Live-Cell Mapping of CRISPR-Cas Genome Editing from Targeting to Repair

项目摘要 CRISPR相关的规则间隔短回文重复序列（CRISPR-associated，CRISPR- 细菌和古细菌中的Cas）系统可以特异性地结合和切割可编程的DNA序列， RNA引导的方式。出于这个原因，CRISPR效应子核酸酶，特别是S.化脓性Cas9（SpyCas 9） 和Acidaminococcus sp.Cas12a（AsCas 12 a）作为单蛋白效应物切割DNA，是有前途的工具 用于治疗性基因组编辑。这些细菌蛋白质可以沿着在人类细胞核中部署， 被称为单向导RNA（sgRNA）的工程化RNA转录物，以特异性靶向细胞中的任何DNA序列。 基因组，条件是它与sgRNA中的20-nt间隔序列互补，并且含有短的 前间区序列邻近基序（PAM）。尽管这些CRISPR-Cas工具有许多 未来可能的用途，控制脱靶切割事件和编辑CRISPR结果的挑战- 必须克服诱导的DNA断裂以实现其治疗潜力。 对活细胞中CRISPR效应子的更好理解将为更好地研究CRISPR效应子提供策略。 CRISPR-Cas在人类细胞核中的控制。一种活细胞成像平台，其中细胞稳定表达核酸酶- 与HaloTag结构域融合的死SpyCas 9已成功用于可视化Cas9 DNA询问， 活的小鼠细胞核。使用这个CRISPR成像平台作为基础，我建议i）构建稳定的 表达融合至HaloTag结构域的SpyCas 9和AsCas 12 a的WT和核酸酶死亡变体的细胞系 ii）比较靶向正交标记的DNA的SpyCas 9和AsCas 12 a的结合保真度 iii）对荧光标记的WT CRISPR效应子和人修复酶进行成像，以映射这些序列。 处理CRISPR诱导的DNA断裂的步骤的顺序和持续时间。具体来说，通过测量 CRISPR效应物的结合与靶DNA上各种单个修复酶的结合之间的时间，I 将时空映射从CRISPR-Cas转移到修复的底物。 这项研究的结果将提供Cas9和Cas 12 a的直接可视化和比较。 细胞中的脱靶结合。这项研究还将确定可能的速率决定步骤，同源性为导向的 修复结果在处理CRISPR诱导的DNA断裂，提供了一个路线图工程高保真 在Cas9或Cas 12 a编辑的细胞中的修复结果。最后，本研究可确定可能的替代方案， 高保真修复结果的途径，这可能有助于治疗突变的癌症患者， BRCA 1和其他已知有助于CRISPR诱导的DNA断裂加工的蛋白质。

Project Summary Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (CRISPR- Cas) systems in bacteria and archaea can specifically bind and cut a sequence of DNA in a programmable, RNA-guided manner. For this reason, CRISPR effector nucleases, particularly S. pyogenes Cas9 (SpyCas9) and Acidaminococcus sp. Cas12a (AsCas12a), which cut DNA as single-protein effectors, are promising tools for therapeutic genome editing. These bacterial proteins can be deployed in human nuclei, along with an engineered RNA transcript known as a single guide RNA (sgRNA), to specifically target any DNA sequence in the genome, provided it is complementary to a 20-nt spacer sequence in the sgRNA and contains a short protospacer adjacent motif (PAM) next to the target sequence. Though these CRISPR-Cas tools have many possible future uses, the challenges of controlling off-target cleavage events and editing outcomes of CRISPR- induced DNA breaks must be overcome to realize their therapeutic potential. Improved understanding of CRISPR effectors in live cells would provide insight into strategies for better control of CRISPR-Cas in human nuclei. A live-cell imaging platform, in which cells stably express nuclease- dead SpyCas9 fused to a HaloTag domain, has been successfully used to visualize Cas9 DNA interrogation in live mouse cell nuclei. Using this CRISPR imaging platform as a foundation, I propose to i) construct stable cell lines expressing WT and nuclease-dead variants of SpyCas9 and AsCas12a, fused to a HaloTag domain for imaging ii) compare binding fidelity of SpyCas9 and AsCas12a targeted to orthogonally labeled DNA sequences and iii) image fluorescently labeled WT CRISPR effectors and human repair enzymes to map the sequence and duration of steps processing CRISPR-induced DNA breaks. Specifically, by measuring the lag time between binding of CRISPR effectors and binding of various individual repair enzymes on target DNA, I will spatiotemporally map substrate transfer from CRISPR-Cas to repair. The findings of this study will provide a direct visualization and comparison of Cas9 and Cas12a on-and off- target binding in cells. This study will also identify possible rate-determining steps for homology-directed repair outcomes in processing CRISPR-induced DNA breaks, providing a road map to engineering high-fidelity repair outcomes in Cas9- or Cas12a- edited cells. Finally, this study may identify possible alternative pathways for high-fidelity repair outcomes, which could be useful in treating cancer patients with mutations in BRCA1 and other proteins known to contribute to CRISPR-induced DNA break processing.