Improved genome editing by an engineered CRISPR-Cas12a

Ma, Enbo; Chen, Kai; Shi, Honglue; Stahl, Elizabeth C.; Adler, Ben; Trinidad, Marena; Liu, Junjie; Zhou, Kaihong; Ye, Jinjuan; Doudna, Jennifer A

doi:10.1093/nar/gkac1192

DOE OSTI · 2470693

Improved genome editing by an engineered CRISPR-Cas12a

Ma, Enbo·Chen, Kai·Shi, Honglue·Stahl, Elizabeth C.·Adler, Ben·Trinidad, Marena·Liu, Junjie·Zhou, Kaihong·Ye, Jinjuan·Doudna, Jennifer A

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Abstract

CRISPR-Cas12a is an RNA-guided, programmable genome editing enzyme found within bacterial adaptive immune pathways. Unlike CRISPR-Cas9, Cas12a uses only a single catalytic site to both cleave target double-stranded DNA (dsDNA) (cis-activity) and indiscriminately degrade single-stranded DNA (ssDNA) (trans-activity). To investigate how the relative potency of cis- versus trans-DNase activity affects Cas12a-mediated genome editing, we first used structure-guided engineering to generate variants of Lachnospiraceae bacterium Cas12a that selectively disrupt trans-activity. The resulting engineered mutant with the biggest differential between cis - and trans -DNase activity in vitro showed minimal genome editing activity in human cells, motivating a second set of experiments using directed evolution to generate additional mutants with robust genome editing activity. Notably, these engineered and evolved mutants had enhanced ability to induce homology-directed repair (HDR) editing by 2–18-fold compared to wild-type Cas12a when using HDR donors containing mismatches with crRNA at the PAM-distal region. Finally, a site-specific reversion mutation produced improved Cas12a (iCas12a) variants with superior genome editing efficiency at genomic sites that are difficult to edit using wild-type Cas12a. This strategy establishes a pipeline for creating improved genome editing tools by combining structural insights with randomization and selection. The available structures of other CRISPR-Cas enzymes will enable this strategy to be applied to improve the efficacy of other genome-editing proteins.

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Ma, Enbo, Chen, Kai, Shi, Honglue, Stahl, Elizabeth C., Adler, Ben, Trinidad, Marena, Liu, Junjie, Zhou, Kaihong, Ye, Jinjuan, Doudna, Jennifer A. 2022-12-20. Improved genome editing by an engineered CRISPR-Cas12a. https://doi.org/10.1093/nar%2Fgkac1192

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