Novel methods to improve nuclease mediated homologous recombination

项目摘要 2020年诺贝尔化学奖授予Emmanuelle Charpentier和Jennifer Doudna博士， 他们开发了革命性的基因编辑工具CRISPR/Cas9。它允许对基因组进行精确编辑 席卷了生命科学领域。它有无数的应用。科学家们希望利用它来开发 用于治疗人类遗传疾病的治疗策略。然而，仍然有几个障碍需要 在实现临床应用之前，主要问题之一是不期望的插入或 在脱靶位点，以及在靶位点的缺失（indel）事件，其中目标是引入 精确的校正或突变。另一个有待进一步改进的方面是， 当使用大尺寸供体片段时，其通常低于1%。在本STTR的第一阶段， 在第一个项目中，我们通过融合BRCA 2外显子编码的36个氨基酸长的肽来改造spCas 9蛋白， 27（Brex 27），据报道其结合RAD 51以增强同源定向修复（HDR）。我们命名 这种新的变体是精细整合spCas 9（mi-spCas 9），它具有以下独特的组合： 期望的特征，包括提高敲入率，减少不期望的脱靶事件，以及减少 不期望的靶向插入或缺失（indel）事件，提供了“一石三鸟”的工具， 基因编辑在这个第二阶段项目中，我们建议研究进一步工程化Brex 27，开发Adeno 相关病毒（AAV）友好的mi-saCas 9，并展示其临床相关应用。具体而言，i） 在目标1中，我们将开发下一代mi-Cas9（mi-spCas 9-v2），以接近完全消除 ii）在目标2中，我们将开发和优化一种对AAV友好的方法， iii）在目标3中，我们将证明mi-Cas9在临床上的优势; 相关应用。我们预期mi-spCas 9-v2和mi-saCas 9导致基因敲除的多倍增加。 在率和接近于零的目标和脱靶的indel率。完成拟议的研究将提高 基因组编辑的安全性和有效性，推动新的mi-Cas9工具更接近新兴的数十亿- 基础研究和治疗的美元市场。

Project Summary The 2020 Nobel Prize in Chemistry was awarded to Drs. Emmanuelle Charpentier and Jennifer Doudna for their development of a revolutionary gene-editing tool, CRISPR/Cas9. It allows precise edits to the genome and has swept through the life science field. It has countless applications. Scientists hope to use it to develop therapeutic strategies for treating human genetic diseases. However, there are still several hurdles that need to be overcome before achieving clinical applications. One of the major concerns is the undesirable insertion or deletion (indel) events at off-target sites, as well as at the on-target site where the goal is to introduction precise correction or mutation. Another aspect that remains to be further improved is the low efficiency of knockin (KI) when a large size donor fragment is used, which is often below 1%. In Phase I of this STTR project, we engineered the spCas9 protein by fusing a 36 amino acid long peptide encoded by BRCA2 Exon 27 (Brex27), which has been reported to bind RAD51 to enhance homology-directed repair (HDR). We named this new variant the meticulous integration spCas9 (mi-spCas9), which possesses a unique combination of desirable features, including improving knock-in rates, reducing undesirable off-target events, and reducing undesirable on-target insertion or deletion (indel) events, providing a “one small stone for three birds” tool in gene editing. In this Phase II project, we propose studies to further engineer Brex27, to develop an Adeno Associated Virus (AAV) friendly mi-saCas9 and demonstrate its clinically relevant applications. Specifically, i) in Aim 1, we will develop next-generation mi-Cas9s (mi-spCas9-v2) towards near-complete abolishment of undesirable on-target and off-target indels; ii) in Aim 2, we will develop and optimize an AAV-friendly mi- saCas9 for in vivo gene editing; iii) in Aim 3, we will demonstrate the advantages of mi-Cas9s in clinically relevant applications. We expect that mi-spCas9-v2 and mi-saCas9 lead to a multi-fold increase in gene knock- in rates and close to zero on-target and off-target indel rates. Completion of the proposed studies will enhance the safety and efficacy of genome editing, propelling novel mi-Cas9 tools closer to an emerging multi-billion- dollar market of basic research and therapeutic.