Doench John G, Hartenian Ella, Graham Daniel B, Tothova Zuzana, Hegde Mudra, Smith Ian, Sullender Meagan, Ebert Benjamin L, Xavier Ramnik J, Root David E
1] Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA. [2].
Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.
Nat Biotechnol. 2014 Dec;32(12):1262-7. doi: 10.1038/nbt.3026. Epub 2014 Sep 3.
Components of the prokaryotic clustered, regularly interspaced, short palindromic repeats (CRISPR) loci have recently been repurposed for use in mammalian cells. The CRISPR-associated (Cas)9 can be programmed with a single guide RNA (sgRNA) to generate site-specific DNA breaks, but there are few known rules governing on-target efficacy of this system. We created a pool of sgRNAs, tiling across all possible target sites of a panel of six endogenous mouse and three endogenous human genes and quantitatively assessed their ability to produce null alleles of their target gene by antibody staining and flow cytometry. We discovered sequence features that improved activity, including a further optimization of the protospacer-adjacent motif (PAM) of Streptococcus pyogenes Cas9. The results from 1,841 sgRNAs were used to construct a predictive model of sgRNA activity to improve sgRNA design for gene editing and genetic screens. We provide an online tool for the design of highly active sgRNAs for any gene of interest.
原核生物成簇的、规律间隔的短回文重复序列(CRISPR)位点的组成部分最近已被重新用于哺乳动物细胞。与CRISPR相关的(Cas)9蛋白可通过单个导向RNA(sgRNA)进行编程,以产生位点特异性DNA断裂,但关于该系统的靶向效率,已知的规则很少。我们创建了一组sgRNA,覆盖六个内源性小鼠基因和三个内源性人类基因的所有可能靶位点,并通过抗体染色和流式细胞术定量评估了它们产生靶基因无效等位基因的能力。我们发现了能提高活性的序列特征,包括对化脓性链球菌Cas9的原间隔序列临近基序(PAM)的进一步优化。1841个sgRNA的结果被用于构建sgRNA活性预测模型,以改进用于基因编辑和遗传筛选的sgRNA设计。我们提供了一个在线工具,用于设计针对任何感兴趣基因的高活性sgRNA。