CRISPR/Cas9基因编辑技术构建靶向敲除小鼠微小RNA-101a基因的一体化载体系统

Construction of all-in-one CRISPR/Cas9 vector system targeting miR-101a gene in mouse hepatic cell line AML12

目的:利用CRISPR/Cas9基因编辑技术构建两种一体化载体,用于敲除AML12细胞系中的微小RNA(microRNA,miR)-101a。方法:设计三条小鼠靶向miR-101a基因的小向导RNA(sgRNA)以及一条靶向绿色荧光蛋白基因的sgRNA,分别构建至一体化载体系统(pENTRY-U6-sgRNA-EF1α-WT Cas9),并将sgRNA1和sgRNA3构建至一体化载体系统(pENTRY-U6-sgRNA-U6-sgRNA-EF1α-Cas9 D10A)。在AML12细胞系中转染构建的一体化质粒,72 h后用T7核酸内切酶Ⅰ(T7EⅠ)检测剪切效率。将pENTRY一体化质粒通过Gateway的方式重组到pAD载体中,再转染293A细胞进行腺病毒包装。在AML12细胞系中进行腺病毒感染,72 h后检测miR-101a成熟体的表达水平以评估敲除效率。结果:构建的pENTRY一体化质粒经测序鉴定后正确。质粒转染AML12细胞系后进行T7EⅠ检测,均发生了明显剪切形成了两条带,表明设计的sgRNA有效果。pENTRY-体化质粒重组到pAD载体中,经酶切鉴定正确。腺病毒转染AML12细胞系后,实时荧光定量PCR检测表明细胞中miR-101a成熟体表达水平较对照组均明显下降(均P<0.01)。结论:构建了具有较高敲除效率的靶向小鼠miR-101a基因的一体化载体系统,为后续微小RNA功能研究奠定了技术支撑和实验基础,同时也为其他微小RNA敲除提供了借鉴方法。

Objective： To develop an all-in-one CRISPR/Cas9 vector system that can efficiently knockdown miR-lOla expression in mice. Methods： Three sgRNAs targeting mouse miR-101a gene and a small guide （sgRNA） targeting green fluorescent protein gene were designed and constructed into an all-in-one vector system （pENTRY- U6-sgRNA-WT Cas9）. Moreover, sgRNA1 and sgRNA3 were selected and constructed into a double-nicking Cas9 vector （pENTRY-U6-sgRNA-U6-sgRNA-Cas9 D10A）. The constructed plasmids were transfeeted into mouse liver AML12 cells for validation by T7 Endo I （TTE I ） 72 h after transfection. The pAD vectors were cloned via the Gateway system, and the recombinant adenovirus vectors were packaged in 293A cells. The virus particles were used to infect AML12 cells and the expression levels of mature miR-101 a were analyzed to monitor the knockout efficiency after 72 h. Results： The constructed pENTRY all-in-one vectors were validated by gene sequencing and TTE I assay, which showed CRISPR/Cas9-mediated mismatches at target sites of miR-101a gene. The adenovirus vectors were constructed successfully. The CRISPR/Cas9 containing adenovirus was introduced to AML12 cells and the quantitative real-time PCR assays indicated that the expression level of mature miR-101a was significantly decreased compared with that of the control （ all P 〈 0.01 ）. Conclusions： We have successfully constructed two ＂all-in-one＂ CRISPR/Cas9 vector systems targeting miR-101a gene in mouse liver AML12 cells with high efficiency. It provides experimental basis for research of microRNA, and a reference method for knockout of other miRNAs.