Institute of Enzymology, Research Centre for Natural Sciences of the Hungarian Academy of Sciences, Budapest, Hungary.
Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary.
Genome Biol. 2017 Oct 6;18(1):190. doi: 10.1186/s13059-017-1318-8.
The propensity for off-target activity of Streptococcus pyogenes Cas9 (SpCas9) has been considerably decreased by rationally engineered variants with increased fidelity (eSpCas9; SpCas9-HF1). However, a subset of targets still generate considerable off-target effects. To deal specifically with these targets, we generated new "Highly enhanced Fidelity" nuclease variants (HeFSpCas9s) containing mutations from both eSpCas9 and SpCas9-HF1 and examined these improved nuclease variants side by side to decipher the factors that affect their specificities and to determine the optimal nuclease for applications sensitive to off-target effects.
These three increased-fidelity nucleases can routinely be used only with perfectly matching 20-nucleotide-long spacers, a matching 5' G extension being more detrimental to their activities than a mismatching one. HeFSpCas9 exhibit substantially improved specificity for those targets for which eSpCas9 and SpCas9-HF1 have higher off-target propensity. The targets can also be ranked by their cleavability and off-target effects manifested by the increased fidelity nucleases. Furthermore, we show that the mutations in these variants may diminish the cleavage, but not the DNA-binding, of SpCas9s.
No single nuclease variant shows generally superior fidelity; instead, for highest specificity cleavage, each target needs to be matched with an appropriate high-fidelity nuclease. We provide here a framework for generating new nuclease variants for targets that currently have no matching optimal nuclease, and offer a simple means for identifying the optimal nuclease for targets in the absence of accurate target-ranking prediction tools.
通过合理设计的提高保真度的变体（eSpCas9；SpCas9-HF1），大大降低了酿脓链球菌 Cas9（SpCas9）的脱靶活性。然而，仍有一部分靶标会产生相当大的脱靶效应。为了专门处理这些靶标，我们生成了新的“高保真度”核酸酶变体（HeFSpCas9s），它们包含了来自 eSpCas9 和 SpCas9-HF1 的突变，并对这些改进的核酸酶变体进行了并排研究，以破译影响它们特异性的因素，并确定最适合对脱靶效应敏感的应用的核酸酶。
这三种提高保真度的核酸酶通常只能与完全匹配的 20 个核苷酸长的间隔物一起使用，与不匹配的碱基相比，5'G 延伸对其活性的损害更大。HeFSpCas9 对那些 eSpCas9 和 SpCas9-HF1 具有更高脱靶倾向的靶标具有显著提高的特异性。还可以根据这些增加保真度的核酸酶的靶标切割率和脱靶效应对靶标进行排序。此外，我们表明，这些变体中的突变可能会降低 SpCas9s 的切割效率，但不会降低其 DNA 结合能力。
没有单一的核酸酶变体表现出普遍优越的保真度；相反，对于最高特异性切割，每个靶标都需要与适当的高保真度核酸酶相匹配。我们在这里提供了一个针对目前没有匹配最佳核酸酶的靶标生成新核酸酶变体的框架，并提供了一种简单的方法来识别在没有准确靶标排序预测工具的情况下靶标的最佳核酸酶。
