Characterizing and improving homology-directed repair in CRISPR/Cas-mediated genome editing

背景CRISPR/Cas基因组编辑正在给生物医学研究带来革命性的变化。RNA引导的Cas9核酸酶可以诱导DNA中的双链断裂(DSB)，通过插入或缺失DNA(INDELs)而导致靶基因的敲除。然而，基因组编辑的大多数应用要求在基因组中引入特定的突变，而不是INDELS。这可以通过使用包含所需突变的模板DNA的同源定向修复(HDR)来实现。然而，在目前的方案中，HDR介导的诱变的产率很低，需要进行选择/纯化步骤。总体目标这项研究计划旨在产生知识和方法，以实现有效的，无疤痕的基因组修改。具体目标和方法1.建立一个基于流式细胞仪的系统，用于监测人类细胞的功能性基因修饰。这将通过建立细胞模型来实现，在该模型中，表面蛋白--表皮生长因子受体(EGFR)被HDR突变，从而导致用流式细胞术进行检测。因此，HDR的效率是通过表达该标记的细胞的百分比来反映的。2.优化HDR介导的修饰。我们将系统地比较几种类型的供体(模板)DNA和在整合不足的慢病毒载体背景下的递送选项的效率。特别是，我们将分析它们的最佳长度和方向，并确定相对于CRISPR/CAS组件的最佳交付时间。3.通过过表达DNA修复蛋白来改善HDR。为了提高HDR的效率，我们将在高表达CRISPR/Cas组件的同时，过表达参与HDR修复DSB的人和非人蛋白。4.通过抑制非同源末端连接(NHEJ)修复改善HDR。NHEJ和HDR竞争DSB的修复，有证据表明抑制NHEJ可以增强HDR。因此，在EGFR模型中，我们将使用NHEJ蛋白DNA-PK的药理抑制剂以及53BP1的显性阴性版本来增加HDR的产量。5.脱靶编辑和DNA不稳定性分析。我们将对修改后的细胞中可能的非靶点位置进行排序，以分析本项目中开发的方案是否会导致非靶点编辑的增加。将通过核型分析来分析基因组的不稳定性。这项研究计划将显著提高我们对人类发展研究成功的决定因素的认识。它将导致新的工具，帮助将基因组修改从基础遗传学转移到农业和人类健康的实际应用中。

BACKGROUND CRISPR/Cas genome editing is revolutionizing biomedical research. RNA-guided Cas9 nuclease can induce targeted double strand breaks (DSBs) in DNA, leading to knockout of the targeted locus by insertion or deletion of DNA (indels). Nevertheless, most applications of genome editing call for the introduction of specific mutations in the genome, rather than indels. This can be achieved by homology-directed repair (HDR) using a template DNA containing the desired mutation. However, the yields of HDR-mediated mutagenesis in current protocols are low, necessitating steps of selection/purification. GENERAL OBJECTIVE This research program aims at generating knowledge and methods toward achieving efficient, scarless genome modification. SPECIFIC OBJECTIVES AND METHODOLOGIES 1. To create a FACS-based system for monitoring functional gene modification in human cells. This will be achieved by the establishment of cellular models in which a surface protein, epidermal growth factor receptor (EGFR), is mutated by HDR, leading to detection by flow cytometry. Hence, the efficiency of HDR is reflected by the % of cells expressing the marker. 2. To optimize HDR-mediated modification. We will methodically compare the efficiencies of several types of donor (template) DNAs and delivery options in the context of integration-deficient lentiviral vectors. In particular, we will analyze their optimal length and orientation, and determine the optimal timing of delivery relative to the CRISPR/Cas components. 3. Improving HDR by over-expression of DNA repair proteins. To increase the efficiency of HDR, we will over-express human and nonhuman proteins involved in repair of DSBs by HDR, concomitantly with the expression of the CRISPR/Cas components. 4. Improving HDR by inhibition of non-homologous end-joining (NHEJ) repair. NHEJ and HDR compete for the repair of DSBs, and there is evidence that inhibition of NHEJ can enhance HDR. Thus, we will use pharmacological inhibitors of the NHEJ protein DNA-PK as well as a dominant negative version of 53BP1 to increase HDR yields in the EGFR model. 5. Analysis of off-target editing and DNA instability. We will sequence likely off-target sites in the modified cells to analyze whether the protocols developed in this project lead to an increase in off-target editing. Genome instability will be analyzed by karyotyping. IMPACT This research program will significantly improve our knowledge of the determinants for successful HDR. It will lead to novel tools to help move genome modification from basic genetics toward practical applications in agriculture and human health.