CRISPR-Cas基因编辑系统脱靶效应SV检测的研究

CRISPR-Cas作为应用最为广泛的基因编辑工具，在纠正致病突变方面有巨大的应用潜力。但是脱靶效应限制了CRISPR-Cas系统的临床应用。申请人团队利用GOTI和RNA-seq技术，发现了单碱基编辑器CBEs会导致以SNV为主的严重的DNA和RNA脱靶效应，并通过蛋白工程手段显著降低了CBEs的脱靶效应。受研究手段限制，CRISPR-Cas是否会导致脱靶效应SV尚不明确，因此亟需建立准确的脱靶效应SV检测方法。本研究拟使用三代长读长测序技术结合GOTI技术建立基因组脱靶效应SV检测方法，同时针对多种基因编辑工具在体外培养细胞和小鼠胚胎内使用不同类型sgRNA全基因组范围内评价基因编辑工具的安全性。阐明不同基因编辑技术是否会产生SV以及与细胞类型和sgRNA的关系，探究SV的形成机制，并尝试对产生脱靶的相应工具进行改进，从而为基础科研和临床应用提供更加安全可靠的工具。

As the most widely used gene-editing tool, CRISPR-Cas technology holds great promise in correcting pathogenic mutations. However, the off-target effects have limited the clinical application of CRISPR-Cas systems. The applicant and team members have previously found that cytosine base editors induced severe DNA and RNA off-target effects using GOTI and RNA-seq techniques, and further remarkably reduced the off-target effects of cytosine base editors by protein engineering. While limited by previous research methods, it is not clear whether CRISPR-Cas systems would induce off-target SVs. Therefore, there is an urgent need to develop accurate methods for the detection of off-target SVs. This research is aimed to develop a genome-wide method for the detection of off-target SVs based on third-generation sequencing and GOTI technology, and systematically evaluate the safety of multiple gene-editing tools with different kinds of sgRNAs in both cell lines and mouse embryos. This research will also explore the potential off-target SVs of different gene-editing tools and their relationship with cell types and sgRNAs. Besides, the mechanism for the production of off-target SVs will be explored, and the precision of CRISPR-Cas systems inducing off-target SVs will be further increased by protein engineering. This study will thus provide reliable tools for basic scientific research and clinical application.