基于CRISPR/Cas9技术构建多重sgRNA实现在人原代T细胞中敲除ADRB2基因

ADRB2 Gene Knockout in Human Primary T Cells by Multiple sgRNAs Construced using CRISPR/Cas9 Technology

目的:利用CRISPR/Cas9技术,通过多重sgRNA策略,实现在人原代T细胞中ADRB2基因的快速、高效敲除。方法:针对ADRB2基因5’端组成性外显子设计6条sgRNA,通过与pGL3-U6-sgRNA-PGK-Puro载体连接,构建6个单一sgRNA表达载体,分别与Cas9表达载体瞬时转染HEK-293T细胞;通过T7EN I酶切检测其介导的切割效率,筛选出4条高活性的配对sgRNA。继而将4条sgRNA有序串联克隆至pGL3-U6-sgRNA-ccdB-EF1α-Puro载体,构建成靶向ADRB2基因的多重sgRNA表达载体。瞬时转染HEK-293T细胞后,经T7EN I酶切和TA克隆测序明确其对ADRB2基因的修饰效率及方式。通过电穿孔技术将多重sgRNA质粒与Cas9质粒导入人原代T细胞。运用流式细胞术(FCM)检测该方法对靶蛋白β2肾上腺素能受体(β2 adrenergic receptor,β2-AR)的敲除效率。结果:T7EN I酶切和TA克隆测序分析的结果均表明,与单一sgRNA相比,多重sgRNA策略可获得更丰富的突变类型和更高的基因编辑效率。突变模式除了碱基的缺失、插入,还有大片段DNA缺失、倒位。随机送检的10个TA克隆全部发生了基因修饰,突变效率高达100%,且均出现了提前终止密码子。FCM检测发现,对照组中β2-AR阳性T细胞比例为43.09%,但转染多重sgRNA/Cas9后β2-AR的阳性表达率下降至25.61%。结论:本研究初步建立了在人原代T细胞中敲除β2-AR的技术方法,且该方法简便、可操作性强。本研究为进一步深入探索β2-AR在人T细胞抗肿瘤免疫中的作用奠定了技术基础。

Objective:To knockout ADRB2 gene rapidly and efficiently in human primary T cells by using CRISPR/Cas9 technology and multiple sgRNAs strategy.Methods:Six paired-sgRNAs,which were designed to target the 5’constitutive coding exons of ADRB2 gene,were cloned into pGL3-U6-sgRNA-PGK-Puro vector separately.The expression vectors containing the single sgRNAs were constructed and transiently co-transfected into HEK-293 T cell line with Cas9 expression vector.The sgRNA-mediated cleavage efficiency was tested by T7EN I digestion assay.Concatenating four highly efficient paired sgRNAs were cloned into pGL3-U6-sgRNA-ccdB-EF1α-Puro expression vector.The recombinant plasmid allows the cells to express 4 sgRNAs,which target different sites on the ADRB2 genomic locus.The cleavage efficiency and mutation model were tested by T7 EN I digest assay and T-A cloning technique.Multiple sgRNAs plasmid and Cas9 plasmid was transiently transferred into human primary T cells by electroporation.Flow cytometry(FCM)was used to detect the knockout efficiency ofβ2 adrenergic receptor(β2-AR).Results:The results of T7 EN I digestion and TA cloning sequencing showed that the multiple sgRNAs strategy could obtain more abundant mutation types and higher gene editing efficiency than single sgRNA.In addition to the deletion and insertion of bases,large fragment DNA deletions and inversions could be observed.All of the random 10 TA clones for detection were genetically modified,thus the mutation efficiency was as high as 100%.FCM assay showed that 43.09%of the cells in the control T cells wereβ2-AR positive,but the proportion ofβ2-AR positive cells in the multiple sgRNAs electrotransformed T cells decreased to 25.61%.Conclusion:A method,which is simple and operable,for knocking outβ2-AR in human primary T cells has been established preliminarily.The results are helpful for the further study of the role ofβ2-AR in human T cells.