转基因啮齿类动物的胚胎干细胞（embryonic stem cells, ESCs）是研究发育模型和疾病模型等的重要工具。本研究基于最新型的基因靶向修饰技术CRISPR-Cas9系统，在小鼠胚胎干细胞系中敲除Ago1和Dicer1基因，以影响RNA诱导的转录后沉默调控（RNA interference, RNAi）。该系统由一个设计好的向导RNA即sgRNA和一种高效核酸内切酶即Cas9蛋白组成，其中sgRNA根据碱基互补配对靶向基因组特异片段，Cas9通过与sgRNA识别而到达特异位点，高效切割DNA形成双链缺口（DSB），利用DNA自身的易错非同源末端修复（NHEJ）产生碱基的随机丢失或插入（indels），从而获得敲除基因的目的。该文根据敲除基因第一和第二外显子中NGG结尾的23bp的spacer序列构建载体，将sgRNA质粒与Cas9-eGFP质粒共同转染小鼠ESCs，流式筛选GFP阳性细胞，培养一段时间后，挑出单克隆，通过T7内切酶酶切法和测序来鉴定基因型。成功得到1个Ago1-/-Dicer1-/-细胞系，该实验为以后探究RNA介导的转录后沉默调控过程与小鼠ESCs多能性维持之间可能存在的关系奠定基础。

Recently a new kind of targeted genome editing, Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated system(CRISPR-cas system), has emerged after ZFN and TALEN. In this system, one or several sgRNAs(single guide RNAs) are designed to target specific genome sites and then guide effective nuclease Cas9 protein to cause DNA double-strand break, and then activates NHEJ(non-homologous end joining ) which is easy to introduce insertion/deletion mutations (indels) or HDR(homology-directed repair ) when there are donor homologous strands. Besides, embryonic stem(ES) cells are derived from ICMs in the blastocyst and cultured in certain condition in vitro to maintain self-renew and have pluripotency to differentiate into cells of all the three germ layers, and that’s why researchers are inclined to study development process, function of special genes and build disease models and so on. What’s more, we have interest in whether Dicer1 and Ago1 are related to self-renewal of ES cells. In the RNAi process, dicer and other ribonucleases convert long double stranded RNAs(dsRNAs) into small 21–25 nt long dsRNAs (siRNAs, miRNAs) . The RISC( RNA induced silencing complex ) consists of siRNAs/miRNAs as guide RNA to target on special cites in mRNAs and Ago (Argonaute) protein as catalytic subunits to cleave mRNAs, which cause post-transcription suppression. Here we report knocking out Ago1 and Dicer1 in mouse embryonic stem cells(ES cells) using CRISPR/Cas9 system. We transfect several sgRNAs plasmids and Cas9-eGFP plasmid into ES cells and sort GFP+ cells by FACS, then pick single clonies 7 days later and genotyping to identify mutated clonies. Finally we get an Ago1-/-Dicer1-/- clony. This work lays the foundation to study whether RNAi relates to self-renewal of ES cells.