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

Fang, L.; Wang, W.; Li, G.; Zhang, L.; Li, J.; Gan, D.; Yang, J.; Tang, Y.; Ding, Z.; Zhang, M.; Zhang, W.; Deng, D.; Song, Z.; Zhu, Q.; Cui, H.; Hu, Y.; Chen, W.

doi:10.1101/2020.10.03.324715

bioRxiv · 10.1101/2020.10.03.324715

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

Fang, L.·Wang, W.·Li, G.·Zhang, L.·Li, J.·Gan, D.·Yang, J.·Tang, Y.·Ding, Z.·Zhang, M.·Zhang, W.·Deng, D.·Song, Z.·Zhu, Q.·Cui, H.·Hu, Y.·Chen, W.

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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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Fang, L., Wang, W., Li, G., Zhang, L., Li, J., Gan, D., Yang, J., Tang, Y., Ding, Z., Zhang, M., Zhang, W., Deng, D., Song, Z., Zhu, Q., Cui, H., Hu, Y., Chen, W.. 2020-10-03. CIGAR-seq, a CRISPR/Cas-based method for unbiased screening of novel mRNA modification regulators. https://doi.org/10.1101/2020.10.03.324715

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