基于CRISPR/Cas9技术的HaCaT的TLR4基因定向敲除及其功能初步研究

Targeted knockout and functional analysis of TLR4 gene in cell line HaCaT by CRISPR/Cas9

[目的]构建敲除Toll Like Receptor 4(TLR4)基因的Ha Ca T细胞株,为后续利用该细胞株进行过敏原性研究提供材料。[方法]利用CRISPR/Cas9系统,根据靶向原理设计并合成4条特异性识别TLR4基因的向导RNA(single-molecule guide RNAs,sgRNAs),构建p X459-sgRNAh TLR4重组质粒,并转入Ha Ca T中,用嘌呤霉素筛选出单克隆阳性细胞。测序确认突变位点,然后利用脂多糖(lipopolysaccharide,LPS)刺激对TLR4进行功能验证来进一步确认敲除效果。[结果]测序结果表明#26单克隆细胞株在靶点附近缺失1 bp,造成TLR4编码基因的移码突变,蛋白翻译提前终止。功能性验证结果表明,在LPS的刺激下,IL-8和CCL20的mRNA水平分别下降约85%和90%,且IL-8的蛋白分泌水平也显著性下调(87%)。[结论]成功构建了敲除TLR4的稳定细胞株,并且验证TLR4的功能缺损。

[Objective]To construct Ha Ca T cell lines knocked out TLR4 gene for future use.[Method] CRISPR/Cas9 system was applied. Four single-guide RNAs( sgRNAs) targeted on TLR4 gene were synthesized and each was inserted into p X459 plasmid. Ha Ca T cells were transfected with the individual recombinant p X459-sgRNAh TLR4 plasmid,and positive clones were screened with puromycin. Mutational sites were verified by sequencing analysis. The knockout cell lines were validated by LPS stimulation.[Result] A TLR4 gene knockout Ha Ca T cell line(#26) was obtained,with a 1 bp deletion mutation in TLR4 gene,leading frameshift mutation and early termination of TLR4 translation. The levels of IL-8( 85%) and CCL20( 90%)mRNA decreased compared to wild type,and the IL-8 protein secretion also decreased significantly( 87%).[Conclusion]A TLR4 gene knockout cell line was constructed by using CRISPR/Cas9 method,and function of TLR4 was validated.