CIGAR-seq, a CRISPR/Cas-based method for unbiased screening of novel mRNA modification regulators.
Mol Syst Biol
; 16(11): e10025, 2020 11.
Article
em En
| MEDLINE
| ID: mdl-33251765
ABSTRACT
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.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
RNA Mensageiro
/
Processamento Pós-Transcricional do RNA
/
Análise de Sequência de RNA
/
Sistemas CRISPR-Cas
/
Metiltransferases
Tipo de estudo:
Diagnostic_studies
/
Screening_studies
Limite:
Humans
Idioma:
En
Revista:
Mol Syst Biol
Assunto da revista:
BIOLOGIA MOLECULAR
/
BIOTECNOLOGIA
Ano de publicação:
2020
Tipo de documento:
Article
País de afiliação:
China