CIGAR-seq, a CRISPR/Cas-based method for unbiased screening of novel mRNA modification regulators

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*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract / Description of output

Cellular RNA is decorated with over 170 types of chemical modifications. Many modifications in mRNA, including m6 A and m5 C, 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 m5 C 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 m5 C sites and together contributed to almost all the m5 C modification in mRNA. Finally, using m1 A as an example, we demonstrated that CIGAR-seq can be easily adapted for identifying regulators of other mRNA modification.
Original languageEnglish
Article numbere10025
JournalMolecular Systems Biology
Volume16
Issue number11
DOIs
Publication statusPublished - 30 Nov 2020

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