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

Improved genome editing by an engineered CRISPR-Cas12a

Journal Article · Mon Dec 19 19:00:00 EST 2022 · Nucleic Acids Research

DOI:https://doi.org/10.1093/nar/gkac1192· OSTI ID:2470693

^[1]; ^[1]; ^[1]; Stahl, Elizabeth C. ^[1]; ^[1]; Trinidad, Marena ^[1]; Liu, Junjie ^[1]; Zhou, Kaihong ^[1]; Ye, Jinjuan ^[1]; Doudna, Jennifer A ^[2]

Univ. of California, Berkeley, CA (United States)
Univ. of California, Berkeley, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

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.

View Accepted Manuscript (DOE)

Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: USDOE; National Science Foundation (NSF); National Institutes of Health (NIH); National Institute of General Medical Sciences (NIGMS)

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 2470693

Journal Information:: Nucleic Acids Research, Journal Name: Nucleic Acids Research Journal Issue: 22 Vol. 50; ISSN 0305-1048

Publisher:: Oxford University PressCopyright Statement

Country of Publication:: United States

Language:: English

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