Discovery and engineering of ChCas12b for precise genome editing

IF 18.8 1区综合性期刊 Q1 MULTIDISCIPLINARY SCIENCES

Science Bulletin Pub Date : 2024-10-30 DOI:10.1016/j.scib.2024.06.012

{"title":"Discovery and engineering of ChCas12b for precise genome editing","authors":"","doi":"10.1016/j.scib.2024.06.012","DOIUrl":null,"url":null,"abstract":"<div><div>Many clustered regularly interspaced short palindromic repeat and CRISPR-associated protein 12b (CRISPR-Cas12b) nucleases have been computationally identified, yet their potential for genome editing remains largely unexplored. In this study, we conducted a GFP-activation assay screening 13 Cas12b nucleases for mammalian genome editing, identifying five active candidates. Candidatus hydrogenedentes Cas12b (ChCas12b) was found to recognize a straightforward WTN (W = T or A) proto-spacer adjacent motif (PAM), thereby dramatically expanding the targeting scope. Upon optimization of the single guide RNA (sgRNA) scaffold, ChCas12b exhibited activity comparable to SpCas9 across a panel of nine endogenous loci. Additionally, we identified nine mutations enhancing ChCas12b specificity. More importantly, we demonstrated that both ChCas12b and its high-fidelity variant, ChCas12b-D496A, enabled allele-specific disruption of genes harboring single nucleotide polymorphisms (SNPs). These data position ChCas12b and its high-fidelity counterparts as promising tools for both fundamental research and therapeutic applications.</div></div>","PeriodicalId":421,"journal":{"name":"Science Bulletin","volume":"69 20","pages":"Pages 3260-3271"},"PeriodicalIF":18.8000,"publicationDate":"2024-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Science Bulletin","FirstCategoryId":"103","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S2095927324004080","RegionNum":1,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"MULTIDISCIPLINARY SCIENCES","Score":null,"Total":0}

引用次数: 0

Abstract

Many clustered regularly interspaced short palindromic repeat and CRISPR-associated protein 12b (CRISPR-Cas12b) nucleases have been computationally identified, yet their potential for genome editing remains largely unexplored. In this study, we conducted a GFP-activation assay screening 13 Cas12b nucleases for mammalian genome editing, identifying five active candidates. Candidatus hydrogenedentes Cas12b (ChCas12b) was found to recognize a straightforward WTN (W = T or A) proto-spacer adjacent motif (PAM), thereby dramatically expanding the targeting scope. Upon optimization of the single guide RNA (sgRNA) scaffold, ChCas12b exhibited activity comparable to SpCas9 across a panel of nine endogenous loci. Additionally, we identified nine mutations enhancing ChCas12b specificity. More importantly, we demonstrated that both ChCas12b and its high-fidelity variant, ChCas12b-D496A, enabled allele-specific disruption of genes harboring single nucleotide polymorphisms (SNPs). These data position ChCas12b and its high-fidelity counterparts as promising tools for both fundamental research and therapeutic applications.

Abstract Image

查看原文本刊更多论文

发现并设计用于精确基因组编辑的 ChCas12b

许多簇状规则间隔短回文重复和CRISPR相关蛋白12b（CRISPR-Cas12b）核酸酶已被计算鉴定，但它们在基因组编辑方面的潜力在很大程度上仍未被开发。在这项研究中，我们进行了一项 GFP 激活试验，筛选出 13 种可用于哺乳动物基因组编辑的 Cas12b 核酸酶，并确定了五种具有活性的候选核酸酶。研究发现，Candidatus hydrogenedentes Cas12b（ChCas12b）能直接识别WTN（W = T或A）原间隔邻接基序（PAM），从而极大地扩展了靶向范围。在对单导 RNA（sgRNA）支架进行优化后，ChCas12b 在九个内源基因位点上表现出与 SpCas9 相当的活性。此外，我们还发现了九种增强 ChCas12b 特异性的突变。更重要的是，我们证明了 ChCas12b 及其高保真变体 ChCas12b-D496A 都能对含有单核苷酸多态性（SNP）的基因进行等位基因特异性破坏。这些数据使 ChCas12b 及其高保真变体成为基础研究和治疗应用的理想工具。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

求助全文

约1分钟内获得全文求助全文

来源期刊

Science Bulletin MULTIDISCIPLINARY SCIENCES-

CiteScore

24.60

自引率

2.10%

发文量

8092

期刊介绍： Science Bulletin (Sci. Bull., formerly known as Chinese Science Bulletin) is a multidisciplinary academic journal supervised by the Chinese Academy of Sciences (CAS) and co-sponsored by the CAS and the National Natural Science Foundation of China (NSFC). Sci. Bull. is a semi-monthly international journal publishing high-caliber peer-reviewed research on a broad range of natural sciences and high-tech fields on the basis of its originality, scientific significance and whether it is of general interest. In addition, we are committed to serving the scientific community with immediate, authoritative news and valuable insights into upcoming trends around the globe.