应用CRISPR/Cas9基因编辑技术构建沉默MARCH2的细胞系

Construction of MARCH2 KO Cell Line by CRISPR/Cas9 Technology

摘要: CRISPR/Cas9系统是一种先进的基因编辑技术，人工设计先导RNA (single-guide RNA, sgRNA)介导外源表达的Cas9蛋白特异性的结合、切割基因组靶点，切割后的DNA有非同源末端连接(non-homologous end joining, NHEJ)和同源重组(homologous recombination)两种修复方式，以构建基因特异性敲除或敲入细胞。The membrane associated RING-CH (MARCH)家族由一系列结构相关的蛋白组成，目前，在人类基因组中至少有11个已知成员(MARCH-1–11)。MARCH2参与囊泡运输的调节，如突触囊泡的形成、细胞内运动、胞外分泌和内吞。在本研究中，我们针对MARCH2基因第二外显子设计了CRISPR/Cas9干扰靶序列，并应用蛋白质印迹方法对其基因剔除的有效性进行了验证，结果显示在两个MARCH2等位基因都含有移码突变，证实我们应用CRISPR/Cas9系统成功构建了MARCH2敲除的细胞系，这将是MARCH2功能及相关机制研究的强有力的工具。

Abstract: CRISPR/Cas9 system is the state-of-the-art genome-editing technology through constitutive ex-pression of nucleases Cas9, which binds to a specific site in the genome mediated by single guide RNA (sgRNA) and induces double-strand breaks (DSBs) at desired genomic loci. DSBs induced by these site-specific nucleases can be repaired by error-prone nonhomologous end joining (NHEJ) or homologous recombination, to generate gene-specific knockout (KO) or knock-in cells. The mem-brane associated RING-CH (MARCH) family comprises a structurally related protein family. Cur-rently, there are at least eleven known MARCH genes (MARCH-1–11) in the human genome. MARCH2 is involved in the regulation of vesicle trafficking, and thus refers to the formation, intracellular movement, exocytosis, and endocytosis of synaptic vesicles. In this study, two sgRNAs targeting the Exon2 of MARCH2 gene were designed, and their gene targeting efficiency was assessed via the western blot analyses. Through dilution plating, a monoclonal MARCH2 KO cell line was isolated and subjected to the sequence analysis, which revealed that it contained frameshift mutations in both MARCH2 alleles. The results obtained demonstrate the successful application of the CRISPR/Cas9 system to the construction of the MARCH2 KO cell line, and strongly suggest that it is a promising tool for studying the functions and related mechanisms of MARCH2.