Ma Enbo, Chen Kai, Shi Honglue, Wasko Kevin M, Esain-Garcia Isabel, Trinidad Marena I, Zhou Kaihong, Ye Jinjuan, Doudna Jennifer A
Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, United States.
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, United States.
Nucleic Acids Res. 2025 Jul 8;53(13). doi: 10.1093/nar/gkaf649.
CRISPR-Cas12a enzymes are versatile RNA-guided genome-editing tools with applications encompassing viral diagnosis, agriculture, and human therapeutics. However, their dependence on a 5'-TTTV-3' protospacer adjacent motif (PAM) next to DNA target sequences restricts Cas12a's gene targeting capability to only ∼1% of a typical genome. To mitigate this constraint, we used a bacterial-based directed evolution assay combined with rational engineering to identify variants of Lachnospiraceae bacterium Cas12a with expanded PAM recognition. The resulting Cas12a variants use a range of noncanonical PAMs while retaining recognition of the canonical 5'-TTTV-3' PAM. In particular, biochemical and cell-based assays show that the variant Flex-Cas12a utilizes 5'-NYHV-3' PAMs that expand DNA recognition sites to ∼25% of the human genome. With enhanced targeting versatility, Flex-Cas12a unlocks access to previously inaccessible genomic loci, providing new opportunities for both therapeutic and agricultural genome engineering.
CRISPR-Cas12a酶是多功能的RNA引导基因组编辑工具，其应用涵盖病毒诊断、农业和人类治疗学。然而，它们对DNA靶序列旁5'-TTTV-3'原间隔相邻基序（PAM）的依赖将Cas12a的基因靶向能力限制在典型基因组的约1%。为了减轻这一限制，我们使用基于细菌的定向进化分析结合理性工程来鉴定具有扩展PAM识别能力的毛螺菌科细菌Cas12a变体。所得的Cas12a变体使用一系列非规范PAM，同时保留对规范5'-TTTV-3' PAM的识别。特别是，生化和基于细胞的分析表明，变体Flex-Cas12a利用5'-NYHV-3' PAM，将DNA识别位点扩展到人类基因组的约25%。随着靶向通用性的增强，Flex-Cas12a开启了对以前无法访问的基因组位点的访问，为治疗和农业基因组工程提供了新机会。
