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

Abstract

Cellular RNA is decorated with over 170 types of chemical modifications. Many modifications in mRNA, including m 6 A and m 5 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 m 5 C methyltransferase. Subsequent mRNA bisulfite sequencing in HAP1 cells without or with NSUN6 and/or NSUN2 knockout showed that NSUN6 and NSUN2 worked on nonoverlapping subsets of mRNA m 5 C sites and together contributed to almost all the m 5 C modification in mRNA. Finally, using m 1 A as an example, we demonstrated that CIGAR-seq can be easily adapted for identifying regulators of other mRNA modification.

Deciphering Epitranscriptome: Modification of mRNA Bases Provides a New Perspective for Post-transcriptional Regulation of Gene Expression

“…Recently, Fang et al (2020) reported a new method C RISPR i ntegrated g RNA a nd r eporter sequencing (CIGAR -seq) by combining pooled CRISPR screen and the reporters associated with RNA modification. Using the CIGAR-seq method, they could discover NSUN6 as a novel m 5 C methyltransferase in mRNA.…”

“…The recent advances in high-throughput next-generation sequencing (NGS) together with the novel chemogenetic RNA-labelng techniques have provided unprecedented opportunities to understand the RNA structure and functions. Such advances provide a better understanding of the presence and dynamics of base modifications like m 6 A ( Zhao et al, 2017b ), m 5 C ( Cui Q. et al, 2017 ; David et al, 2017 ; Fang et al, 2020 ), 5-hydroxymethylcytidine (hm 5 C) ( Huber et al, 2015 ; Delatte et al, 2016 ; Zhang et al, 2016 ), and m 1 A ( Dominissini et al, 2016 ; Li et al, 2016a ; Shen et al, 2016 ; Xiong et al, 2018 ) in RNAs. Base modifications, such as addition of 5′ cap (e.g., N 7 -methylguanosine, m 7 G-cap), and RNA editing are vital for mRNA stability ( Kiledjian, 2018 ), translation ( Topisirovic et al, 2011 ; Holstein et al, 2016 ) and functional diversity ( Peng et al, 2018 ).…”

Advances in mRNA 5-methylcytosine modifications: Detection, effectors, biological functions, and clinical relevance

“…Therefore, the function of NSUN2 in m 5 C modifications may be closely related to the position of the methylation or the specific modified target gene. Fang et al 38 discovered NSUN6, a new mRNA m 5 C methyltransferase, using CRISPR integrated gRNA and reporter sequencing (CIGAR-seq), a CRISPR-Cas9-based method for the unbiased screening of novel mRNA modification effectors. Using mRNA bisulfite sequencing in NSUN6-and/or NSUN2-knockout a near-haploid human leukemia cell line (HAP1) cells, they reported that the mRNA m 5 C sites targeted by the two methylases do not overlap and that these enzymes are responsible for nearly all of the m 5 C modifications in the mRNA.…”

“…In mRNA, m 5 C modifications, along with multiple effector enzymes, such as NOP2/Sun RNA methyltransferase 2 (NSUN2), 37 NSUN6, 38 tRNA aspartic acid methyltransferase 1 (TRDMT1), 39 and Aly/REF export factor (ALYREF), 40 perform numerous functions, including the promotion of mRNA nuclear-cytoplasmic trafficking; 40 viral protein expression; 41 DNA damage repair; 42 mRNA stability; 43 cell tolerance, 44 proliferation and migration; 39 development, differentiation, and reprogramming of stem cells; 45,46 and regulation of mRNA splicing. 47 In addition, the distribution of m 5 C varies among cell types.…”

The epitranscriptome: At the crossroad of cancer prognosis

“…Furthermore, the authors identified that m5C level correlated with decreased expression of the methyltransferase NOP2/Sun RNA Methyltransferase 6 (NSUN6) [4] . The importance of its activity was recently determined by observing augmented translation of those mRNAs, that were methylated by NSUN6 [ 5 , 6 ]. The emerging role of NSUN6, and m5C methylation mediated by its activity, has been also found in gastrointestinal and head and neck cancers [ 7 , 8 ].…”