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.

抽象的 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，这是使用 saCas9 的一个限制因素。 NNGRRT 的严格 PAM 要求 此外，减少 saCas9 的努力有限。 在目标 1 中，我们提出了一种放松 saCas9 的 PAM 的计算方法。 我们认为关键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， 通过破坏 KPR 残基与 DNA 侧链的相互作用来突变 KPR 残基，同时 引入与 DNA 主链的有利相互作用将放宽 PAM 要求。 在前期工作中论证了向 NNNRRT 放宽 PAM 要求的可行性。 在这里，我们将开发和优化一种计算方法 UniDesign 来设计 saCas9s 进一步放宽 NNNRRN PAM (rr-saCas9s) 在目标 2 中，我们建议改进 rr-saCas9。 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 提高了敲入率并减少了脱靶率 重要的是，Brex27 可以用作“即插即用”事件。 改进其他基因编辑核酸酶的模块，只要它们的作用机制取决于 在这里，我们将 Brex27 与 rr- 融合。 saCas9（mirr-saCas9），并进行实验来表征mirr-saCas9，进行比较 与 rr-saCas9 和 saCas9 一起，重点关注 PAM 要求、靶向编辑功效以及 所提出的 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，可靶向序列增加了 16 倍 （NNNRRN 与 NNGRRT）；以及 (ii) 由于 HDR 增强而降低了脱靶插入缺失率 此外，经过验证的计算模型适用于 改进其他 CRISPR 变体以进行 PAM 修饰和其他所需的工程。