Targeted mRNA demethylation using an engineered dCas13b-ALKBH5 fusion protein

被引：185

作者：

Li, Jiexin ^{[1
]}

Chen, Zhuojia ^{[2
,3
]}

Chen, Feng ^{[1
]}

Xie, Guoyou ^{[1
]}

Ling, Yuyi ^{[1
]}

Peng, Yanxi ^{[1
]}

Lin, Yu ^{[1
,4
]}

Luo, Nan ^{[1
]}

Chiang, Cheng-Ming ^{[5
,6
]}

Wang, Hongsheng ^{[1
]}

机构：

[1] Sun Yat Sen Univ, Sch Pharmaceut Sci, Guangdong Key Lab Chiral Mol & Drug Discovery, Guangzhou 510006, Guangdong, Peoples R China

[2] Sun Yat Sen Univ, Canc Ctr, Guangzhou 510060, Peoples R China

[3] Collaborat Innovat Ctr Canc Med, State Key Lab Oncol South China, Guangzhou 510060, Peoples R China

[4] Southern Med Univ, Nanfang Hosp, Dept Gastroenterol, Guangdong Prov Key Lab Gastroenterol, Guangzhou 510006, Guangdong, Peoples R China

[5] Univ Texas Southwestern Med Ctr Dallas, Simmons Comprehens Canc Ctr, Dept Pharmacol, 5323 Harry Hines Blvd, Dallas, TX 75390 USA

[6] Univ Texas Southwestern Med Ctr Dallas, Dept Biochem, 5323 Harry Hines Blvd, Dallas, TX 75390 USA

来源：

NUCLEIC ACIDS RESEARCH | 2020年 / 48卷 / 10期

基金：

中国博士后科学基金; 中国国家自然科学基金;

关键词：

DNA METHYLATION; CANCER-CELLS; TRANSLATION; EXPRESSION; EFFICIENT; REVEALS; ALKBH5;

D O I：

10.1093/nar/gkaa269

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

Studies on biological functions of N-6-methyladenosine (m(6)A) 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 dm(6)ACRISPR, was created by linking a catalytically inactive Type VI-B Cas13 enzyme from Prevotella sp. P5-125 (dPspCas13b) to m(6)A demethylase AlkB homolog 5 (ALKBH5). dm(6)ACRISPR specifically demethylates m(6)A of targeted mRNA such as cytochrome b5 form A (CYB5A) to increase its mRNA stability. It can also demethylate beta-catenin-encoding CTNNB1 mRNA that contains multiple m(6)A sites to trigger its translation. In addition, the dm(6)ACRISPR 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.

引用

页码：5684 / 5694

页数：11

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