利用Crispr/Cas9技术构建影响先天性心脏病关键基因的表达载体

Construction of congenital heart disease key gene expression vector by Crispr/Cas9 technology

目的利用Crispr/Cas9技术构建影响先天性心脏病关键基因表达载体,为后续基因编辑工作做好前期分子实验基础。方法利用NCBI数据库检索调控先天性心脏病关键基因NKX2-5、GATA4、TBX5,通过生物软件在线设计3个基因对应的sgRNA。对慢性病毒载体lentiCRISPRv2进行BsmB I单酶切,通过胶回收获得线性化载体。利用T4连接酶将处理后的双链sgRNA和载体连接,通过大肠杆菌转化、质粒提取和测序最终得到lentiCRISPRv2-NKX2-5、lentiCRISPRv2-GATA4、lentiCRISPRv2-TBX5 3种病毒表达载体。结果获得评分最高且碱基数量合适的sgRNA,通过对表达载体的线性化酶切,成功去除载体中2 000 bp左右的置换片段,对连接后的载体测序显示双链sgRNA与病毒表达载体置换片段序列一致。结论利用Crispr/Cas9可成功构建先天性心脏病关键基因NKX2-5、GATA4、TBX5病毒表达载体。

Objective Crispr/Cas9 technology was used to construct the expression vector of key genes affecting congenital heart disease and the basis of preliminary molecular experiments was carried out for subsequent gene editing experiments. Methods The key genes NKX2-5, GATA4 and TBX5 of congenital heart disease were searched by NCBI database, and the corresponding sgRNA of three genes was designed online by biosoftware. The chronic virus vector lentiCRISPRv2 was digested by BsmB I single enzyme, the linearized vector was obtained by gel recovery. T4 ligase was used to connect the treated double-stranded sgRNA with the vector. Three viral expression vectors, lentiCRISPRv2-NKX2-5, lentiCRISPRv2-GATA4 and lentiCRISPRv2-TBX5, were finally obtained by Escherichia coli transformation, plasmid extraction and sequencing. Results The sgRNA with the highest score and the right number of bases was obtained, through linear enzymatic digestion of the expression vector, the displaced fragments of about 2 000 bp in the vector were successfully removed. Sequencing of the linked vectors showed that the sequence of double-stranded sgRNA was identical with that of the replacement fragment of the viral expression vector. Conclusion The expression vectors of the key genes of congential heart disase NKX2-5, GATA4 and TBX5 were successfully constructed using Crispr/Cas9.