A cleavage rule for selection of increased-fidelity SpCas9 variants with high efficiency and no detectable off-targets.
Streptococcus pyogenes Cas9 (SpCas9) has been employed as a genome engineering tool with a promising potential within therapeutics. However, its off-target effects present major safety concerns for applications requiring high specificity. Approaches developed to date to mitigate this effect, including any of the increased fidelity (i.e., high-fidelity) SpCas9 variants, only provide efficient editing on a relatively small fraction of targets without detectable off-targets. Upon addressing this problem, we have revealed a rather unexpected cleavability ranking of target sequences, and a cleavage rule that governs the on-target and off-target cleavage of increased fidelity SpCas9 variants but not that of SpCas9-NG or xCas9. As a consequence of this rule, each target needs a matching-fidelity, optimal variant for efficient cleavage without detectable off-target effects. By exploiting this finding, we have developed an extended set of increased fidelity variants spanning across a wide range, with differences in fidelity small enough to comprise an optimal variant for any target, irrespective of its cleavability ranking. We have demonstrated efficient editing with maximum specificity even on those targets that have been challenged but failed in previous studies.