Novel methods to improve nuclease mediated homologous recombination

改进核酸酶介导的同源重组的新方法

• 批准号: 9345608
• 负责人: Jifeng Zhang
• 金额: $ 22.5万
• 依托单位: ATGC, INC.
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2019-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9345608
• 关键词: ATPase Domain; Address; Animal Model; BRCA2 gene; Binding; Biomedical Research; Biotechnology; Cell Line; Cell Therapy; Cell model; Cells; Chimeric Proteins; Clustered Regularly Interspaced Short Palindromic Repeats; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; DNA; DNA Damage; DNA Repair; Elements; Embryo; Emerging Technologies; Enhancers; Exons; Filament; Gene Targeting; Gene-Modified; Genes; Genome; Human; Knock-in; Mediating; Messenger RNA; Methods; Michigan; Modeling; Mus; Mutation; Nature; Nonhomologous DNA End Joining; Nucleoproteins; Organism; Oryctolagus cuniculus; Phase; Play; Process; Proteins; Recruitment Activity; Regenerative Medicine; Reporter; Reporting; Role; Serine; Site; Stem cells; Supplementation; Support System; System; Testing; Therapeutic; Threonine; Universities; Work; arm; base; design; direct application; experimental study; gene correction; homologous recombination; human pluripotent stem cell; improved; in vivo; inhibitor/antagonist; knockout gene; mutant; novel; nuclease; nuclease I; plasmid DNA; repaired; response; success; tool; transcription activator-like effector nucleases; treatment group; zinc finger nuclease

Abstract Gene correction therapy is one of the most important application directions in regenerative medicine. Emerging technologies such as CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR associated protein 9), Zinc Finger Nuclease (ZFN), and Transcription Activator-Like Effector Nuclease (TALEN) have enabled efficient and precise gene editing in a wide spectrum of species, and hold promises for eventually achieving gene correction/therapy in therapeutic settings. However, several major challenges remain to be addressed, including low knock-in efficiency, off-targeting effect and lack of an efficient delivery system in vivo. The present proposal focuses on the challenge of low knock-in efficiency. Recently we reported that RS-1, a homology directed repair (HDR) enhancer improves the efficiency of Cas9 or TALEN mediated knock-in in rabbit embryos. Microinjecting human RAD51 (hRAD51) mRNA to the embryos mimicked the beneficial effects of RS-1 treatment. In the present project, we propose experiments to further improve nuclease mediated HR rates. In Aim 1, we will first develop a RAD51 augmentation method to improve Cas9 mediated HR. On RAD51, Threonine 13 (T13) and Serine 14 (S14) are the two best known sites that are phosphorylated/activated in DNA repair processes. So we hypothesize that the replacement of T13 and S14 with their phosphomimetics (T13E and S14D) and the use of such mutant RAD51 mRNAs will lead to consecutively active RAD51 which leads to enhanced Cas9-mediated HR rate. BRCA2 is a key player in HR. It is recruited to processed double strand breaks (DSBs), and facilitates the assembly of RAD51. In Aim 2, we will develop a TALE and BRCA2 exon27 fusion protein (TALE-BE27) to help recruiting RAD51 at the DSB to further improve the HR rate. In Aim 3, we will validate these HR improving methods in rabbit embryos. The proposal aims to address a bottleneck problem in regenerative medicine (i.e. low knock-in efficiency). Its success will have significant impacts on the entire field, as a majority of stem cell based therapy will require targeted gene modifications.

摘要 基因矫正治疗是再生医学最重要的应用方向之一。新兴 CRISPR（clustered regularly interspaced short palindromic repeats）/Cas9 （CRISPR相关蛋白9）、锌指核酸酶（ZFN）和转录激活因子样效应物 核酸酶（TALEN）已经在广泛的物种中实现了高效和精确的基因编辑，并保持了 有望最终在治疗环境中实现基因校正/治疗。然而，几大 但仍存在一些挑战，包括敲入效率低、脱靶效应和缺乏免疫抑制剂。 有效的体内递送系统。本提案集中于低敲入效率的挑战。 最近我们报道了RS-1（一种同源定向修复（HDR）增强子）提高了Cas9的效率 或TALEN介导的敲入。将人RAD 51（hRAD 51）mRNA显微注射到胚胎中 模拟RS-1治疗的有益效果。在本项目中，我们提出了进一步的实验， 提高核酸酶介导HR速率。在目标1中，我们将首先开发一种RAD 51增强方法， 在RAD 51上，苏氨酸13（T13）和丝氨酸14（S14）是两个已知的最好的位点，它们是Cas9介导的HR。 在DNA修复过程中磷酸化/活化。所以我们假设T13和S14的替换 与它们的磷酸模拟物（T13 E和S14 D）结合，并且使用这样的突变体RAD 51 mRNA将导致 连续活跃的RAD 51导致Cas9介导的HR速率增强。BRCA 2是人力资源中的关键角色。它 被募集到加工的双链断裂（DSB），并促进RAD 51的组装。在目标2中， 将开发一种TALE和BRCA 2外显子27融合蛋白（TALE-BE 27），以帮助在DSB招募RAD 51， 进一步提高了HR。在目标3中，我们将在兔胚胎中验证这些HR改善方法。 该提案旨在解决再生医学的瓶颈问题（即低敲入效率）。其 成功将对整个领域产生重大影响，因为大多数基于干细胞的治疗将需要 定向基因修饰