RT Journal Article
SR Electronic
T1 Suppression of unwanted CRISPR/Cas9 editing by co-administration of catalytically inactivating truncated guide RNAs
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 597849
DO 10.1101/597849
A1 John C. Rose
A1 Nicholas A. Popp
A1 Christopher D. Richardson
A1 Jason J. Stephany
A1 Julie Mathieu
A1 Cindy T. Wei
A1 Jacob E. Corn
A1 Dustin J. Maly
A1 Douglas M. Fowler
YR 2019
UL http://biorxiv.org/content/early/2019/04/03/597849.abstract
AB 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 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 novel and flexible approach for suppressing unwanted editing of both off-targets and HDR-corrected sites.