Rapid DNA unwinding accelerates genome editing by engineered CRISPR-Cas9

Eggers, Amy R.; Chen, Kai; Soczek, Katarzyna M.; Tuck, Owen T.; Doherty, Erin E.; Xu, Bryant; Trinidad, Marena I.; Thornton, Brittney W.; Yoon, Peter H.; Doudna, Jennifer A.

doi:10.1016/j.cell.2024.04.031

Title: Rapid DNA unwinding accelerates genome editing by engineered CRISPR-Cas9

Journal Article · 22 May 2024 · Cell

DOI: https://doi.org/10.1016/j.cell.2024.04.031 · OSTI ID:2447084

Eggers, Amy R. ^[1]; Chen, Kai ^[1]; Soczek, Katarzyna M. ^[1]; Tuck, Owen T. ^[2]; Doherty, Erin E. ^[2]; Xu, Bryant ^[1]; Trinidad, Marena I. ^[2]; Thornton, Brittney W. ^[1]; Yoon, Peter H. ^[1];

^[3]

University of California, Berkeley, CA (United States); Innovative Genomics Institute, Berkeley, CA (United States)
Innovative Genomics Institute, Berkeley, CA (United States); University of California, Berkeley, CA (United States)
University of California, Berkeley, CA (United States); Innovative Genomics Institute, Berkeley, CA (United States); Gladstone Institutes, San Francisco, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Thermostable clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas9) enzymes could improve genome-editing efficiency and delivery due to extended protein lifetimes. However, initial experimentation demonstrated Geobacillus stearothermophilus Cas9 (GeoCas9) to be virtually inactive when used in cultured human cells. Laboratory-evolved variants of GeoCas9 overcome this natural limitation by acquiring mutations in the wedge (WED) domain that produce >100-fold-higher genome-editing levels. Cryoelectron microscopy (cryo-EM) structures of the wild-type and improved GeoCas9 (iGeoCas9) enzymes reveal extended contacts between the WED domain of iGeoCas9 and DNA substrates. Biochemical analysis shows that iGeoCas9 accelerates DNA unwinding to capture substrates under the magnesium-restricted conditions typical of mammalian but not bacterial cells. These findings enabled rational engineering of other Cas9 orthologs to enhance genome-editing levels, pointing to a general strategy for editing enzyme improvement. Together, these results uncover a new role for the Cas9 WED domain in DNA unwinding and demonstrate how accelerated target unwinding dramatically improves Cas9-induced genome-editing activity.

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

Sponsoring Organization:: Apple Tree Partners (ATP); National Institutes of Health (NIH); National Science Foundation (NSF); USDOE Office of Science (SC)

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 2447084

Journal Information:: Cell, Journal Name: Cell Journal Issue: 13 Vol. 187; ISSN 0092-8674

Publisher:: ElsevierCopyright Statement

Country of Publication:: United States

Language:: English

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Title: Rapid DNA unwinding accelerates genome editing by engineered CRISPR-Cas9

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