重症β地贫食蟹猴胚胎构建的高效突变基因敲入技术研究

重症β地贫是我国南方地区高发的严重致死/致残的单基因遗传病。基因治疗是最有前景的根治手段，但在临床试验前应利用非人灵长类动物模型验证其有效性、安全性。申请者前期构建食蟹猴地贫模型时发现受精卵的基因敲入效率极低，针对发现的问题进行研究可极大提升食蟹猴地贫模型构建成功率。本项目拟利用Cas9连接53BP1特异突变体提高靶位点同源重组修复率、利用链霉亲和素与生物素高亲和性募集供体模板至靶位点、引入先导编辑技术等策略改进基因编辑技术，并通过荧光泛素化指示剂结合高内涵成像系统检测食蟹猴早期胚胎细胞周期时序以指导编辑时机，使β珠蛋白突变基因敲入效率提高至5-10%。在此基础上优化脱靶检测方法，从基因组、转录组等层面分析非特异性遗传修饰效应。阐明跨越受精卵基因敲入效率极低的内在机制，突破构建动物模型时受精卵基因编辑的技术瓶颈，建立安全、高效和精准的受精卵/早期胚胎编辑工作体系。

Severe β-thalassemia is a single gene genetic disease with Serious death/disability in southern China, with a huge need for treatment. The most promising cure mean is the gene therapy, in order to verify the safety and efficiency，it’s urgent to build the non-human primate disease model same to human mutant genotypes of severe β-thalassemia. When we constructed the cynomolgus monkey thalassemia model earlier, we found that the gene knock-in efficiency of the zygotes is extremely low.There is room for improvement worth exploring.Accordingly, this project intends to take advantage of some improved methods to increase knock-in rate to 5-10% in fertilized oocytes of cynomolgus monkey, such as increasing homologous recombination rate of target site by combination of Cas9 with special mutation of 53BP1, collecting biotinylated donor template to target site using the high affinity between streptavidin and biotin, introducing prime edit technology, guiding edit moment by monitoring cell cycle schedule of early embryos with the methods of fluorescent ubiquitination–based cell-cycle indicator combined to the high-connotation imaging system. Basing on this, the off-target detection method will be optimized and the effect of no special genetic modification will be analyzed by genome and transcriptome sequencing methods. Break through and cross the internal mechanism of extremely low efficiency of gene input of zygotes, break through the technical means of gene editing of zygotes when establish animal models, and establish a safe, efficient and accurate editing system for zygotes/early embryo.