CIGAR‐seq,a CRISPR/Cas‐based method for unbiased screening of novel mRNA modification regulators |
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| Authors: | Liang Fang Wen Wang Guipeng Li Li Zhang Jun Li Diwen Gan Jiao Yang Yisen Tang Zewen Ding Min Zhang Wenhao Zhang Daqi Deng Zhengyu Song Qionghua Zhu Huanhuan Cui Yuhui Hu Wei Chen |
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| Institution: | 1. Department of Biology, Southern University of Science and Technology, Shenzhen Guangdong, China ; 2. Academy for Advanced Interdisciplinary Studies, Southern University of Science and Technology, Shenzhen Guangdong, China ; 3. Harbin Institute of Technology, Harbin Heilongjiang, China |
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| Abstract: | Cellular RNA is decorated with over 170 types of chemical modifications. Many modifications in mRNA, including m6A and m5C, have been associated with critical cellular functions under physiological and/or pathological conditions. To understand the biological functions of these modifications, it is vital to identify the regulators that modulate the modification rate. However, a high‐throughput method for unbiased screening of these regulators is so far lacking. Here, we report such a method combining pooled CRISPR screen and reporters with RNA modification readout, termed CRISPR integrated gRNA and reporter sequencing (CIGAR‐seq). Using CIGAR‐seq, we discovered NSUN6 as a novel mRNA m5C methyltransferase. Subsequent mRNA bisulfite sequencing in HAP1 cells without or with NSUN6 and/or NSUN2 knockout showed that NSUN6 and NSUN2 worked on non‐overlapping subsets of mRNA m5C sites and together contributed to almost all the m5C modification in mRNA. Finally, using m1A as an example, we demonstrated that CIGAR‐seq can be easily adapted for identifying regulators of other mRNA modification. |
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| Keywords: | CIGAR‐ seq m5C modification mRNA modification NSUN6 pooled CRISPR screen |
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