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.