Development of a CRISPR‐Cas9 Based Luciferase Turn‐On System as Nonhomologous End Joining Pathway Reporter
Development of a CRISPR‐Cas9 Based Luciferase Turn‐On System as Nonhomologous End Joining Pathway Reporter
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DOI：
10.1002/cbic.202100128
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发表时间：
2021-04
期刊：
影响因子：
3.2
通讯作者：
Yi Wang;Yanjie Zhao;Weijun Su;Xiaojing Guo;Shuai Li
中科院分区：
文献类型：
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作者：
Yi Wang;Yanjie Zhao;Weijun Su;Xiaojing Guo;Shuai Li
需要一种非同源末端连接 (NHEJ) 通路报告系统来促进 NHEJ 化学抑制剂的筛选和发现。在这项研究中，我们开发了一种基于 CRISPR-Cas9 的荧光素酶开启系统作为 NHEJ 通路报告基因。通过用 ATC 取代核苷酸 205C，我们在荧光素酶编码区中引入了阅读框移位和前终止密码子，从而生成了生物发光信号静音 HEK293T 报告细胞系。然后，将表达靶向荧光素酶编码区的向导RNA的CRISPR-Cas9质粒引入报告细胞系中，以生成NHEJ相关的插入缺失，以恢复阅读框并随后打开生物发光信号。我们观察到 CRISPR-Cas9 载体转染后信号增加了三千多倍。 NHEJ 通路的不同已知化学抑制剂，如 NU7441、KU0060648 和 KU55933，可以显着抑制 CRISPR-Cas9 靶向产生的生物发光信号。此外，我们通过高通量测序验证了我们的系统。
There is a need of a non‐homologous end joining (NHEJ) pathway reporter system that facilitates screening and discovery of NHEJ chemical inhibitors. In this study, we developed a CRISPR‐Cas9 based luciferase turn‐on system as a NHEJ pathway reporter. By substituting nucleotide 205C with ATC, we introduced a reading‐frame shift and a pre‐stop codon into the luciferase coding region and thereby generated a bioluminescent signal mute HEK293T reporter cell line. Then, a CRISPR‐Cas9 plasmid expressing a guide RNA targeting luciferase coding region was introduced into the reporter cell line to generate NHEJ‐associated indel to restore the reading frame and subsequently turn on the bioluminescent signal. We observed over three‐thousand fold increase in signal after CRISPR‐Cas9 vector transfection. Different known chemical inhibitors of the NHEJ pathway, such as NU7441, KU0060648, and KU55933, could significantly inhibit the bioluminescent signal generated by CRISPR‐Cas9 targeting. In addition, we validated our system by high throughput sequencing.