CRISPR–Cas9 nucleases are now becoming widely used for genome-editing, but can induce unwanted off-target mutations, i.e. there easily may be other genes targeted unintentionally that one would not want targeted. Existing strategies for decreasing genome-wide off-target effects of the widely used Streptococcus pyogenes Cas9 (SpCas9) are imperfect, possessing only partial or unproven efficacies and other limitations that constrain their use.
In the attached article and editorial, authors describe SpCas9-HF1, a high-fidelity variant harboring alterations designed to reduce non-specific DNA splicing. SpCas9-HF1
retains on-target activities comparable to wild-type SpCas9 with >85% of single-guide RNAs (sgRNAs) tested in human cells. Notably, with sgRNAs targeted to standard non-repetitive sequences, SpCas9-HF1 rendered all, or almost all, off-target events undetectable by genome-wide break-capture and targeted-sequencing methods. Even for atypical, repetitive target sites, the vast majority of off-target mutations induced by wild-type SpCas9 were not detected with SpCas9-HF1.
With its exceptional precision, SpCas9-HF1 provides an attractive alternative to wild-type SpCas9 for research and therapeutic applications. More broadly, the authors suggest a general strategy for optimizing genome-wide specificities of other CRISPR-RNA-guided nucleases in the future.
Nature 28 Jan 2016; 529: 490–495 and editorial (pp 408–409)