Open AccessCIGAR‐seq, a CRISPR/Cas‐based method for unbiased screening of novel mRNA modification regulators
Author(s) -
Fang Liang,
Wang Wen,
Li Guipeng,
Zhang Li,
Li Jun,
Gan Diwen,
Yang Jiao,
Tang Yisen,
Ding Zewen,
Zhang Min,
Zhang Wenhao,
Deng Daqi,
Song Zhengyu,
Zhu Qionghua,
Cui Huanhuan,
Hu Yuhui,
Chen Wei
Publication year - 2020
Publication title -
molecular systems biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 8.523
H-Index - 148
ISSN - 1744-4292
DOI - 10.15252/msb.202010025
Subject(s) - biology , crispr , computational biology , trans activating crrna , genetics , genome editing , gene
Cellular RNA is decorated with over 170 types of chemical modifications. Many modifications in mRNA, including m 6 A and m 5 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 C RISPR i ntegrated g RNA a nd r eporter sequencing (CIGAR‐seq). Using CIGAR‐seq, we discovered NSUN6 as a novel mRNA m 5 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 m 5 C sites and together contributed to almost all the m 5 C modification in mRNA. Finally, using m 1 A as an example, we demonstrated that CIGAR‐seq can be easily adapted for identifying regulators of other mRNA modification.