Abstract：
CRISPR/Cas9 is a powerful tool for gene editing in various cell types and organisms. However, it is still challenging to screen genetically modified cells from an excess of unmodified cells. Our previous studies demonstrated that surrogate reporters can be used for efficient screening of genetically modified cells. Here, we developed two novel traffic light screening reporters, puromycin-mCherry-EGFP (PMG) based on single-strand annealing (SSA) and homology-directed repair (HDR), respectively, to measure the nuclease cleavage activity within transfected cells and to select genetically modified cells. We found that the two reporters could be self-repaired coupling the genome editing events driven by different CRISPR/Cas nucleases, resulting in a functional puromycin-resistance and EGFP selection cassette that can be afforded to screen genetically modified cells by puromycin selection or FACS enrichment. We further compared the novel reporters with different traditional reporters at several endogenous loci in different cell lines, for the enrichment efficiencies of genetically modified cells. The results indicated that the SSA-PMG reporter exhibited improvements in enriching gene knockout cells, while the HDR-PMG system was very useful in enriching knock-in cells. These results provide robust and efficient surrogate reporters for the enrichment of CRISPR/Cas9-mediated editing in mammalian cells, thereby advancing basic and applied research.
摘要：
CRISPR/Cas9是在各种细胞类型和生物体中进行基因编辑的强大工具。然而,从过量的未修饰细胞中筛选遗传修饰细胞仍然具有挑战性。我们以前的研究表明，替代报告基因可用于有效筛选转基因细胞。这里,我们开发了两个新颖的交通灯筛选记者，基于单链退火（SSA）和同源定向修复（HDR）的嘌呤霉素-mCherry-EGFP（PMG），分别,以测量转染细胞内的核酸酶切割活性并选择遗传修饰的细胞。我们发现，这两个报告基因可以自我修复，耦合由不同的CRISPR/Cas核酸酶驱动的基因组编辑事件，产生功能性嘌呤霉素抗性和EGFP选择盒,其可以被提供用于通过嘌呤霉素选择或FACS富集来筛选遗传修饰的细胞。我们进一步比较了新的记者与不同的传统记者在几个内源位点在不同的细胞系，遗传修饰细胞的富集效率。结果表明，SSA-PMG报告基因在富集基因敲除细胞方面表现出改善，而HDR-PMG系统在富集敲入细胞中非常有用。这些结果为哺乳动物细胞中CRISPR/Cas9介导的编辑的富集提供了强大而有效的替代报告基因。从而推进基础研究和应用研究。
