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

表征和改进 CRISPR/Cas 介导的基因组编辑中的同源定向修复

• 批准号: DDG-2015-00016
• 负责人: Berthoux, Lionel
• 金额: $ 0.73万
• 依托单位: Université du Québec à Trois-Rivières
• 依托单位国家: 加拿大
• 项目类别: Discovery Development Grant
• 财政年份: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=612420
• 关键词: Characterizing; improving; homology; directed; repair

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.

CRISPR/Cas基因组编辑正在彻底改变生物医学研究。rna引导的Cas9核酸酶可以诱导DNA中的靶向双链断裂（DSBs），通过插入或删除DNA （indels）来敲除目标位点。然而，大多数基因组编辑的应用都要求在基因组中引入特定的突变，而不是插入。这可以通过使用含有所需突变的模板DNA的同源定向修复（HDR）来实现。然而，在目前的方案中，hdr介导的诱变的产量很低，需要选择/纯化步骤。本研究计划旨在为实现高效、无疤痕的基因组修饰提供知识和方法。具体目标和方法建立一个基于facs的系统，用于监测人类细胞中的功能性基因修饰。这将通过建立细胞模型来实现，其中表面蛋白表皮生长因子受体（EGFR）被HDR突变，从而通过流式细胞术进行检测。因此，HDR的效率由表达该标记物的细胞百分比来反映。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和递送选择在整合缺陷慢病毒载体的背景下的效率。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组件的最佳递送时间。3. 通过DNA修复蛋白的过度表达改善HDR。为了提高HDR的效率，我们将在表达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相互竞争修复dsb，有证据表明抑制NHEJ可以增强HDR。因此，我们将使用NHEJ蛋白DNA-PK的药理学抑制剂以及53BP1的显性阴性版本来增加EGFR模型中的HDR产量。5. 脱靶编辑与DNA不稳定性分析。我们将对修饰细胞中可能的脱靶位点进行测序，以分析该项目中开发的方案是否会导致脱靶编辑的增加。基因组不稳定性将通过核型分析。影响：该研究项目将显著提高我们对成功的HDR决定因素的认识。它将带来新的工具，帮助将基因组修饰从基础遗传学转向农业和人类健康的实际应用。