{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,22]],"date-time":"2026-10-22T17:32:23Z","timestamp":1792690343779,"version":"4.5.0"},"reference-count":39,"publisher":"Oxford University Press (OUP)","issue":"22","license":[{"start":{"date-parts":[[2022,12,20]],"date-time":"2022-12-20T00:00:00Z","timestamp":1671494400000},"content-version":"vor","delay-in-days":11,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000001","name":"National Science Foundation","doi-asserted-by":"publisher","award":["1817593"],"award-info":[{"award-number":["1817593"]}],"id":[{"id":"10.13039\/100000001","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["RM1HG009490"],"award-info":[{"award-number":["RM1HG009490"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000057","name":"National Institute of General Medical Sciences","doi-asserted-by":"publisher","award":["F32GM140637-01"],"award-info":[{"award-number":["F32GM140637-01"]}],"id":[{"id":"10.13039\/100000057","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2022,12,9]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>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\u201318-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.<\/jats:p>","DOI":"10.1093\/nar\/gkac1192","type":"journal-article","created":{"date-parts":[[2022,11,29]],"date-time":"2022-11-29T17:28:57Z","timestamp":1669742937000},"page":"12689-12701","source":"Crossref","is-referenced-by-count":113,"title":["Improved genome editing by an engineered CRISPR-Cas12a"],"prefix":"10.1093","volume":"50","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4374-796X","authenticated-orcid":false,"given":"Enbo","family":"Ma","sequence":"first","affiliation":[{"name":"Innovative Genomics Institute, University of California , Berkeley , CA \u00a094720, USA"},{"name":"Department of Molecular and Cell Biology, University of California , Berkeley , CA \u00a094720, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3325-3536","authenticated-orcid":false,"given":"Kai","family":"Chen","sequence":"additional","affiliation":[{"name":"Innovative Genomics Institute, University of California , Berkeley , CA \u00a094720, USA"},{"name":"Department of Molecular and Cell Biology, University of California , Berkeley , CA \u00a094720, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3847-1652","authenticated-orcid":false,"given":"Honglue","family":"Shi","sequence":"additional","affiliation":[{"name":"Innovative Genomics Institute, University of California , Berkeley , CA \u00a094720, USA"},{"name":"California Institute for Quantitative Biosciences (QB3), University of California , Berkeley , CA \u00a094720, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elizabeth C","family":"Stahl","sequence":"additional","affiliation":[{"name":"Innovative Genomics Institute, University of California , Berkeley , CA \u00a094720, USA"},{"name":"California Institute for Quantitative Biosciences (QB3), University of California , Berkeley , CA \u00a094720, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7488-3040","authenticated-orcid":false,"given":"Ben","family":"Adler","sequence":"additional","affiliation":[{"name":"Innovative Genomics Institute, University of California , Berkeley , CA \u00a094720, USA"},{"name":"Department of Molecular and Cell Biology, University of California , Berkeley , CA \u00a094720, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marena","family":"Trinidad","sequence":"additional","affiliation":[{"name":"Innovative Genomics Institute, University of California , Berkeley , CA \u00a094720, USA"},{"name":"Department of Molecular and Cell Biology, University of California , Berkeley , CA \u00a094720, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Junjie","family":"Liu","sequence":"additional","affiliation":[{"name":"Innovative Genomics Institute, University of California , Berkeley , CA \u00a094720, USA"},{"name":"Department of Molecular and Cell Biology, University of California , Berkeley , CA \u00a094720, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kaihong","family":"Zhou","sequence":"additional","affiliation":[{"name":"Howard Hughes Medical Institute, University of California , Berkeley , CA \u00a094720, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jinjuan","family":"Ye","sequence":"additional","affiliation":[{"name":"Howard Hughes Medical Institute, University of California , Berkeley , CA \u00a094720, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jennifer\u00a0A","family":"Doudna","sequence":"additional","affiliation":[{"name":"Innovative Genomics Institute, University of California , Berkeley , CA \u00a094720, USA"},{"name":"Department of Molecular and Cell Biology, University of California , Berkeley , CA \u00a094720, USA"},{"name":"California Institute for Quantitative Biosciences (QB3), University of California , Berkeley , CA \u00a094720, USA"},{"name":"Howard Hughes Medical Institute, University of California , Berkeley , CA \u00a094720, USA"},{"name":"Department of Chemistry, University of California , Berkeley , CA , USA"},{"name":"MBIB Division, Lawrence Berkeley National Laboratory , Berkeley , CA \u00a094720, USA"},{"name":"Gladstone Institutes, University of California , San Francisco , CA \u00a094114, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2022,12,20]]},"reference":[{"key":"2023052317251306000_B1","doi-asserted-by":"crossref","first-page":"209","DOI":"10.1146\/annurev-micro-091014-104441","article-title":"CRISPR-Cas: new tools for genetic manipulations from bacterial immunity systems","volume":"69","author":"Jiang","year":"2015","journal-title":"Annu. 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