Directed evolution expands CRISPR-Cas12a genome editing capacity

Abstract

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 Cas12as 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 (LbCas12a) with expanded PAM recognition. The resulting Cas12a variants use a range of non-canonical 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.