Versatile and efficient mammalian genome editing with Type I-C CRISPR System of Desulfovibrio vulgaris.

IF 8 2区 生物学 Q1 BIOLOGY Science China Life Sciences Pub Date : 2024-11-01 Epub Date: 2024-08-07 DOI:10.1007/s11427-023-2682-5
Pan Li, Dingcai Dong, Fei Gao, Yuyang Xie, Honglin Huang, Siwei Sun, Zhao Ma, Cheng He, Jinsheng Lai, Xuguang Du, Sen Wu
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Abstract

CRISPR-Cas tools for mammalian genome editing typically rely on single Cas9 or Cas12a proteins. While type I CRISPR systems in Class I may offer greater specificity and versatility, they are not well-developed for genome editing. Here, we present an alternative type I-C CRISPR system from Desulfovibrio vulgaris (Dvu) for efficient and precise genome editing in mammalian cells and animals. We optimized the Dvu type I-C editing complex to generate precise deletions at multiple loci in various cell lines and pig primary fibroblast cells using a paired PAM-in crRNA strategy. These edited pig cells can serve as donors for generating transgenic cloned piglets. The Dvu type I-C editor also enabled precise large fragment replacements with homology-directed repair. Additionally, we adapted the Dvu-Cascade effector for cytosine and adenine base editing, developing Dvu-CBE and Dvu-ABE systems. These systems efficiently induced C-to-T and A-to-G substitutions in human genes without double-strand breaks. Off-target analysis confirmed the high specificity of the Dvu type I-C editor. Our findings demonstrate the Dvu type I-C editor's potential for diverse mammalian genome editing applications, including deletions, fragment replacement, and base editing, with high efficiency and specificity for biomedicine and agriculture.

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利用普通脱硫弧菌的 I-C 型 CRISPR 系统进行多功能、高效的哺乳动物基因组编辑。
用于哺乳动物基因组编辑的 CRISPR-Cas 工具通常依赖单个 Cas9 或 Cas12a 蛋白。虽然I类CRISPR系统可能具有更高的特异性和多功能性,但它们在基因组编辑方面并不发达。在这里,我们介绍了一种来自Desulfovibrio vulgaris(Dvu)的I-C型CRISPR系统,用于在哺乳动物细胞和动物中进行高效、精确的基因组编辑。我们对 Dvu I-C 型编辑复合物进行了优化,利用成对的 PAM-in crRNA 策略在各种细胞系和猪原代成纤维细胞中生成多个位点的精确缺失。这些经过编辑的猪细胞可作为供体,用于产生转基因克隆仔猪。Dvu I-C 型编辑器还能通过同源定向修复实现大片段的精确替换。此外,我们还将 Dvu 级联效应器用于胞嘧啶和腺嘌呤碱基编辑,开发出了 Dvu-CBE 和 Dvu-ABE 系统。这些系统能有效地诱导人类基因中的 C 到 T 和 A 到 G 的置换,而不会发生双链断裂。脱靶分析证实了 Dvu I-C 型编辑器的高度特异性。我们的研究结果表明,Dvu I-C 型编辑器可用于多种哺乳动物基因组编辑应用,包括缺失、片段置换和碱基编辑,在生物医学和农业领域具有高效率和特异性。
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来源期刊
CiteScore
15.10
自引率
8.80%
发文量
2907
审稿时长
3.2 months
期刊介绍: Science China Life Sciences is a scholarly journal co-sponsored by the Chinese Academy of Sciences and the National Natural Science Foundation of China, and it is published by Science China Press. The journal is dedicated to publishing high-quality, original research findings in both basic and applied life science research.
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