绵羊胚胎FGF5基因单碱基突变体系的建立

研究旨在利用单碱基编辑技术定点修饰绵羊（Ovis aries）成纤维细胞生长因子5（fibroblast growth factor 5，FGF5）基因第1外显子以引入终止密码子，获得定点编辑类型的绵羊胚胎，为培育具有长毛性状的绵羊提供试验材料。首先设计合成4个单导向RNA （single guide RNA，sgRNA）寡核苷酸链（sgRNA-T1~sgRNA-T4），构建4组不同的重组表达载体；将构建好的pGL3-U6-sgRNA-PGK-puromycin和pCMV-AncBE4max-P2A-GFP质粒以共转染的方法分别转入4组绵羊成纤维细胞，随后用Cruiser^TM酶对转染的细胞进行活性检测并在胚胎水平进行测序验证。结果显示，sgRNA-T1和sgRNA-T4组细胞的PCR产物可被Cruiser^TM酶酶切，且测序结果表明都具有靶向效果，编辑效率分别为68.75%和47.37%。利用显微注射技术将不同浓度的AncBE4max mRNA与有效sgRNA混合注射到绵羊孤雌激活胚胎中，并检测胚胎卵裂率、囊胚率和编辑效率，结果显示，胚胎水平的最佳注射浓度组合为AncBE4max （ng/μL）∶sgRNA （ng/μL）=100∶50，从该浓度组中随机挑选的单枚胚胎测序结果显示，引入终止密码子的编辑效率为80%。而sgRNA-T1在不同浓度组合的注射胚胎中均未检测到编辑。本研究针对FGF5基因的第1外显子，通过在成纤维细胞转染表达载体，成功筛选到高效靶向绵羊FGF5基因的2个sgRNA （T1、T4）；通过显微注射绵羊孤雌激活胚胎，成功在胚胎上实现FGF5基因第1外显子打靶位点C→T的转变，为后期FGF5基因定点编辑羊的生产奠定基础。

Establishment of Single Base Mutation System of FGF5 Gene in Sheep Embryos

The purpose of this study was to modify the exon 1 of sheep (Ovis aries) fibroblast growth factor 5 (FGF5) gene by single base editing technique to introduce the stop codon, to obtain sheep embryos with site-directed editing, and to provide experimental materials for breeding sheep with long wool traits. Firstly, four single guide RNA (sgRNA) oligonucleotide chains (sgRNA-T1 to sgRNA-T4) were designed and synthesized, and four groups of recombinant expression vectors were constructed. The constructed pGL3-U6-sgRNA-PGK-puromycin and pCMV-AncBE4max-P2A-GFP plasmids were transferred into four groups of sheep fibroblasts by co-transfection, and then the activity of the transfected cells was detected by Cruiser^TM enzyme and sequenced at embryos level. The results showed that the PCR products of sgRNA-T1 and sgRNA-T4 groups with targeted effects were screened at cell level and could be cleaved by Cruiser^TM enzyme, and the sequencing results showed that both groups had targeted effects, with editing efficiency of 68.75% and 47.37%, respectively. Different concentrations of AncBE4max mRNA and effective sgRNA were mixed and transforred into sheep parthenogenetic activated embryos by microinjection technique, and the cleavage rate, blastocyst rate and editing efficiency of the embryos were detected. The results showed that at the embryo level, the best injection concentration combination of AncBE4max(ng/μL):sgRNA(ng/μL) was 100:50. The sequencing results of single embryos randomly selected from this concentration group showed that the editing efficiency of introducing stop codons was 80%. However, no editing was detected in the injected embryos with different concentrations of sgRNA-T1. In this study, aiming at the exon 1 of FGF5 gene, two sgRNA (T1, T4) targeting sheep FGF5 gene were successfully screened by transfection expression vector in fibroblasts, and the transformation of the target site C→T of exon 1 of FGF5 gene was successfully realized in embryos by microinjection of sheep parthenogenetic activated embryos, which laid a foundation for the production of sheep with FGF5 gene targeted editing in the later stage.