利用CRISPR/Cas9技术建立OXTR基因敲除猪胎儿成纤维细胞系

doi:10.13560/j.cnki.biotech.bull.1985.2021-1432

摘要/Abstract

摘要：

利用CRISPR/Cas9系统建立催产素受体（oxytocin receptor,OXTR）基因敲除的巴马猪胎儿成纤维细胞系（porcine fetal fibroblasts,PFFs）,为构建OXTR基因敲除巴马猪模型提供供体细胞。首先,对人和猪的OXTR基因进行了生物学信息分析。其次,利用在线设计工具设计了2个靶向猪OXTR外显子区的sgRNA,并克隆至pX330骨架质粒中,最后将打靶质粒和Neomycin抗性质粒共转染至PFFs中,用G418药物筛选出抗性单克隆细胞,测序鉴定其基因型。生物信息学分析显示人和猪OXTR基因进化距离较近,氨基酸序列一致性达到91%,相似性为93%,三维结构的RMSD值为0.009。成功构建OXTR基因的Cas9/sgRNA打靶载体,转染PFFs细胞后,药物筛选获得OXTR基因敲除的单克隆细胞并测序证实了基因突变型。人和猪OXTR基因具有高度同源性;构建的Cas9/sgRNA表达载体高效实现了OXTR基因编辑,成功获得基因敲除的单细胞克隆,为后续构建OXTR敲除的巴马猪模型奠定了前期基础。

关键词: CRISPR/Cas9, OXTR基因, 生物信息分析, 猪胎儿成纤维细胞

Abstract:

This work is aimed to construct OXTR（oxytocin receptor）- knocked porcine fetal fibroblasts（PFFs）via CRISPR/Cas9,which could be used as donor cells to generate OXTR disrupted Bama miniature pig models. Firstly,the pig OXTR and its human counterpart were analyzed by bioinformatics. Next,two sgRNAs targeting the exon region of pig OXTR were designed using online tools（http://www.rgenome.net/cas-offinder/）and ligated into the pX330 plasmid. Finally,the gene targeting plasmids were co-transfected with a neomycin-expression plasmid into early passage of primary PFFs. G418 screening was used to obtain the resistant single-cell colonies,and the Sanger sequencing was used to determine their genotypes. Bioinformatics analysis showed that the OXTR genes of humans and pigs had a close evolutionary distance. The identity and similarity of the amino acid sequence of human and pig OXTR were 91% and 93%,respectively. The RMSD value of the three-dimensional structure was 0.009. The Cas9/sgRNA targeting vectors of gene OXTR were constructed successfully. After the transfection of PFFs cells,drug screening was used to obtain OXTR knockout monoclonal cells,and they were sequenced,as well as the gene mutant type was confirmed. In conclusion,the human and porcine OXTR are evolutionarily conserved and highly homologous. The constructed Cas9/sgRNA expression vector may allow the OXTR gene editing achieved. The OXTR-knocked monoclonal cells were successfully obtained,which paves the early foundation for the generation of OXTR-knocked Bama pig model.

Key words: CRISPR/Cas9, OXTR gene, bioinformatics analysis, porcine fetal fibroblasts

刘静静, 刘晓蕊, 李琳, 王盈, 杨海元, 戴一凡. 利用CRISPR/Cas9技术建立OXTR基因敲除猪胎儿成纤维细胞系[J]. 生物技术通报, 2022, 38(6): 272-278.

LIU Jing-jing, LIU Xiao-rui, LI Lin, WANG Ying, YANG Hai-yuan, DAI Yi-fan. Establishment of Porcine Fetal Fibroblasts with OXTR-knockout Using CRISPR/Cas9[J]. Biotechnology Bulletin, 2022, 38(6): 272-278.

图/表 8

表1 OXTR基因exon 2序列扩增引物

Table 1 Amplification primers for exon 2 sequene of OXTR gene

名称 Primer name	引物序列 Primer sequence（5'-3'）	产物大小 Fragment length
OXTR-F	ACCTGCCCAAGAAGTCTCAG	748 bp
OXTR-R	CAGCAGCAGGTAAGTGGAAG	748 bp

图1 不同物种OXTR序列构建的系统进化树

Fig.1 Phylogenetic tree of OXTR sequences of different species

图2 人/猪OXTR的一级、二级、三级结构分析 A：人/猪OXTR氨基酸序列同源性分析,相同氨基酸用蓝色阴影表示;B：人/猪蛋白质二级结构,红色表示a螺旋,绿色表示β折叠,蓝色表示β转角;C：人/猪蛋白质三维建模结果,红色为人类OXTR结构,绿色为猪OXTR结构

Fig.2 Analysis of primary,secondary,and tertiary structures of OXTR between humans and pigs A：Amino acid sequence homology analysis of human and pig OXTR,and the identical amino acids are blue shaded. B：Secondary structures of human and pig proteins. Red indicates alpha-helix;green indicates beta-sheet;and blue indicates beta-turn. C：Tertiary structures of human and pig proteins. Red indicates the structure of human OXTR,and green indicates the structure of pig OXTR

图3 猪OXTR关键残基和结构域的鉴别 A：猪OXTR的结构域和催化残基示意图;B：OXTR的多重序列比对结果,＃表示与配体结合的关键氨基酸残基

Fig. 3 Identification of domains and key residues of porcine OXTR A：Schematic diagram of domains and catalytic residues of pig OXTR. B：Multiple sequence alignment of OXTR,＃ denotes critical amino acid residues binding to ligand

图4 OXTR基因靶点和重组载体测序 A：巴马猪OXTR基因靶点示意图,灰色表示非编码区,黑色表示编码区;B：pX330载体示意;C、D：重组载体示意图,黑色划线处为插入序列

Fig.4 Target of OXTR gene and sequencing of recombinant vectors A：Schematic diagram of OXTR gene target in Bama miniature pig. Gray indicates the non-coding area and black indicates the coding areas. B：Schematic diagram of pX330 vector. C and D：Sequencing map of recombinant vector. Black underlined is the insertion sequence

表2 OXTR基因靶向位点的sgRNA寡核苷酸序列

Table 2 Oligonucleotide sequences of sgRNAs at OXTR targeting sites

sgRNA	序列Sequence（5'-3'）
sgRNA-1-F	CACCGGTGCTGCCTCAGCTACTGT
sgRNA-1-R	AAACACAGTAGCTGAGGCAGCACC
sgRNA-2-F	CACCGAACTTGCGGCTCAAGACTG
sgRNA-2-R	AAACCAGTCTTGAGCCGCAAGTTC

图5 OXTR敲除PFFs的测序结果

Fig.5 Sequencing results of OXTR-knockout PFFs

表3 OXTR 敲除PFFs的基因型

Table 3 Genotypes of OXTR-knockout PFFs

编号 ID name	目的片段 Target fragment	突变型 Indels
WT	CAGGTGCTGCCTCAGCTACTGTGGG//GGCAGAACTTGCGGCTCAAGACTGCG	WT
1	CAGGTGCTGCCTCAGCTAC-----// ----------------------CTGCGG	-411 bp
2	CAGGTGCTGCCTCAGCTAC-----// ----------------------CTGCGG	-411 bp
3	CAGGTGCTGCCTCAGCTAC-----// ----------------------CTGCGG	-411 bp
4	CAGGTGCTGCCTCAGCTACTG---// -------------------------CGG	-412 bp
5	CAGGTGCTGCCTCAGCTACTCTTGA//AGCGGAAGGTGATATCCCACACTGCGG	-289,+289 bp

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