Abstract
Studies on biological functions of N6-methyladenosine (m6A) modification in mRNA have drawn significant attention in recent years. Here we describe the construction and characterization of a CRISPR–Cas13b-based tool for targeted demethylation of specific mRNA. A fusion protein, named dm6ACRISPR, was created by linking a catalytically inactive Type VI-B Cas13 enzyme from Prevotella sp. P5–125 (dPspCas13b) to m6A demethylase AlkB homolog 5 (ALKBH5). dm6ACRISPR specifically demethylates m6A of targeted mRNA such as cytochrome b5 form A (CYB5A) to increase its mRNA stability. It can also demethylate β-catenin-encoding CTNNB1 mRNA that contains multiple m6A sites to trigger its translation. In addition, the dm6ACRISPR system incurs efficient demethylation of targeted epitranscriptome transcripts with limited off-target effects. Targeted demethylation of transcripts coding for oncoproteins such as epidermal growth factor receptor (EGFR) and MYC can suppress proliferation of cancer cells. Together, we provide a programmable and in vivo manipulation tool to study mRNA modification of specific genes and their related biological functions.
Highlights
As the prominent dynamic mRNA modification, N6-methyladenosine (m6A) has been identified since the 1970s 1, while thousands of RNA transcripts contain m6A modifications with unique distribution patterns as well 2. m6A was governed by methyltransferase complex (“writers”), demethylases (“erasers”) and RNA-binding proteins (‘readers’) 3
U6 promoter-driven gRNA transcription system was cloned into the pC0043-PspCas13b gRNA backbone by use of BbsI 23
In HEK293T cells, the transfection of both fusion proteins can significantly decrease global m6A of mRNA, suggesting the in vivo demethylation function of ALKBH5 fusion proteins (Figure S1 B). The efficiency of both dCas13b-ALKBH5 and ALKBH5-dCas13b was less than that of ALKBH5 (Figure S1 B), which might be due to the increase of steric hindrance of fusion proteins that impairs their catalytic activities
Summary
As the prominent dynamic mRNA modification, N6-methyladenosine (m6A) has been identified since the 1970s 1, while thousands of RNA transcripts contain m6A modifications with unique distribution patterns as well 2. m6A was governed by methyltransferase complex (“writers”), demethylases (“erasers”) and RNA-binding proteins (‘readers’) 3. We constructed and characterized a CRISPR-Cas13b-based tool for the first time that targeted m6A methylation of mRNA by fusing the catalytically dead Type VI-B Cas[13] enzyme from Prevotella sp.P5–125 (dPspCas13b) with the m6A demethylase ALKBH5 via a six amino-acid (GSGGGG) linker. This tool combines the ease of Cas[13] protein using short complementary single guide RNA (sgRNA) with the already established ALKBH5 activity in fusion constructs 30. This work provides a programmable and in vivo manipulation tool to study mRNA modification and its potential biological functions of specific genes
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