An efficient and precise method for generating knockout cell lines based on CRISPR-Cas9 system.
An efficient and precise method for generating knockout cell lines based on CRISPR-Cas9 system.
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基于CRISPR-Cas9系统的高效精准基因敲除细胞系生成方法
DOI：
10.1002/elsc.202000032
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发表时间：
2020-12
影响因子：
2.7
通讯作者：
Huang W
中科院分区：
文献类型：
--
作者：
Lu X;Guo Y;Gu S;Tan D;Cheng B;Li Z;Huang W
尽管CRISPR-Cas9系统的效率和通用性比传统的基因组编辑方法如锌指或TALEN有了很大的提高，但由于指导RNA（gRNA）的靶向位置或功效的差异，筛选敲除/敲入细胞克隆仍然耗时费力。在这里，我们采用了CRISPR-Cas9系统的靶向敲入策略，并表征了产生单或双敲除细胞系的效率。具体来说，将基于同源臂的供体盒与gRNA表达单元共转染，该供体盒由编码荧光蛋白的基因和由组成型启动子驱动的抗生素选择标记物组成。基于FACS分选和抗生素药物选择，通过基因分型和蛋白质表达水平确认阳性细胞克隆。结果表明，通过细胞分选和选择鉴定的超过70%的分析克隆在单敲除实验和双敲除实验中均被成功靶向。该程序需要不到三周的时间才能获得敲除细胞系。我们相信，这种方法可以在大多数细胞系中以高效率产生敲除细胞克隆中是适用的和通用的。
Although the efficiency and versatility of CRISPR‐Cas9 system has been greatly improved over conventional genome editing methods such as zinc finger or TALEN, it is still time‐consuming and labor‐intensive for screening knockout/knock‐in cell clones due to differences of the targeted location or efficacies of guide RNAs (gRNAs). Here, we adapted a targeted knock‐in strategy with CRISPR‐Cas9 system and characterized the efficiency for generating single or double knockout cell lines. Specifically, a homology‐arm based donor cassette consisting of genes encoding a fluorescence protein and antibiotic selection marker driven by a constitutive promoter was co‐transfected with a gRNA expressing unit. Based on FACS sorting and antibiotic drug selection, positive cell clones were confirmed by genotyping and at the protein expression level. The results indicated that more than 70% of analyzed clones identified by cell sorting and selection were successfully targeted in both single and double knockout experiments. The procedure takes less than three weeks to obtain knockout cell lines. We believe that this methodology could be applicable and versatile in generating knockout cell clones with high efficiency in most cell lines.
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影响因子：
3.5
作者：
Imai-Nishiya H;Mori K;Inoue M;Wakitani M;Iida S;Shitara K;Satoh M
通讯作者：
Satoh M
影响因子：
3.7
作者：
Li K;Wang G;Andersen T;Zhou P;Pu WT
通讯作者：
Pu WT
DOI：
10.1093/dnares/dsx029
发表时间：
2017-12-01
期刊：
DNA research : an international journal for rapid publication of reports on genes and genomes
影响因子：
--
作者：
Tálas A;Kulcsár PI;Weinhardt N;Borsy A;Tóth E;Szebényi K;Krausz SL;Huszár K;Vida I;Sturm Á;Gordos B;Hoffmann OI;Bencsura P;Nyeste A;Ligeti Z;Fodor E;Welker E
通讯作者：
Welker E
影响因子：
4.8
作者：
Wassef, M.;Luscan, A.;Margueron, R.
通讯作者：
Margueron, R.
Kindlin-2 将机械环境与脯氨酸合成和肿瘤生长联系起来
影响因子：
16.6
作者：
Guo, Ling;Cui, Chunhong;Wu, Chuanyue
通讯作者：
Wu, Chuanyue