Abstract：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 CRISPR-derived 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 MSTN-miRNA-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, MSTN-miRNA-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 MSTN-miRNA-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 animals and large livestocks.
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. CRISPR/Cas13a和MicroRNA介导的牛MSTN基因RNA干扰效率的比较[J]. 农业生物技术学报, 2024, 32(1): 147-157.
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. RNA Interference Efficiency Comparison of Bovine (Bos taurus) MSTN Gene Mediated by CRISPR-Cas13a and MicroRNA. 农业生物技术学报, 2024, 32(1): 147-157.
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