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 genom...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.展开更多
基金funded by the National Key R&D Program of China(2021YFA0805900,2023YFF1000200,2023YFF1000900,and 2023YFC3402004)the China Postdoctoral Science Foundation(2021M703521).
文摘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.
