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

Nat Commun. 2020 Jun 1;11(1):2697. doi: 10.1038/s41467-020-16542-9.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Base Sequence
  • Binding Sites / genetics
  • Biocatalysis
  • CRISPR-Cas Systems*
  • Cell Line, Tumor
  • Cells, Cultured
  • DNA Repair
  • Gene Editing / methods*
  • HEK293 Cells
  • Humans
  • Mice
  • Models, Genetic
  • Mutation*
  • RNA, Guide, CRISPR-Cas Systems / genetics*
  • RNA, Guide, CRISPR-Cas Systems / metabolism

Substances

  • RNA, Guide, CRISPR-Cas Systems