Liu Yangyang, Han Xiao, Yuan Junting, Geng Tuoyu, Chen Shihao, Hu Xuming, Cui Isabelle H, Cui Hengmi
From the Institute of Epigenetics and Epigenomics.
College of Animal Science and Technology.
J Biol Chem. 2017 Apr 7;292(14):5624-5633. doi: 10.1074/jbc.M116.769034. Epub 2017 Feb 14.
The type II bacterial CRISPR/Cas9 system is a simple, convenient, and powerful tool for targeted gene editing. Here, we describe a CRISPR/Cas9-based approach for inserting a poly(A) transcriptional terminator into both alleles of a targeted gene to silence protein-coding and non-protein-coding genes, which often play key roles in gene regulation but are difficult to silence via insertion or deletion of short DNA fragments. The integration of 225 bp of bovine growth hormone poly(A) signals into either the first intron or the first exon or behind the promoter of target genes caused efficient termination of expression of , (protein-coding genes), and (non-protein-coding gene). Both and were used as markers in the selection of clonal cell lines with biallelic integration of a poly(A) signal. Genotyping analysis indicated that the cell lines displayed the desired biallelic silencing after a brief selection period. These combined results indicate that this CRISPR/Cas9-based approach offers an easy, convenient, and efficient novel technique for gene silencing in cell lines, especially for those in which gene integration is difficult because of a low efficiency of homology-directed repair.
II型细菌CRISPR/Cas9系统是一种用于靶向基因编辑的简单、便捷且强大的工具。在此，我们描述了一种基于CRISPR/Cas9的方法，用于将聚腺苷酸（poly(A)）转录终止子插入目标基因的两个等位基因中，以沉默蛋白质编码基因和非蛋白质编码基因，这些基因在基因调控中通常起着关键作用，但通过插入或缺失短DNA片段很难实现沉默。将225bp的牛生长激素poly(A)信号整合到目标基因的第一个内含子、第一个外显子或启动子之后，可有效终止 、 （蛋白质编码基因）和 （非蛋白质编码基因）的表达。 和 均用作筛选具有双等位基因poly(A)信号整合的克隆细胞系的标记。基因分型分析表明，经过短暂的筛选期后，这些细胞系表现出预期的双等位基因沉默。这些综合结果表明，这种基于CRISPR/Cas9的方法为细胞系中的基因沉默提供了一种简单、便捷且高效的新技术，特别是对于那些由于同源定向修复效率低而难以进行基因整合的细胞系。
