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bioRxiv · 10.1101/2022.09.02.506401

Improved Genome Editing by an Engineered CRISPR-Cas12a

Ma, E.·Chen, K.·Shi, H.·Stahl, E. C.·Adler, B. A.·Liu, J.·Zhou, K.·Ye, J.·Doudna, J. 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 (LbCas12a) 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 depending on the genomic locus. Finally, we found that 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 of coupled rational engineering and directed evolution establishes a pipeline for creating improved genome editing tools by combining structural insights with randomization and selection. The availability of experimental and predicted 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, E., Chen, K., Shi, H., Stahl, E. C., Adler, B. A., Liu, J., Zhou, K., Ye, J., Doudna, J. A.. 2022-09-02. Improved Genome Editing by an Engineered CRISPR-Cas12a. https://doi.org/10.1101/2022.09.02.506401

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