应用CRISPR/Cas 基因组编辑技术敲除德保猪MSTN基因的研究

CRISPR/Cas9 has been widely used in generating site-specific genetically modified animal models. Myostatin (MSTN) is encoded by MSTN gene involved in the inhibition of muscle differentiation and growth. Debao pig has high disease resistance, and tender, delicious meat, and are not selected for fast growth. In this study, We determine the efficiency of the CRISPR/Cas9 system to edit MSTN in pig and generate knock-out (KO) Debao pig with the aim to promote muscle development and body growth. Firstly, we design more than five target sites of CRISPR/Cas9 in the conserved domain of MSTN gene of pig combined with flow cytometry detection screening effective gRNA loci using RGS-CR reporter in HEK293T cells. The editing efficiency will be tested in fibroblast and embryos of pig respectively. The Cas9 mRNA/sgRNA obtained by transcript in vitro will be co-microinjected into somatic nuclear transfer embryos of pig, comparing the embryos development efficiency and editing rate in different target sites, the concentration of Cas9 mRNA/sgRNA and injection time are all parameters which be concerned. In addition, the small molecular compounds about changing chromosomes and inhibiting DNA damage repair pathway NHEJ, and over-expression of HR relative factors are planed be applied to improve the CRISPR/Cas9 system-mediated efficiency of genetic modification. Finally, The somatic nuclear transfer embryos of high quality which injected with Cas9 mRNA/sgRNA will be selected and transplanted into the surrogate to obtain individual MSTN gene knockout Debao pig.

CRISPR/Cas9基因编辑技术的广泛应用大大促进了建立基因改造动物模型的速度。广西地方猪种德保猪肉质好但生长速度慢。本课题拟应用CRISPR/Cas9系统对肌肉生长负调控基因MSTN进行定向编辑以提高德保猪生长速度。首先在MSTN基因重要功能域设计多个打靶位点，应用RGS-CR报告系统在293T细胞检测筛选有效gRNA位点，并在细胞和胚胎水平分别验证基因组切割效率。将体外转录sgRNA和Cas9 mRNA分别注入核移植胚胎，比较多因素对胚胎发育、基因组编辑效率的影响。同时根据文献资料以及实验室工作推测，我们将添加与染色体状态改变相关或抑制DNA损伤修复相关的小分子化合物或过表达同源重组因子有可能提高CRISPR/Cas系统介导的基因修饰效率。应用经过优化的方案，将体外转录Cas9的mRNA和sgRNA注射德保猪体细胞核移植胚胎，通过ET技术获得MSTN基因敲除的德保猪个体。

CRISPR/Cas9基因编辑技术的广泛应用大大促进了建立基因改造动物模型的速度。广西地方猪种德保猪肉质好但生长速度慢。而 MNST 基因是一种肌肉生长抑制因子，MSTN 基因的自然或者人为突变，能导致动物或者人肌肉肥大的表型。本项目系统探讨了影响德保猪、兔和水牛体细胞基因组编辑效率的因素，初步建立了基于CRISPR/Cas9 系统的高效家畜基因组编辑研究平台。研究获得了基于 CRISPR/Cas9 基因编辑德保猪体细胞株和核移植胚胎，获得了基于 CRISPR/Cas9 基因编辑水牛体细胞株和核移植胚胎，获得了MSTN基因编辑兔新品系，基因编辑兔生长速度明显提升，表现出双肌臀特征，利用优化的基因编辑系统我们还获得了浅黄色新毛色基因编辑兔。