CRISPR/Cas9靶点滞留在DNA双链断裂修复及基因编辑产物异质性形成中的作用及机制

CRISPR基因编辑产物异质性是领域内亟需解决的一个重要问题，而决定基因编辑产物异质性的因子除靶点DNA序列外还有DNA双链断裂（DSB）修复途径的选择。前期研究发现，CRISPR/Cas9靶点结合能力影响非同源末端连接修复中核心因子的招募及同源重组修复途径的利用。我们推测CRISPR/Cas9的靶点滞留或DSB末端的滞留势必影响修复途径的选择，特别是，末端滞留的CRISPR复合物可能因为滞留状况的不同，有的自由解离，有的则需要转录或DNA复制帮助解离，从而产生不同的末端构象，启动不同的DSB修复途径，增加修复产物异质性。本项目拟利用可控的转录和复制元件，结合多种DSB修复报告系统及内源基因组位点，分析CRISPR末端滞留对DSB修复途径选择的影响，阐明转录和复制的作用。这不仅将帮助揭示基因编辑产物异质性产生的分子机制，而且有助于改良CRISPR基因编辑技术以解决基因编辑产物异质性问题。

Mutation spectra at a target site vary widely in CRISPR/Cas9 genome editing with highly heterogeneous repair products, even at the same locus at different time or in different organisms, limiting application of precise gene editing. Along with the DNA sequence of the target itself, the repair pathway choices at a specific CRISPR/Cas9-induced DNA double strand break (DSB) may determine the spectrum of mutations in CRISPR genome editing. Our preliminary studies have indicated that the residence of CRISPR/Cas9 at a target even after DNA cleavage reduces the engagement of core non-homologous end joining (NHEJ) factors in CRISPR/Cas9-induced NHEJ and increases the usage of homologous recombination (HR). We hypothesize that the residence strength and duration of the CRISPR/Cas9 complex at a cleaved target regulate the DSB repair pathway choices for this CRISPR/Cas9-induced DSB. It is likely that CRISPR/Cas9 bound with the cleaved target might encounter transcription or replication locally and be released from the target, thereby exposing DNA ends of the DSB for proper and timely repair. However, different CRISPR/Cas9-target disassociation modules, free or passive, may generate different end configurations, which engage different repair pathways. In this proposal, to test the hypothesis above, we will: 1) Analyze the effect of the CRISPR/Cas9-target binding affinity on the choices of DSB repair pathways; 2) Asses the impact of local transcription in repair of CRISPR/Cas9-induced DSBs; 3) Study the role of local DNA replication in repair of CRISPR/Cas9-induced DSBs. With the DSB reporter systems coupled with proper transcription and replication control, we can analyze the repair pathway choices determined by the target residence of CRISPR/Cas9 and elucidate the molecular mechanism of transcription-coupled or replication-coupled DSB repair in CRISPR/Cas9 genome editing. This will not only better our understanding of sources and mechanisms for the heterogeneity of repair products in CRISPR genome editing but also help to improve the technology by reducing this heterogeneity.