CRISPR/Cas9介导HDR-SSA两步法猪IGF2基因“无缝编辑”新技术研究

Recent years, the safety issue against transgenosis has been a social concern. There is a safe and feasible strategy for editing SNP site with potential breeding importance, and the breeding may be achieved by converting the SNP site of target gene from wild type into a natural mutant. However, there are still some difficulties when conducting precise genome editing, such as the off-target effect, low efficiency for HDR-based recombination, poor selection for positive cell clones, deletion of selecting cassette and the low biallelic targeting efficiency. Hence, to edit the SNP site (G>A) within the intron of porcine IGF2 gene, we proposed the “seamless editing” technique based on our previous studies, which uses CRISPR/Cas9 twice to mediate the knock-in of donor DNA (with selecting cassette and the intent mutation) by HDR and subsequently the complete deletion of selecting cassette by SSA repair mechanism. In this project, the strategies including Cas9 nickase, sgRNA-shRNA tandem structure, recombination related factor-Cas9 fusion as well as paired donor vectors will be further applied to establish the novel, secure and efficient platform for the “precise editing” of porcine genome, which will facilitate the genome editing breeding research in the future.

鉴于近年来转基因安全问题备受关注，针对具有重要育种价值的SNP位点进行“精确编辑”，使其从野生型向自然存在的突变型转变，被认为是一种安全可行的基因组编辑育种策略。然而在基因组“精确编辑”研究中，尚存在着脱靶效应、HDR重组效率低、阳性细胞克隆筛选难、筛选标记基因再删除以及双等位打靶效率低等问题。因此，我们在前期一系列相关研究的基础上，针对猪IGF2基因内含子SNP位点（G>A）的“精确编辑”，提出了CRISPR/Cas9介导HDR-SSA两步法的“无缝编辑”技术：通过HDR和SSA修复机制先后实现供体DNA的敲入（含筛选标记及目标突变）和筛选标记基因组件的无痕迹删除，同时整合Cas9切口酶、sgRNA-shRNA串联表达、重组因子-Cas9融合表达以及双供体载体等优化策略，建立一个新型的、安全高效的猪基因组“精确编辑”技术平台，为猪基因组编辑育种研究奠定基础。

鉴于近年来转基因安全问题备受关注，针对具有重要育种价值的SNP位点进行“精确编辑”，使其从野生型向自然存在的突变型转变，被认为是一种安全可行的基因组编辑育种策略。然而在基因组“精确编辑”研究中，尚存在着HDR重组效率低、阳性细胞克隆筛选难、筛选标记基因再删除以及双等位打靶效率低等问题。因此，本项目课题组针对猪IGF2基因内含子SNP位点（G>A）的“精确编辑”，开发并优化了CRISPR/Cas9介导HDR-SSA两步法的“无缝编辑”技术，建立了相应的双等位无缝编辑系统，并实现了猪IGF2基因的无缝编辑。为了提高筛选标记再删除效率，课题组进一步开发了CRISPR/Cas9-PiggyBac系统；为了实现无筛选标记整合的基因编辑，课题组进一步开发了HDR-USR和acNHEJ-USR基因编辑阳性细胞报告筛选系统。这些CRISPR/Cas9配套系统的开发为以后的家畜尤其是猪基因组编辑育种研究提供了有效的技术手段。