Programmable m6A modification of cellular RNAs with a Cas13-directed methyltransferase

Abstract

N 6 -methyladenosine (m 6 A) is the most widespread internal mRNA modification in humans. Despite recent progress in understanding the biological roles of m 6 A, the inability to install m 6 A site-specifically in individual transcripts has hampered efforts to elucidate causal relationships between the presence of a specific m 6 A and phenotypic outcomes. Here we demonstrate that nucleus-localized dCas13 fusions with a truncated METTL3 methyltransferase domain and cytoplasm-localized fusions with a modified METTL3:METTL14 methyltransferase complex can direct site-specific m 6 A incorporation in distinct cellular compartments, with the former fusion protein having particularly low off-target activity. Independent cellular assays across multiple sites confirm that this targeted RNA methylation (TRM) system mediates efficient m 6 A installation in endogenous RNA transcripts with high specificity. Finally, we show that TRM can induce m 6 A-mediated changes to transcript abundance and alternative splicing. These findings establish TRM as a tool for targeted epitranscriptome engineering to help reveal the effect of individual m 6 A modifications and dissect their functional roles.

Roles of N6-Methyladenosine (m6A) in Stem Cell Fate Decisions and Early Embryonic Development in Mammals

“…New or refined m 6 A-seq technologies with low amounts of RNA samples input (>500 ng total RNA) or antibody-independent methods (MAZTER-seq, m 6 A-REF-seq, DART-seq, m 6 A-SEAL, and m 6 A-label-seq) have great application potential in the study of the m 6 A epitranscriptome in relation to disease treatment and embryonic development ( Zeng et al, 2018 ; Meyer, 2019 ; Garcia-Campos et al, 2019 ; Zhang Z. et al, 2019 ; Shu et al, 2020 ; Wang et al, 2020 ). Moreover, recent studies have developed m 6 A editing technologies that enable methylation or demethylation at a single site in transcripts through CRISPR-dCas9 and CRISPR-dCas13b systems ( Rauch et al, 2018 ; Liu X. M. et al, 2019 ; Wilson et al, 2020 ; Li J. et al, 2020 ). We believe that m 6 A modification has great potential for application in regenerative and precision medicine, including cancer treatment, organ transplantation and reproductive development.…”

CRISPR-Cas13 mediated Knock Down inDrosophilacultured cells

“…Here, we describe the use and generation of a new toolkit to perturb gene expression in Drosophila. The CRISPR-Cas13 system can be used for RNA knockdown with greater accuracy than RNAi 13,19 , but also to image RNA molecules 12,28 , manipulate the epitranscriptome 29,30 , edit transcripts 14 , and alter splicing 19,31 , but the use of this system in Drosophila has been limited by the potential toxicity of the nucleases 22 Our Drosophila cell-line is compatible with large-scale pooled screening, and therefore could be used to perform pooled, variable-dose screening 33 . Last but not least, we achieved tunable expression of PspCas13b by decreasing its translation through the addition of upstream ORFs of different lengths.…”

“…Last but not least, we achieved tunable expression of PspCas13b by decreasing its translation through the addition of upstream ORFs of different lengths. This strategy has successfully been employed in many biological contexts, including for balancing Cas9 activity and toxicity in Cas13 fusions have been used to precisely edit the epi-transcriptome or alter splicing 14,21,[29][30][31] . Utilizing these reagents in Drosophila could reveal the developmental need for epi-transcriptome modification of transcripts in time and space and could be used to determine the developmental function of particular splicing changes.…”

Advances in biological functions of RNA chemical modification m<sup>6</sup>A

“…利用CRISPR-Cas9系统最大的问 题在于需要PAM(PAMer)序列, 然而利用不需要PAM 序列的Cas13则可以进一步拓宽序列的选择性. 利用 CRISPR-dCas13与ALKBH5或METTL3-METTL14的 融合蛋白也可以实现对m 6 A的定向编辑 [109,110] . 通过这 些定向m 6 [62,111] .…”