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

作　　者：Pan Li Dingcai Dong Fei Gao Yuyang Xie Honglin Huang Siwei Sun Zhao Ma Cheng He Jinsheng Lai Xuguang Du Sen Wu 

机构地区：[1]State Key Laboratory of Animal Biotech Breeding,College of Biological Sciences,National Engineering Laboratory for Animal Breeding,Frontiers Science Center for Molecular Design Breeding,China Agricultural University,Beijing 100193,China [2]College of Veterinary Medicine,China Agricultural University,Beijing 100193,China [3]Sanya Institute of China Agricultural University,Sanya 572025,China [4]State Key Laboratory of Maize Bio-breeding,National Maize Improvement Center,Department of Plant Genetics and Breeding,China Agricultural University,Beijing 100193,China [5]Frontiers Science Center for Molecular Design Breeding,China Agricultural University,Beijing 100193,China [6]Center for Crop Functional Genomics and Molecular Breeding,China Agricultural University,Beijing 100193,China

出　　处：《Science China(Life Sciences)》2024年第11期2471-2487,共17页中国科学（生命科学英文版）

基　　金：funded by the National Key R&D Program of China(2021YFA0805900,2023YFF1000200,2023YFF1000900,and 2023YFC3402004);the China Postdoctoral Science Foundation(2021M703521).

摘　　要：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.

关 键 词：type I CRISPR systems Desulfovibrio vulgaris Dvu type I-C editor define deletion large fragment replacement base editing 

分 类 号：Q78[生物学—分子生物学]