Structure-based computational engineering of saCas9 PAM requirement

saCas9 PAM 要求的基于结构的计算工程

• 批准号: 10696610
• 负责人: Xiaoqiang Huang
• 金额: $ 27.5万
• 依托单位: ATGC, INC.
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10696610
• 关键词: 2019-nCoV; ACE2; Address; Affinity; Amino Acid Sequence; Amino Acids; Apoptosis; BIRC4 gene; Binding; Businesses; CASP9 gene; COVID-19 treatment; Cells; Chimeric Proteins; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Computer Models; Computing Methodologies; Cultured Cells; DNA; Development; Engineering; Event; Generations; Genes; Genome; Guide RNA; Human; Knock-in; Licensing; Methods; Michigan; Modeling; Modification; Molecular Conformation; Mutate; Names; Noise; Nucleic Acids; Nucleotides; Peptides; Phase; Play; Process; Protein Engineering; Proteins; Relaxation; Reporting; Safety; Sampling; Side; Staphylococcus aureus; Structure; Tertiary Protein Structure; Test Result; Testing; Universities; Variant; Work; adeno-associated viral vector; clinically relevant; computational platform; design; experimental study; improved; in vivo; inhibitor; insertion/deletion mutation; nanomolar; novel; nuclease; off-target site; prototype; repaired; structural biology; tool; translational genomics

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.

摘要 SaCas 9是一种主要的基因编辑核酸酶，由于其在体内应用中的优势，其是优选的。 与spCas 9相比，其尺寸相对较小。使用saCas 9的一个限制因素是 其NNGRRT的严格PAM要求。此外，在减少saCas 9的表达方面存在有限的努力。 脱靶编辑率。在目标1中，我们提出了一种计算方法来放松saCas 9的PAM 要求。我们假设关键PAM识别（KPR） saCas 9的残基和PAM核苷酸的侧链决定了PAM的需求， 通过破坏KPR残基与DNA侧链的相互作用， 引入与DNA主链的有利的相互作用将放松PAM的要求。我们 已在初步工作中证明了将PAM要求放宽到NNNRRT的可行性。 在这里，我们将开发和优化一种计算方法UniDesign来设计saCas 9， 进一步松弛的NNNRRN PAM（rr-saCas 9 s）。在目标2中，我们提出改进rr-saCas 9 安全为代价的最近，我们报道了mispCas 9的发展，其中小尺寸（36个氨基酸）的 将HDR（同源定向修复）促进肽Brex 27融合至spCas 9。 与spCas 9相比，mispCas 9导致增加的敲入率以及减少的脱靶 插入和缺失（indel）事件。重要的是，Brex 27可以作为“即插即用” 模块来改善其他基因编辑核酸酶，只要它们的作用机制取决于 基因组双链断裂（DSB）的产生。在这里，我们将Brex 27融合到RR- saCas 9（mirr-saCas 9），并进行实验以表征mirr-saCas 9， 使用rr-saCas 9和saCas 9，重点关注PAM要求、靶向编辑功效和 脱靶率如果成功，所提出的mirr-saCas 9将为saCas 9增加有利的功能 包括：（i）由于放宽的PAM要求， （NNNRRN vs NNGRRT）;和（ii）由于在NNGRRT时增强的HDR，脱靶插入缺失率降低。 非目标站点。此外，经验证的计算模型适用于 用于PAM修饰和其它期望工程改造的其它CRISPR变体的改进。