0018","DOIUrl":"10.1089/crispr.2024.0018","url":null,"abstract":"<p><p>The CRISPR-Cas9 system has been applied for clinical applications of gene therapy. Most CRISPR-based gene therapies are derived from <i>Streptococcus pyogenes</i> Cas9, which is challenging to package into a single adeno-associated virus vector and limits its clinical applications. <i>Campylobacter jejuni</i> Cas9 (CjCas9) is one of the smallest Cas9 proteins. CjCas9-mediated base editing (CjBE) efficiency varies across genomic sites, while CjCas9-mediated prime editing (CjPE) efficiency is less than 5% on average. Here we developed enhanced cytosine base editors (enCjCBEs) and adenine base editors (enCjABEs) by engineered CjCas9<sup>P47K</sup>. We demonstrated the robust C-to-T conversion (70% on average) by enCjCBE or A-to-G conversion (76% on average) by enCjABE. Meanwhile, we applied the CjCas9<sup>P47K</sup> variant to generate enhanced CjPE (enCjPE), which increases the editing efficiency 17-fold at the <i>PRNP</i> site over wild-type CjPE. Fusing nonspecific DNA binding protein Sso7d to enCjCas9 and MS2 stem-loop RNA aptamer to the 3-terminal of cognate pegRNA resulted in 12% editing efficiency on average with a 24-fold increase over wild-type CjPE, and we termed it SsenCjPE. The SsenCjPE can also be combined with hMLH1dn to further increase the editing efficiency and MMLV RTaseΔRnH to reduce size. Finally, we introduced an additional mutation D829R into SsenCjPE and generated SsenCjPE-M2 with a 61-fold increase of PE efficiency over wild-type at the <i>PRNP</i> site. In summary, enCjBEs, SsenCjPEs, or SsenCjPE-M2 are compact Cas9-derived BE or prime editors in biological research or biomedical applications.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":"395-405"},"PeriodicalIF":3.7,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142649684","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}
CRISPR JournalPub Date : 2024-12-01Epub Date: 2024-09-30DOI: 10.1089/crispr.2024.0047
Kotaro Kamata, Nils Birkholz, Marijn Ceelen, Robert D Fagerlund, Simon A Jackson, Peter C Fineran
{"title":"Repurposing an Endogenous CRISPR-Cas System to Generate and Study Subtle Mutations in Bacteriophages.","authors":"Kotaro Kamata, Nils Birkholz, Marijn Ceelen, Robert D Fagerlund, Simon A Jackson, Peter C Fineran","doi":"10.1089/crispr.2024.0047","DOIUrl":"10.1089/crispr.2024.0047","url":null,"abstract":"<p><p>While bacteriophage applications benefit from effective phage engineering, selecting the desired genotype after subtle modifications remains challenging. Here, we describe a two-phase endogenous CRISPR-Cas-based phage engineering approach that enables selection of small defined edits in <i>Pectobacterium carotovorum</i> phage ZF40. We designed plasmids containing sequences homologous to ZF40 and a mini-CRISPR array. The plasmids allowed genome editing through homologous recombination and counter-selection against non-recombinant phage genomes using an endogenous type I-E CRISPR-Cas system. With this technique, we first deleted target genes and subsequently restored loci with modifications. This two-phase approach circumvented major challenges in subtle phage modifications, including inadequate sequence distinction for CRISPR-Cas counter-selection and the requirement of a protospacer-adjacent motif, limiting sequences that can be modified. Distinct 20-bp barcodes were incorporated through engineering as differential target sites for programmed CRISPR-Cas activity, which allowed quantification of phage variants in mixed populations. This method aids studies and applications that require mixtures of similar phages.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":"343-354"},"PeriodicalIF":3.7,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142332259","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}
CRISPR JournalPub Date : 2024-12-01Epub Date: 2024-10-10DOI: 10.1089/crispr.2024.0003
Brett M Sansbury, Sophia B Masciarelli, Salma Kaouser, Olivia M Tharp, Kelly H Banas, Eric B Kmiec
{"title":"Mutation-Specific CRISPR Targeting with SaCas9 and AsCas12a Restores Therapeutic Sensitivity in Treatment-Resistant Melanoma.","authors":"Brett M Sansbury, Sophia B Masciarelli, Salma Kaouser, Olivia M Tharp, Kelly H Banas, Eric B Kmiec","doi":"10.1089/crispr.2024.0003","DOIUrl":"10.1089/crispr.2024.0003","url":null,"abstract":"<p><p><b>Background:</b> Melanoma remains one of the most challenging cancers to treat effectively with drug resistant remaining a constant concern, primarily with activating <i>BRAF</i> mutations. Mutations in the <i>BRAF</i> gene appear in approximately 50% of patients, 90% of which are V600E. Two frontline <i>BRAF</i> inhibitors (BRAFi), vemurafenib and dabrafenib, are frequently used to treat unresectable or metastatic <i>BRAF</i> V600E melanoma. Initial response rates are high, but soon thereafter, 70-80% of patients develop resistance to treatment within a year. A major mechanism of resistance is the generation of a secondary Q61K mutation in the <i>NRAS</i> gene. <b>Methods:</b> We have developed an approach in which a CRISPR-Cas complex can be designed to distinguish between mutant genes enabling resistance to standard care in tumor cells and normal genomes of healthy cells. For the first time, we demonstrated the utility of two CRISPR-directed mutation-specific editing approaches to restore BRAFi sensitivity in <i>BRAF</i><sup>V600E</sup>/<i>NRAS</i><sup>Q61K</sup> resistant A375 cells. <b>Results:</b> We utilize an AsCas12a protospacer adjacent motif site created by the <i>NRAS</i> Q61K mutation and the Q61K mutation in the critical seed region of an SaCas9 sgRNA for Q61K-selective targeting. We show here that both approaches allow for effective <i>NRAS</i> targeting of only mutated-Q61K and after CRISPR-directed Q61K-targeting, previously resistant A375 cells are re-sensitized to BRAFi treatment. <b>Conclusion:</b> Our data support the feasibility of the development of CRISPR-Cas therapeutic approaches to the treatment of melanoma. Successful therapeutic CRISPR-directed gene editing would enable both specific and efficient editing of a mutation-specific targeting approach eliminate concern for on- and off-target damage to the genomes of healthy cells.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":"366-373"},"PeriodicalIF":3.7,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142480624","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}
