Suppression of unwanted CRISPR-Cas9 editing by co-administration of catalytically inactivating truncated guide RNAs

John C. Rose (), Nicholas A. Popp, Christopher D. Richardson, Jason J. Stephany, Julie Mathieu, Cindy T. Wei, Jacob E. Corn, Dustin J. Maly () and Douglas M. Fowler ()
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John C. Rose: University of Washington
Nicholas A. Popp: University of Washington
Christopher D. Richardson: University of California, Berkeley
Jason J. Stephany: University of Washington
Julie Mathieu: Institute for Stem Cell and Regenerative Medicine, University of Washington
Cindy T. Wei: University of Washington
Jacob E. Corn: University of California, Berkeley
Dustin J. Maly: University of Washington
Douglas M. Fowler: University of Washington

Nature Communications, 2020, vol. 11, issue 1, 1-11

Abstract: Abstract CRISPR-Cas9 nucleases are powerful genome engineering tools, but unwanted cleavage at off-target and previously edited sites remains a major concern. Numerous strategies to reduce unwanted cleavage have been devised, but all are imperfect. Here, we report that off-target sites can be shielded from the active Cas9•single guide RNA (sgRNA) complex through the co-administration of dead-RNAs (dRNAs), truncated guide RNAs that direct Cas9 binding but not cleavage. dRNAs can effectively suppress a wide-range of off-targets with minimal optimization while preserving on-target editing, and they can be multiplexed to suppress several off-targets simultaneously. dRNAs can be combined with high-specificity Cas9 variants, which often do not eliminate all unwanted editing. Moreover, dRNAs can prevent cleavage of homology-directed repair (HDR)-corrected sites, facilitating scarless editing by eliminating the need for blocking mutations. Thus, we enable precise genome editing by establishing a flexible approach for suppressing unwanted editing of both off-targets and HDR-corrected sites.

Date: 2020
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DOI: 10.1038/s41467-020-16542-9

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