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CRISPR/dCas9-Mediated DNA Methylation Editing on emx2 in Chinese Tongue Sole (Cynoglossus semilaevis) Testis Cells

文献类型： 外文期刊

作者： Sun, Yanxu 1 ; Wang, Hong-Yan 2 ; Liu, Binghua 2 ; Yue, Bowen 1 ; Liu, Qian 2 ; Liu, Yuyan 2 ; Rosa, Ivana F. 3 ; Doretto, Lucas B. 2 ; Han, Shenglei 2 ; Lin, Lei 2 ; Gong, Xiaoling 1 ; Shao, Changwei 2 ;

作者机构： 1.Shanghai Ocean Univ, Coll Fisheries & Life Sci, Shanghai 201306, Peoples R China

2.Chinese Acad Fishery Sci, Yellow Sea Fisheries Res Inst, State Key Lab Mariculture Biobreeding & Sustainabl, Qingdao 266071, Peoples R China

3.Sao Paulo State Univ UNESP, Inst Biosci, Dept Struct & Funct Biol, BR-01049010 Botucatu, Brazil

4.Shanghai Ocean Univ, Key Lab Explorat & Utilizat Aquat Genet Resources, Minist Educ, Shanghai 201306, Peoples R China

5.Shanghai Ocean Univ, Natl Demonstrat Ctr Expt Fisheries Sci Educ, Shanghai 201306, Peoples R China

6.Qingdao Marine Sci & Technol Ctr, Lab Marine Fisheries Sci & Food Prod Proc, Qingdao 266237, Peoples R China

关键词： CRISPR/dCas9; DNA methylation; emx2; Cynoglossus semilaevis

期刊名称：INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES （ 影响因子：4.9； 五年影响因子：5.6 ）

ISSN： 1661-6596

年卷期： 2024 年 25 卷 14 期

页码：

收录情况： SCI

摘要： DNA methylation is a key epigenetic mechanism orchestrating gene expression networks in many biological processes. Nonetheless, studying the role of specific gene methylation events in fish faces challenges. In this study, we validate the regulation of DNA methylation on empty spiracles homeobox 2 (emx2) expression with decitabine treatment in Chinese tongue sole testis cells. We used the emx2 gene as the target gene and developed a new DNA methylation editing system by fusing dnmt3a with catalytic dead Cas9 (dCas9) and demonstrated its ability for sequence-specific DNA methylation editing. Results revealed that utilizing dCas9-dnmt3a to target emx2 promoter region led to increased DNA methylation levels and decreased emx2 expression in Chinese tongue sole testis cells. More importantly, the DNA methylation editing significantly suppressed the expression of MYC proto-oncogene, bHLH transcription factor (myc), one target gene of emx2. Furthermore, we assessed the off-target effects of dCas9-dnmt3a and confirmed no significant impact on the predicted off-target gene expression. Taken together, we developed the first DNA methylation editing system in marine species and demonstrated its effective editing ability in Chinese tongue sole cells. This provides a new strategy for both epigenetic research and molecular breeding of marine species.

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