CRISPR JournalPub Date : 2024-10-01Epub Date: 2024-10-16DOI: 10.1089/crispr.2024.0016
Zhang Xinyue, Siwei Li, Wang Yujie, Dai Yingcai, Bi Changhao, Zhang Xueli
{"title":"Engineering of HEK293T Cell Factory for Lentiviral Production by High-Throughput Selected Genes.","authors":"Zhang Xinyue, Siwei Li, Wang Yujie, Dai Yingcai, Bi Changhao, Zhang Xueli","doi":"10.1089/crispr.2024.0016","DOIUrl":"10.1089/crispr.2024.0016","url":null,"abstract":"<p><p>Lentiviral vectors (LVs) are crucial tools in gene therapy and bioproduction, but high-yield LV production systems are urgently needed. Using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 high-throughput screening, we identified nine critical genes (<i>LDAH, GBP3, BPIFC, NHLRC1, NHLRC3, ZNF425, TTC37, LRRC4B</i>, and <i>SPINK6</i>) from 17,501 genes that limit LV packaging and formation. Knocking out these genes in HEK293T cells significantly increased virus production, with <i>LDAH</i> knockout exhibiting a 6.63-fold increase. Studies on multigene knockouts demonstrated that the cumulative effects of different gene knockouts can significantly enhance lentivirus production in HEK293T cells. Triple knockout of <i>GBP3, BPIFC</i>, and <i>LDAH</i> increased LV titer by ∼8.33-fold, and knockout (or knockdown) of <i>GBP3, NHLRC1,</i> and <i>NHLRC3</i> increased LV titer by ∼6.53-fold. This study established HEK293T cell lines with multiple genes knockout for efficient LV production, providing reliable technical support for LV production and application and offering new perspectives for studying LV packaging mechanisms and related virus research.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":"7 5","pages":"272-282"},"PeriodicalIF":3.7,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142480626","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}
CRISPR JournalPub Date : 2024-10-01DOI: 10.1089/crispr.2024.55675.rfs2023
Suchita Nety
{"title":"Rosalind Franklin Society Proudly Announces the 2023 Award Recipient for <i>The CRISPR Journal</i>.","authors":"Suchita Nety","doi":"10.1089/crispr.2024.55675.rfs2023","DOIUrl":"https://doi.org/10.1089/crispr.2024.55675.rfs2023","url":null,"abstract":"","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":"7 5","pages":"211"},"PeriodicalIF":3.7,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142808608","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}
CRISPR JournalPub Date : 2024-10-01DOI: 10.1089/crispr.2024.0081
Kevin Davies, Alex Philippidis, Rodolphe Barrangou
{"title":"Five Years of Progress in CRISPR Clinical Trials (2019-2024).","authors":"Kevin Davies, Alex Philippidis, Rodolphe Barrangou","doi":"10.1089/crispr.2024.0081","DOIUrl":"10.1089/crispr.2024.0081","url":null,"abstract":"<p><p>In July 2019, Victoria Gray became the first patient with sickle cell disease to receive a CRISPR-based cell therapy as a volunteer in the exa-cel clinical trial, sponsored by Vertex Pharmaceuticals and CRISPR Therapeutics. Barely four years later, the ensuing therapy, branded as Casgevy, received approval from regulatory agencies in Europe, the United States, and the Middle East, ushering in a new era of CRISPR-based medicines. During this period, scores of other clinical trials have been launched, including many actively recruiting patients across phase 1, phase 2, and phase 3 clinical trials around the world. In this brief Perspective, we collate the latest information on therapeutic clinical trials featuring CRISPR, base and prime editing, across a range of both <i>in vivo</i> and <i>ex vivo</i> gene and cell therapies.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":"7 5","pages":"227-230"},"PeriodicalIF":3.7,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142480628","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}
CRISPR JournalPub Date : 2024-10-01Epub Date: 2024-09-26DOI: 10.1089/crispr.2024.0011
Riya Mohan, Susanne B Haga
{"title":"Characterization of Research Support of Genome Editing Technologies and Transition to Clinical Trials.","authors":"Riya Mohan, Susanne B Haga","doi":"10.1089/crispr.2024.0011","DOIUrl":"10.1089/crispr.2024.0011","url":null,"abstract":"<p><p>Genome editing technologies have become widely used research tools. To assess the rate of growth with respect to federal funding of gene editing projects, we analyzed publicly available data retrieved from the NIH RePORTER and Clinicaltrials.gov databases. We identified 6,111 awards between 1977 and 2023, the majority being extramural, investigator-driven R (noneducational) awards (66.7%). There was an average growth rate of 40% between 2008 and 2022, and the biggest increase in awards was observed between 2017 and 2018 (doubling from 140 to 280). Five administering institutes/centers accounted for more than 60% of awards with the highest number of awards from the National Cancer Institute (20.0%). The majority of clinical trials involving some type of genome editing (75%) started in or after 2020. This analysis illuminates the rapid and widespread growth of gene editing research across disciplines and the eventual launch of clinical trials using gene editing tools.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":"249-257"},"PeriodicalIF":3.7,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142332257","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}