CRISPR/Cas13a和MicroRNA介导的牛MSTN基因RNA干扰效率的比较  

RNA Interference Efficiency Comparison of Bovine(Bos taurus)MSTN Gene Mediated by CRISPR-Cas13a and MicroRNA

作　　者：雷佳茹 王淞 狄安琪 安常所 孙雪松 刘明成 袁宏敏 杨磊 李光鹏 LEI Jia-Ru;WANG Song;DI An-Qi;AN Chang-Suo;SUN Xue-Song;LIU Ming-Cheng;YUAN Hong-Min;YANG Lei;LI Guang-Peng(State Key Laboratory of Reproductive Regulation&Breeding of Grassland Livestock,Inner Mongolia University,Hohhot 010070,China)

机构地区：[1]内蒙古大学省部共建草原家畜生殖调控与繁育国家重点实验室,呼和浩特010070

出　　处：《农业生物技术学报》2024年第1期147-157,共11页Journal of Agricultural Biotechnology

基　　金：内蒙古自治区科技领军人才团队(2022LJRC0006);内蒙古自治区揭榜挂帅项目(2022JBGS0025);内蒙古自治区科技重大专项(2021ZD0009;2021ZD0008;2022ZD0008);农业农村部农业重大科技项目(NK2022130203);内蒙古自治区青年科技英才支持项目(NJYT23138);中央引导地方科技发展资金(2022ZY0212)。

摘　　要：RNA干扰(RNA interference,RNAi)主要由小干扰RNA(small interfering RNA,siRNA)、短发夹RNA(short hairpin RNA,shRNA)、微小RNA(microRNA,miRNA)和CRISPR/Cas13a介导。miRNA通过与靶标mRNA碱基互补配对介导RNAi。CRISPR/Cas13a通过crRNA(CRISPR-derived RNA)和Cas13a蛋白的共同作用介导RNAi。关于CRISPR/Cas13a和miRNA介导的RNAi效率比较目前尚无报道。本研究根据肌肉生长抑制素基因(myostatin,MSTN)在西门塔尔牛(Bos taurus)和小鼠(Mus musculous)的mRNA同源序列,设计牛和小鼠通用的MSTN-crRNA-1/2/3和MSTN-miRNA-1/2/3。为了排除由Cas13a蛋白表达不稳定引起的CRISPR/Cas13a干扰效率波动,首先制备稳定表达Cas13a蛋白的牛肌肉卫星细胞(satellite cells,SCs)和小鼠成肌细胞系C2C12,将MSTN-crRNA-1/2/3和MSTN-miRNA-1/2/3分别转染牛SCs和小鼠C2C12。转染48 h后,采用qRT-PCR检测CRISPR/Cas13a和miRNA介导的MSTN干扰效率;此外,采用MTT法和细胞增殖检测试剂盒CCK-8(Cell Counting Kit-8)检测CRISPR/Cas13a和miRNA介导的MSTN基因敲低对牛SCs和小鼠C2C12活力和增殖的影响。结果显示,MSTN-crRNA-1/2/3在牛Cas13a-SCs和小鼠Cas13a-C2C12中的平均干扰效率和最高干扰效率分别为45%和54%,MSTNmiRNA-1/2/3在牛SCs和小鼠C2C12中的平均干扰效率和最高干扰效率分别为30%和33%,说明CRISPR/Cas13a干扰效率高于miRNA;MSTN-miRNA-1/2/3使牛SCs和小鼠C2C12细胞活力和增殖能力分别下降了12%和12.5%(P<0.05),而MSTN-crRNA-1/2/3对细胞活力和增殖能力没有影响。上述结果说明,基于CRISPR/Cas13a构建的MSTN干扰载体优于miRNA。本研究为模式动物和大家畜的基因编辑研究与应用提供基础资料。RNA interference(RNAi)is mainly mediated by small interfering RNA(siRNA),short hairpin RNA(shRNA),microRNA(miRNA)and CRISPR/Cas13a.miRNA mediates RNAi through complementary pairing with target mRNA bases.CRISPR/Cas13a mediates RNAi through the combined action of CRISPRderived RNA(crRNA)and Cas13a proteins.Comparison of CRISPR/Cas13a-and miRNA-mediated RNAi efficiency has not been reported.In this study,according to the mRNA homologous sequence of myostatin gene(MSTN)in Simmental cattle(Bos taurus)and mouse(Mus musculous),MSTN-crRNA-1/2/3 and MSTNmiRNA-1/2/3 were designed.In order to eliminate the fluctuations of CRISPR/Cas13a interference efficiency caused by unstable Cas13a protein expression,firstly,cattle muscle satellite cell lines(SCs)and mouse myoblast cell lines C2C12 with stable expression of Cas13a protein were prepared.Then,MSTN-crRNA-1/2/3 were transfected into cattle Cas13a-SCs and mouse Cas13a-C2C12,respectively.Meanwhile,MSTNmiRNA-1/2/3 were transfected into cattle SCs and mouse C2C12,respectively.The efficiency of CRISPR/Cas13a-and miRNA-mediated MSTN interference was detected by qRT-PCR after 48 h transfection.In addition,MTT assay and Cell Counting Kit-8(CCK-8)assay were used to detect the effects of CRISPR/Cas13a-and miRNA-mediated MSTN knockdown on cell activity and proliferation.The results showed that the average and highest interference efficiency of MSTN-crRNA-1/2/3 in cattle Cas13a-SCs and mouse Cas13a-C2C12 were 45%and 54%,respectively.The average and highest interference efficiency of MSTNmiRNA-1/2/3 in cattle SCs and mouse C2C12 were 30%and 33%,respectively,indicating that CRISPR/Cas13a had higher interference efficiency than miRNA in cattle SCs and mouse C2C12.In addition,the cell viability and proliferation ability of MSTN-miRNA-1/2/3 in cattle SCs and mouse C2C12 decreased by 12%and 12.5%(P<0.05),respectively,while the cell viability and proliferation ability of MSTN-crRNA-1/2/3 had no effect.This study provides basic data for the research and application of gene editing in model ani

关 键 词：RNA干扰(RNAI) CRISPR/Cas13a 微小RNA(miRNA) 肌肉生长抑制素(MSTN) 

分 类 号：S813.3[农业科学—畜牧学]