Directed evolution expands CRISPR–Cas12a genome-editing capacity

Journal Article · Mon Jul 07 20:00:00 EDT 2025 · Nucleic Acids Research (NAR)

DOI:https://doi.org/10.1093/nar/gkaf649· OSTI ID:2583302

Ma, Enbo; Chen, Kai; Shi, Honglue; Wasko, Kevin M; Esain-Garcia, Isabel; Trinidad, Marena I; Zhou, Kaihong; Ye, Jinjuan; Doudna, Jennifer A

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.

• Research Organization: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
• Sponsoring Organization: US Department of Energy; USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23), Biological Systems Science Division (SC-23.2 )
• Grant/Contract Number: AC02-05CH11231
• OSTI ID: 2583302
• Journal Information: Nucleic Acids Research (NAR), Journal Name: Nucleic Acids Research (NAR) Journal Issue: 13 Vol. 53
• Country of Publication: United States
• Language: English