Application of counter-selectable marker PIGA in engineering designer deletion cell lines and characterization of CRISPR deletion efficiency.
Application of counter-selectable marker PIGA in engineering designer deletion cell lines and characterization of CRISPR deletion efficiency.
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DOI：
10.1093/nar/gkab035
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发表时间：
2021-03-18
影响因子：
14.9
通讯作者：
Boeke JD
中科院分区：
文献类型：
--
作者：
Li D;Sun X;Yu F;Perle MA;Araten D;Boeke JD
CRISPR/Cas9系统是一种基因组工程技术，已被应用于基因的indel突变以及靶向基因的缺失和替换。在这里，我们描述了人类X染色体PIGA位点上的配对gRNA缺失，范围从17 kb到2 Mb。我们发现缺失大小和缺失效率之间没有明显的线性相关性，拓扑相关结构域对缺失频率没有实质性的影响。利用这种精确的缺失技术，我们设计了一系列的设计缺失细胞系，包括两个x染色体反选择（负选择）标记PIGA和HPRT1缺失的细胞系，以及每个缺失的附加细胞系。PIGA编码糖基磷脂酰肌醇（GPI）锚定生物合成装置的一个组成部分。PIGA基因反选择标记具有独特的特征，包括现有的PIGA功能和功能丧失的单细胞水平检测，以及有效的反选择剂proaerolysin的存在，我们经常使用它来选择表达PIGA的细胞。这些设计细胞系可以作为具有多种选择标记的通用平台，对于诸如基因组计划-写入（GP-write）之类的大规模基因组工程项目尤其有用。
The CRISPR/Cas9 system is a technology for genome engineering, which has been applied to indel mutations in genes as well as targeted gene deletion and replacement. Here, we describe paired gRNA deletions along the PIGA locus on the human X chromosome ranging from 17 kb to 2 Mb. We found no compelling linear correlation between deletion size and the deletion efficiency, and there is no substantial impact of topologically associating domains on deletion frequency. Using this precise deletion technique, we have engineered a series of designer deletion cell lines, including one with deletions of two X-chromosomal counterselectable (negative selection) markers, PIGA and HPRT1, and additional cell lines bearing each individual deletion. PIGA encodes a component of the glycosylphosphatidylinositol (GPI) anchor biosynthetic apparatus. The PIGA gene counterselectable marker has unique features, including existing single cell level assays for both function and loss of function of PIGA and the existence of a potent counterselectable agent, proaerolysin, which we use routinely for selection against cells expressing PIGA. These designer cell lines may serve as a general platform with multiple selection markers and may be particularly useful for large scale genome engineering projects such as Genome Project-Write (GP-write).
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影响因子：
20.3
作者：
Endo, M;Ware, RE;Parker, CJ
通讯作者：
Parker, CJ
影响因子：
11.4
作者：
Aylon, Y;Liefshitz, B;Kupiec, M
通讯作者：
Kupiec, M
影响因子：
2.8
作者：
Bryce, Steven M.;Bemis, Jeffrey C.;Dertinger, Stephen D.
通讯作者：
Dertinger, Stephen D.
影响因子：
16
作者：
Araujo, Ana Rita;Gelens, Lendert;Sheriff, Rahuman S. M.;Santos, Silvia D. M.
通讯作者：
Santos, Silvia D. M.
影响因子：
3.2
作者：
HOWARD, SP;BUCKLEY, JT
通讯作者：
BUCKLEY, JT