TY - JOUR
T1 - CIGAR-seq, a CRISPR/Cas-based method for unbiased screening of novel mRNA modification regulators
AU - Fang, Liang
AU - Wang, Wen
AU - Li, Guipeng
AU - Zhang, Li
AU - Li, Jun
AU - Gan, Diwen
AU - Yang, Jiao
AU - Tang, Yisen
AU - Ding, Zewen
AU - Zhang, Min
AU - Zhang, Wenhao
AU - Deng, Daqi
AU - Song, Zhengyu
AU - Zhu, Qionghua
AU - Cui, Huanhuan
AU - Hu, Yuhui
AU - Chen, Wei
PY - 2020/11/30
Y1 - 2020/11/30
N2 - 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.
AB - 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.
U2 - 10.15252/msb.202010025
DO - 10.15252/msb.202010025
M3 - Article
SN - 1744-4292
VL - 16
JO - Molecular Systems Biology
JF - Molecular Systems Biology
IS - 11
M1 - e10025
ER -