Targeted mRNA demethylation using an engineered dCas13b-ALKBH5 fusion protein

Jiexin Li; Zhuojia Chen; Feng Chen; Guoyou Xie; Yuyi Ling; Yanxi Peng; Yu Lin; Nan Luo; Cheng Ming Chiang; Hongsheng Wang

doi:10.1093/NAR/GKAA269

Targeted mRNA demethylation using an engineered dCas13b-ALKBH5 fusion protein

Jiexin Li, Zhuojia Chen, Feng Chen, Guoyou Xie, Yuyi Ling, Yanxi Peng, Yu Lin, Nan Luo, Cheng Ming Chiang, Hongsheng Wang

Research output: Contribution to journal › Article › peer-review

131 Scopus citations

Abstract

Studies on biological functions of N⁶methyladenosine (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.

Original language	English (US)
Pages (from-to)	5684-5694
Number of pages	11
Journal	Nucleic acids research
Volume	48
Issue number	10
DOIs	https://doi.org/10.1093/NAR/GKAA269
State	Published - 2020

ASJC Scopus subject areas

Genetics

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title = "Targeted mRNA demethylation using an engineered dCas13b-ALKBH5 fusion protein",

abstract = "Studies on biological functions of N6methyladenosine (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.",

author = "Jiexin Li and Zhuojia Chen and Feng Chen and Guoyou Xie and Yuyi Ling and Yanxi Peng and Yu Lin and Nan Luo and Chiang, {Cheng Ming} and Hongsheng Wang",

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T1 - Targeted mRNA demethylation using an engineered dCas13b-ALKBH5 fusion protein

AU - Li, Jiexin

AU - Chen, Zhuojia

AU - Chen, Feng

AU - Xie, Guoyou

AU - Ling, Yuyi

AU - Peng, Yanxi

AU - Lin, Yu

AU - Luo, Nan

AU - Chiang, Cheng Ming

AU - Wang, Hongsheng

PY - 2020

Y1 - 2020

N2 - Studies on biological functions of N6methyladenosine (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.

AB - Studies on biological functions of N6methyladenosine (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.

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Targeted mRNA demethylation using an engineered dCas13b-ALKBH5 fusion protein

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