Validation Study to Determine the Accuracy of Widespread Promoterless EGFP Reporter at Assessing CRISPR/Cas9-Mediated Homology Directed Repair.
Validation Study to Determine the Accuracy of Widespread Promoterless EGFP Reporter at Assessing CRISPR/Cas9-Mediated Homology Directed Repair.
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确定广泛使用的无启动子 EGFP 报告基因在评估 CRISPR/Cas9 介导的同源定向修复方面的准确性的验证研究
DOI：
10.3390/cimb44040116
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发表时间：
2022-04-12
影响因子：
3.1
通讯作者：
Huang, Lizhen
中科院分区：
文献类型：
--
作者：
Xu, Wanqing;Zuo, Qingxia;Feng, Dongyan;He, Changsheng;Lin, Cailing;Huang, Dongchao;Wan, Yanbin;Chen, Feng;Mo, Guosheng;Sun, Qi;Du, Hongli;Huang, Lizhen
关键词：
评估同源定向修复（HDR）的准确视觉报告系统是评估Cas9介导的精确基因编辑效率的关键先决条件。在本文中，我们测试了广泛的无启动子的EGFP报告基因的效用，以通过荧光表达评估CRISPR/Cas9介导的同源重组的效率。我们首先建立了一个靶向猪GAPDH位点的无启动子EGFP报告供体，以研究CRISPR/Cas9介导的猪细胞同源重组。奇怪的是，在有或没有Cas9/sgRNA的情况下，EGFP在猪细胞中从无启动子供体以出乎意料的高水平表达。当单独转染猪供体时，在人细胞和其他物种的细胞中检测到甚至更高的EGFP表达。因此，EGFP可以在单独用无启动子的EGFP报告基因转染的各种细胞中以一定水平表达，使其成为用于测量Cas9介导的HDR事件的低分辨率报告基因。总之，广泛存在的无启动子的EGFP报告基因可能不是HDR筛选的理想测量，并且迫切需要开发更可靠的高分辨率HDR筛选系统以更好地探索提高哺乳动物细胞中Cas9介导的HDR的效率的策略。
An accurate visual reporter system to assess homology-directed repair (HDR) is a key prerequisite for evaluating the efficiency of Cas9-mediated precise gene editing. Herein, we tested the utility of the widespread promoterless EGFP reporter to assess the efficiency of CRISPR/Cas9-mediated homologous recombination by fluorescence expression. We firstly established a promoterless EGFP reporter donor targeting the porcine GAPDH locus to study CRISPR/Cas9-mediated homologous recombination in porcine cells. Curiously, EGFP was expressed at unexpectedly high levels from the promoterless donor in porcine cells, with or without Cas9/sgRNA. Even higher EGFP expression was detected in human cells and those of other species when the porcine donor was transfected alone. Therefore, EGFP could be expressed at certain level in various cells transfected with the promoterless EGFP reporter alone, making it a low-resolution reporter for measuring Cas9-mediated HDR events. In summary, the widespread promoterless EGFP reporter could not be an ideal measurement for HDR screening and there is an urgent need to develop a more reliable, high-resolution HDR screening system to better explore strategies of increasing the efficiency of Cas9-mediated HDR in mammalian cells.
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影响因子：
16.6
作者：
Carlson-Stevermer J;Abdeen AA;Kohlenberg L;Goedland M;Molugu K;Lou M;Saha K
通讯作者：
Saha K
影响因子：
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Kuehn, Ralf
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影响因子：
14.9
作者：
Brinkman EK;Chen T;Amendola M;van Steensel B
通讯作者：
van Steensel B