Novel methods to improve nuclease mediated homologous recombination

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(成簇的规则间隔短回文重复序列)/Cas9等技术 (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)在广泛的物种中实现了高效和精确的基因编辑，并持有 有望最终在治疗环境中实现基因纠正/治疗。然而，几个主要的 仍有挑战需要解决，包括敲入效率低、脱离目标效应和缺乏 体内高效的递送系统。本提案的重点是敲入效率低的挑战。 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介导的兔胚胎基因敲除。人RAD51(HRAD51)基因的胚胎显微注射 模拟RS-1处理的有益效果。在本项目中，我们建议进行实验以进一步 提高核酸酶介导的心率。在目标1中，我们将首先开发一种RAD51增强方法来改进 Cas9介导的HR。在RAD51上，苏氨酸13(T13)和丝氨酸14(S14)是最知名的两个位点 在DNA修复过程中被磷酸化/激活。所以我们假设T13和S14的替换 与他们的磷仿制(T13E和S14D)和使用这种突变的RAD51 mRNAs将导致 持续激活RAD51，导致Cas9介导的心率增加。BRCA2是人力资源领域的关键人物。它 被招募到处理双链断裂(DSB)，并促进RAD51的组装。在目标2中，我们 将开发一种TALE和BRCA2外显子27融合蛋白(TALE-BE27)，以帮助在DSB招募RAD51 进一步提高人力资源率。在目标3中，我们将在兔胚胎上验证这些HR改良方法。 该提案旨在解决再生医学中的一个瓶颈问题(即低敲入效率)。它的 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疗法都需要 有针对性的基因改造。

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.