Structure-based computational engineering of saCas9 PAM requirement

摘要 SaCas9是一种主要的基因编辑核酸酶，由于其 与spCas9相比，尺寸相对较小。使用saCas9的一个限制因素是 NNGRRT对其严格的PAM要求。此外，减少saCas9的努力有限，S 偏离目标的编辑率。在目标1中，我们提出了一种松弛saCas9的S PAM的计算方法 要求。我们假设关键的PAM识别(KPR)之间的相互作用 SaCas9的残基和PAM核苷酸的侧链决定了PAM的需求量， 通过破坏KPR残基与DNA侧链的相互作用而突变KPR残基 引入与DNA主链的有利相互作用将放宽对PAM的要求。我们 在前期工作中论证了将PAM要求放宽到NNNNRRT的可行性。 在这里，我们将开发和优化一种计算方法UniDesign来使用 进一步松弛NNNRRN PAM(rr-saCas9)。在目标2中，我们建议改进rr-saCas9的S 安全。最近我们报道了mispCas9的开发，其中一个小尺寸(36个氨基酸) 酸)HDR(同源定向修复)促进肽Brex27与spCas9融合。 与spCas9相比，mispCas9导致了更高的敲击率以及更少的偏离目标 插入和删除(INDel)事件。重要的是，英国退欧27可以作为一种“即插即用”方式 模块来改进其他基因编辑核酸酶，只要它们的作用机制取决于 关于基因组双链断裂(DSB)的产生。在这里，我们将把英国退欧27与rr- SaCas9(Mirr-saCas9)，并进行对比实验来表征Mirr-saCas9 使用rr-saCas9和saCas9，重点关注PAM需求、目标编辑效率和 偏离目标的利率。拟议的Mirr-saCas9如果成功，将为saCas9添加有利的功能 包括：(I)由于放宽了PAM要求，目标序列增加了16倍 (NNNRRN与NNGRRT)；以及(Ii)由于在 偏离目标的地点。此外，验证后的计算模型适用于 其他CRISPR变体的改进，用于PAM的改性和其他所需的工程。

ABSTRACT SaCas9 is a major gene editing nuclease that is preferred for in vivo applications thanks to its relatively smaller size compared to that of spCas9. One limiting factor for the use of saCas9 is its strict PAM requirement of NNGRRT. In addition, there are limited efforts to reduce saCas9’s off-target editing rates. In Aim 1, we propose a computational approach to relax saCas9’s PAM requirement. We hypothesize that the interactions between key PAM recognition (KPR) residues of saCas9 and the side chains of PAM nucleotides determine the PAM requirement, and that mutating KPR residues by destroying their interactions with the DNA side chain while introducing favorable interactions with the DNA main chain will relax the PAM requirement. We have demonstrated the feasibility to relax the PAM requirement to NNNRRT in preliminary work. Here we will develop and optimize a computational method UniDesign to engineer saCas9s with further relaxed NNNRRN PAM (rr-saCas9s). In Aim 2, we propose to improve rr-saCas9’s safety. Recently we reported the development of mispCas9, in which a small size (36 amino acids) HDR (homology-directed repair)-promoting peptide Brex27 was fused to spCas9. Compared to spCas9, mispCas9 leads to increased knock-in rates as well as reduced off-target insertion and deletion (indel) events. Importantly, Brex27 can be used as a “plug and play” module to improve other gene-editing nucleases as long as their mechanism of action depends on the generation of double-strand breaks (DSB) of the genome. Here we will fuse Brex27 to rr- saCas9 (mirr-saCas9), and conduct experiments to characterize mirr-saCas9, in comparison with rr-saCas9 and saCas9, with a focus on PAM requirement, on-target editing efficacy, and off-target rates. The proposed mirr-saCas9, if successful, will add favorable features to saCas9 including: (i) 16 times more targetable sequences as a result of the relaxed PAM requirement (NNNRRN vs NNGRRT); and (ii) reduced off-target indel rates as a result of enhanced HDR at off-target sites. Furthermore, the validated computational model is adaptable for the improvement of other CRISPR variants for PAM modification and other desirable engineering.