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.

化脓链球菌Cas9（Streptococcus pyogenes Cas9, SpCas9）已被用作基因组编辑工具，在治疗领域展现出极具前景的应用潜力。然而，其脱靶效应给对特异性要求较高的应用带来了重大安全隐患。迄今为止为缓解该问题所开发的各类方法，包括各类高保真（high-fidelity）SpCas9变体，仅能在占比相对较少的无明确可检测脱靶效应的靶标序列上实现高效编辑。针对这一难题，本研究揭示了靶标序列一种颇为出人意料的切割活性排序，以及一条调控高保真SpCas9变体的靶标切割与脱靶切割的规则，但该规则并不适用于SpCas9-NG或xCas9。受此规则影响，每一条靶标序列都需要匹配对应保真度的最优变体，才能在无明确可检测脱靶效应的前提下实现高效切割。基于这一发现，我们开发了一套覆盖宽泛保真度范围的扩展型高保真变体集，其变体间的保真度差异足够细微，足以针对任意靶标序列提供最优变体，而无需考虑其切割活性排序。我们已证实，即便是此前研究中难以实现高效编辑的靶标，本方法也能实现兼具最高特异性的高效编辑。