Precise and broad scope genome editing based on high-specificity Cas9 nickases

RNA-guided nucleases (RGNs) based on CRISPR systems permit installing short and large edits within eukaryotic genomes. However, precise genome editing is often hindered due to nuclease off-target activities and the multiple-copy character of the vast majority of chromosomal sequences. Dual nicking RGNs and high-specificity RGNs both exhibit low off-target activities. Here, we report that high-specificity Cas9 nucleases are convertible into nicking Cas9(D10A) variants whose precision is superior to that of the commonly used Cas9(D10A )nickase. Dual nicking RGNs based on a selected group of these Cas9(D10A) variants can yield gene knockouts and gene knock-ins at frequencies similar to or higher than those achieved by their conventional counterparts. Moreover, high-specificity dual nicking RGNs are capable of distinguishing highly similar sequences by 'tiptoeing' over pre-existing single base-pair polymorphisms. Finally, high-specificity RNA-guided nicking complexes generally preserve genomic integrity, as demonstrated by unbiased genome-wide high-throughput sequencing assays. Thus, in addition to substantially enlarging the Cas9 nickase toolkit, we demonstrate the feasibility in expanding the range and precision of DNA knockout and knock-in procedures. The herein introduced tools and multi-tier high-specificity genome editing strategies might be particularly beneficial whenever predictability and/or safety of genetic manipulations are paramount.

Automated summary

The study demonstrates that high-specificity Cas9 nucleases can be converted into nicking Cas9(D10A) variants with superior precision compared to the commonly used Cas9(D10A)nickase. Dual nicking RGNs based on these Cas9(D10A) variants can achieve gene knockouts and knock-ins at frequencies similar to or higher than conventional methods. Additionally, the high-specificity dual nicking RGNs are able to distinguish highly similar sequences and preserve genomic integrity.