利用CRISPR技术生成突变肾细胞系
Generating Mutant Renal Cell Lines Using CRISPR Technologies.
作者信息
Perretta-Tejedor Nuria, Freke Grace, Seda Marian, Long David A, Jenkins Dagan
机构信息
Developmental Biology and Cancer, Great Ormond Street Institute of Child Health, University College London, London, UK.
Genetics and Genomic Medicine Programmes, Great Ormond Street Institute of Child Health, University College London, London, UK.
出版信息
Methods Mol Biol. 2020;2067:323-340. doi: 10.1007/978-1-4939-9841-8_20.
Gene editing using the CRISPR/Cas9 system is an extremely efficient approach for generating mutations within the genomic DNA of immortalized cell lines. This procedure begins with a straightforward cloning step to generate a single plasmid encoding the Cas9 enzyme as well as a synthetic guide RNA (sgRNA) which is selected to target specific sites within the genome. This plasmid is transfected into cells either alone, in order to generate random insertion-deletion alleles ("indels") at the desired locus via the nonhomologous end-joining pathway, or in conjunction with a homology-directed repair template oligonucleotide to generate a specific point mutation. Here we describe a procedure to perform gene editing in IMCD3 and HEK293 cells and to subsequently isolate clonal cell lines carrying mutations of interest.
使用CRISPR/Cas9系统进行基因编辑是在永生化细胞系的基因组DNA内产生突变的一种极其有效的方法。该过程始于一个简单的克隆步骤，以生成一个编码Cas9酶以及合成引导RNA（sgRNA）的单一质粒，该sgRNA被选择用于靶向基因组内的特定位点。该质粒可以单独转染到细胞中，以便通过非同源末端连接途径在所需位点产生随机插入缺失等位基因（“indels”），或者与同源定向修复模板寡核苷酸一起转染以产生特定的点突变。在这里，我们描述了一种在IMCD3和HEK293细胞中进行基因编辑并随后分离携带感兴趣突变的克隆细胞系的方法。
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