Chiang Ting-Wei Will, le Sage Carlos, Larrieu Delphine, Demir Mukerrem, Jackson Stephen P
Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge CB2 1QN, UK.
Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, UK.
Sci Rep. 2016 Apr 15;6:24356. doi: 10.1038/srep24356.
The RNA-guided Cas9 nuclease is being widely employed to engineer the genomes of various cells and organisms. Despite the efficient mutagenesis induced by Cas9, off-target effects have raised concerns over the system's specificity. Recently a "double-nicking" strategy using catalytic mutant Cas9(D10A) nickase has been developed to minimise off-target effects. Here, we describe a Cas9(D10A)-based screening approach that combines an All-in-One Cas9(D10A) nickase vector with fluorescence-activated cell sorting enrichment followed by high-throughput genotypic and phenotypic clonal screening strategies to generate isogenic knockouts and knock-ins highly efficiently, with minimal off-target effects. We validated this approach by targeting genes for the DNA-damage response (DDR) proteins MDC1, 53BP1, RIF1 and P53, plus the nuclear architecture proteins Lamin A/C, in three different human cell lines. We also efficiently obtained biallelic knock-in clones, using single-stranded oligodeoxynucleotides as homologous templates, for insertion of an EcoRI recognition site at the RIF1 locus and introduction of a point mutation at the histone H2AFX locus to abolish assembly of DDR factors at sites of DNA double-strand breaks. This versatile screening approach should facilitate research aimed at defining gene functions, modelling of cancers and other diseases underpinned by genetic factors, and exploring new therapeutic opportunities.
RNA引导的Cas9核酸酶正被广泛用于改造各种细胞和生物体的基因组。尽管Cas9能高效诱导突变，但脱靶效应引发了人们对该系统特异性的担忧。最近，一种使用催化突变体Cas9（D10A）切口酶的“双切口”策略已被开发出来，以尽量减少脱靶效应。在此，我们描述了一种基于Cas9（D10A）的筛选方法，该方法将一体化Cas9（D10A）切口酶载体与荧光激活细胞分选富集相结合，随后采用高通量基因型和表型克隆筛选策略，以高效产生等基因敲除和敲入，同时使脱靶效应最小化。我们通过在三种不同的人类细胞系中靶向DNA损伤反应（DDR）蛋白MDC1、53BP1、RIF1和P53以及核结构蛋白核纤层蛋白A/C的基因来验证了这种方法。我们还使用单链寡脱氧核苷酸作为同源模板，高效获得了双等位基因敲入克隆，用于在RIF1位点插入EcoRI识别位点，并在组蛋白H2AFX位点引入点突变，以消除DDR因子在DNA双链断裂位点的组装。这种通用的筛选方法应有助于开展旨在确定基因功能、对由遗传因素引起的癌症和其他疾病进行建模以及探索新治疗机会的研究。
