Target-matched increased fidelity SpCas9 variants can edit any target without genome-wide off-target effects

Abstract

Streptococcus pyogenes Cas9 (SpCas9) has been employed as a genome engineering tool with a promising potential within therapeutics. However, its off-target effects pose a major limitation 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.

SuperFi-Cas9 exhibits remarkable fidelity but severely reduced activity yet works effectively with ABE8e

“…To have meaningful results for these assays we selected targets on which SuperFi (similar to these higher fidelity IFNs) is expected to exhibit a reasonable on-target activity. This rationale is based on our recent study 25 showing that targets have different cleavability, and low activity/high-fidelity IFNs can cleave only high cleavability targets. SuperFi exhibited higher specificity than Blackjack-HypaR-and evoSpCas9 on 3 and 2 of the 4 examined targets, respectively (Fig.…”

“…In all experiments the following plasmids were used for SpCas9, base and prime editor variant expression (Addgene# provided): pX330-Flag-WT_SpCas9 (without sgRNA; with silent mutations) (#126753), pX330-Flag-SpCas9-HF1 (without sgRNA; with silent mutations) (#126755), pX330-Flag-HypaSpCas9 (without sgRNA; with silent mutations) (#126756), pX330-Flag-evoSpCas9 (without sgRNA; with silent mutations) (#126758) 11 , B-HypaR-SpCas9 (#126764) 25 , pAT9676-ABE (#162997) for ABE7, pAT9749-dABE (#162998) for deadABE7, pAT9993-Hypa-ABE (#163001) for Hypa-ABE7; pAT9675-CBE (#163007) for CBE; pAT15069-HeF-CBE (#163007) for HeF-CBE; pAT15482_ABE8e (#174120) for ABE8e; pAT15488_HeF-ABE8e (#174126) for HeF-ABE8e 32 and pCMV-PEmax 35 (#174820) for PEmax expression. pET-Cas9-NLS-6xHis (Addgene #62933) 39 , pET-SpCas9-HF1-NLS-6xHis, pET-SuperFi-Cas9-NLS-6xHis were used for WT, -HF1 and SuperFi-SpCas9 bacterial expression, respectively.…”