碱基编辑器介导的猪IGF2基因高效定点突变

doi:10.16431/j.cnki.1671-7236.2020.11.002

中国畜牧兽医 ›› 2020, Vol. 47 ›› Issue (11): 3427-3435.doi: 10.16431/j.cnki.1671-7236.2020.11.002

碱基编辑器介导的猪IGF2基因高效定点突变

王煜¹, 宋瑞高², 赵建国², 王彦芳¹

1. 中国农业科学院北京畜牧兽医研究所, 北京 100193;
2. 中国科学院动物研究所, 北京 100101

收稿日期:2020-05-27 出版日期:2020-11-20 发布日期:2020-11-20
通讯作者: 王彦芳 E-mail:wangyanfang@caas.cn
作者简介:王煜(1995-),男,山东临沂人,硕士生,研究方向:动物遗传育种与繁殖,E-mail:wangyu950306@163.com
基金资助:
国家自然科学基金（31672387）；中央级公益性科研院所基本科研业务费专项资金（2020-YWF-YB-04）

Efficient Site-directed Mutation of Porcine IGF2 Gene via Base Editors

WANG Yu¹, SONG Ruigao², ZHAO Jianguo², WANG Yanfang¹

1. Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China;
2. Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China

Received:2020-05-27 Online:2020-11-20 Published:2020-11-20

摘要/Abstract

摘要： 本研究旨在利用碱基编辑器在巴马猪胎儿成纤维细胞（porcine fetal fibroblast cells，PFFs）中对生长性状相关基因胰岛素样生长因子2（insulin-like growth factor 2，IGF2）进行高效、精确的定点编辑。通过PCR扩增和测序对巴马猪和大白猪的IGF2基因序列进行鉴定，构建靶向巴马猪的sgRNA-IGF2表达载体，进而通过细胞转染、混合细胞编辑效率检测、单克隆细胞筛选及基因型鉴定等技术手段研究不同碱基编辑器对猪IGF2基因靶点的编辑效率及突变类型。结果显示，巴马猪和大白猪IGF2基因第3内含子存在第3 072 bp处的单核苷酸多态性（SNP）位点，设计靶向巴马猪IGF2基因的单链寡核苷酸序列。单链寡核苷酸经退火后与BsaⅠ线性化的pGL3-U6-sgRNA质粒进行重组连接，构建sgRNA-IGF2表达质粒，重组质粒测序结果表明，sgRNA序列已经精确连入U6启动子和sgRNA骨架之间。将rA1-BE3、hA3A-BE3、hA3A-BE3-Y130F和hA3A-eBE-Y130F 4种胞嘧啶碱基编辑器分别与sgRNA-IGF2表达质粒共转染至猪胎儿成纤维细胞中，对混合细胞编辑效率进行检测结果表明，对于IGF2基因靶点，hA3A-BE3系列碱基编辑器C到T的编辑效率显著高于rA1-BE3（P<0.05）。流式分选、单克隆细胞培养及鉴定结果表明，虽然有71.43%的hA3A-BE3单克隆细胞已被编辑，但有42.86%的细胞存在插入和缺失（indels）；hA3A-BE3-Y130F和hA3A-eBE-Y130F的编辑效率（56.86%和40.38%）虽低于hA3A-BE3（71.43%），但是indels的效率也较低（31.37%和21.15%）。测序结果分析表明，利用碱基编辑器高效筛选到IGF2基因靶点纯合点突变的单克隆细胞。碱基编辑器作为新的基因编辑工具，可以对猪基因组中与经济性状关联的SNP位点进行高效、精确的基因修饰，为加速猪经济性状的遗传改良提供理论与实践基础。

关键词: 碱基编辑器; IGF2基因; 定点突变; 猪胎儿成纤维细胞(PFFs)

Abstract: This study was aimed to efficiently and accurately edit the growth trait-related gene insulin-like growth factor 2 (IGF2) in porcine fetal fibroblasts cells (PFFs) of Bama pigs using base editors.The IGF2 gene sequences of Bama pigs and Large White pigs were identified by PCR amplification and sequencing,and the expression vector of sgRNA-IGF2 targeting Bama pigs was constructed.Then the editing efficiency and product types at IGF2 gene via different base editors were studied by cell transfection,detection of mixed cell editing efficiency,screening of single cell colonies and genotyping.The results showed that there was a single nucleotide polymorphism (SNP) site at 3 072 bp in intron 3 of IGF2 gene between Bama pigs and Large White pigs,and a single-stranded oligonucleotide sequence targeting the IGF2 gene was designed.The single-stranded oligonucleotide was annealed and ligated with the BsaⅠ-linearized pGL3-U6-sgRNA plasmid to construct the sgRNA-IGF2 expression plasmid.The sequencing results of the recombinant plasmid showed that the sgRNA sequence was accurately inserted between the U6 promoter and the sgRNA scaffold.Four different cytosine base editors (rA1-BE3,hA3A-BE3,hA3A-BE3-Y130F and hA3A-eBE-Y130F) were co-transfected with sgRNA-IGF2-expressing plasmid into PFFs,respectively.The editing efficiency in mixed cells showed hA3A-BE3-based CBEs could introduce higher efficiency of C-to-T mutation than rA1-BE3 (P<0.05).The results of flow cytometry,single cell culture and genotyping showed that 71.43% of single cell colonies were mutant in hA3A-BE3 group,but 42.86% of them had insertions/deletions (indels).The editing efficiency of hA3A-BE3-Y130F (56.86%) and hA3A-eBE-Y130F (40.38%) were lower than hA3A-BE3(71.43%),but the indels of them were also lower (31.37% and 21.15%).In addition,single cell colonies with homozygous mutation at IGF2 gene site were achieved by base editors in this study.Base editors,as new gene editing tools,could efficiently and accurately modify SNP associated with economic traits in pig genome and accelerate genetic improvement.

Key words: base editor; IGF2 gene; targeted mutation; porcine fetal fibroblast cells (PFFs)

中图分类号:

王煜, 宋瑞高, 赵建国, 王彦芳. 碱基编辑器介导的猪IGF2基因高效定点突变[J]. 中国畜牧兽医, 2020, 47(11): 3427-3435.

WANG Yu, SONG Ruigao, ZHAO Jianguo, WANG Yanfang. Efficient Site-directed Mutation of Porcine IGF2 Gene via Base Editors[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(11): 3427-3435.

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碱基编辑器介导的猪IGF2基因高效定点突变

Efficient Site-directed Mutation of Porcine IGF2 Gene via Base Editors

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