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

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

PROVIDER: PRJNA661068 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR12573801_1.fastq.gz	Fastqsanger.gz
	SRR12573801_2.fastq.gz	Fastqsanger.gz
	SRR12573802_1.fastq.gz	Fastqsanger.gz
	SRR12573802_2.fastq.gz	Fastqsanger.gz
	SRR12573803_1.fastq.gz	Fastqsanger.gz

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

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Project description: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.

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