Abstract:

CRISPR/Cas9， as a powerful gene editing tool， has been widely applied in porcine genetic improvement. However， editing a single gene does not meet the requirements for simultaneous multi-trait enhancement. To establish a multi-gene editing system in porcine embryonic fibroblasts （PEFs） and generate embryos with concurrent modifications in MSTN， pAPN and CD163 genes using the CRISPR/Cas9 system， based on the functional domains of porcine CD163， MSTN and pAPN proteins， exon 1 of MSTN， exon 7 of CD163， and exon 2 of pAPN were selected as target regions. 2 pairs of sgRNAs were designed and cloned into the pX330 backbone vector， and electroporated into PEF cells to select the sgRNA with the higher editing efficiency for each gene. Homologous recombination was employed to assemble the individual sgRNA expression cassettes for CD163， MSTN and pAPN into an integrated tandem expression system， which was transformed into Fast-T1 competent cells and verified by Sanger sequencing. The three-gene knockout vector was then electroporated into PEF cells， and selection was performed with 2.5 μg·mL^-1 puromycin. Single-cell clones were isolated， followed by PCR amplification and Sanger sequencing to verify the target sequences of MSTN， CD163 and pAPN in each clone. The results indicated that the editing efficiency of sgRNA1 was higher than that of sgRNA2 for CD163， MSTN and pAPN genes， and the sgRNA1 with higher efficiency was selected for the construction of integrated plasmid. Plasmid sequencing confirmed that the three sgRNA expression cassettes were successfully integrated into the pX330 backbone vector. Among 45 single-cell clones transfected with the three-gene editing vector， the mutation rates for MSTN， pAPN and CD163 were 62%， 26% and 11%， respectively， with 4 clones achieving simultaneous editing of all three genes （efficiency of 8.9%）. Furthermore， embryos produced via somatic cell nuclear transfer （SCNT） using the edited cells showed no significant differences in cell fusion rate， cleavage rate， or blastocyst formation rate compared to embryos derived from non-edited cells. Sequencing results demonstrated that the embryonic target modifications were consistent with those in the somatic cells. In conclusion， PEF cells and embryos were successfully generated with concurrent modifications in the porcine MSTN， pAPN and CD163 genes using CRISPR/Cas9 technology， which laid the foundation and provided reference for the production of multi-gene edited cloned pigs.

Key words: CRISPR/Cas9, MSTN, pAPN, CD163, multiplex editing, PEF, embryo

摘要：

CRISPR/Cas9作为一款强大的基因编辑工具，在猪的遗传改良中已经广泛应用。然而，单一基因的编辑无法满足多性状的同步改良。为利用CRISPR/Cas9基因编辑体系构建MSTN、pAPN和CD163多基因编辑猪胚胎成纤维细胞（porcine embryonic fibroblasts， PEF）并制备胚胎，首先，根据猪CD163、MSTN和pAPN蛋白功能结构域，选定MSTN外显子1、CD163外显子7及pAPN外显子2作为打靶区域，各设计2对sgRNAs，连接至骨架pX330载体，通过电转染至PEF，筛选每个基因编辑效率较高的sgRNA；然后，利用同源重组法将CD163、MSTN、pAPN单个基因的sgRNA表达盒组装成一体化串联表达系统，转化至Fast-T1感受态细胞并进行Sanger测序鉴定；最后，利用电转染方式将3基因敲除载体转染至PEF，用2.5 μg·mL^-1的嘌呤霉素进行药筛，挑选单克隆细胞，PCR扩增后进行Sanger测序，鉴定单克隆细胞MSTN、CD163、pAPN基因的打靶序列。结果显示，CD163、MSTN、pAPN基因的sgRNA1编辑效率高于sgRNA2，因此选择较高效率sgRNA1用于构建一体化质粒。质粒测序显示，3个基因的sgRNA表达盒成功连接到骨架pX330载体上。挑取45株转染3基因编辑质粒的单克隆细胞，测序结果显示，MSTN、pAPN、CD163基因的突变率分别为62%、26%、11%，其中有4株细胞实现了3个基因同时编辑（效率为8.9%）。进一步用编辑细胞进行体细胞核移植生产胚胎，与未编辑细胞生产的胚胎在体细胞融合率、胚胎卵裂率和囊胚率上均无显著差异；测序结果显示，胚胎水平的打靶结果与体细胞一致。综上，利用CRISPR/Cas9技术制备猪MSTN、pAPN、CD163基因同时编辑的PEF和胚胎，为多基因编辑克隆猪的制备奠定基础和提供参考。

关键词: CRISPR/Cas9, MSTN, pAPN, CD163, 多基因编辑, PEF, 胚胎