Programmable targeted epigenetic editing using CRISPR system in Bombyx mori

Yue Liu; Sanyuan Ma; Jiasong Chang; Tong Zhang; Xiaoxu Chen; Yan Liang; Qingyou Xia

doi:10.1016/j.ibmb.2019.04.013

Programmable targeted epigenetic editing using CRISPR system in Bombyx mori

Insect Biochem Mol Biol. 2019 Jul:110:105-111. doi: 10.1016/j.ibmb.2019.04.013. Epub 2019 Apr 22.

Authors

Yue Liu¹, Sanyuan Ma², Jiasong Chang¹, Tong Zhang¹, Xiaoxu Chen¹, Yan Liang¹, Qingyou Xia³

Affiliations

¹ Biological Science Research Center, Southwest University, Chongqing, 400716, China.
² Biological Science Research Center, Southwest University, Chongqing, 400716, China; Chongqing Engineering and Technology Research Center for Novel Silk Materials, Southwest University, Chongqing, 400716, China; Chongqing Key Laboratory of Sericulture, Southwest University, Chongqing, 400716, China.
³ Biological Science Research Center, Southwest University, Chongqing, 400716, China; Chongqing Engineering and Technology Research Center for Novel Silk Materials, Southwest University, Chongqing, 400716, China; Chongqing Key Laboratory of Sericulture, Southwest University, Chongqing, 400716, China. Electronic address: xiaqy@swu.edu.cn.

PMID: 31022512
DOI: 10.1016/j.ibmb.2019.04.013

Abstract

DNA methylation has been proven to play roles in regulating gene expression, cell fate, disease determination, and chromatin architecture organization in mammals and plants, and is a significant component of epigenetic modification. Compared to mammals or plants, the status and function of DNA methylation are poorly understood in insects, which is partially due to the lack of efficient manipulation tools. In this study, we show that fusion protein of catalytically inactive Cas9 (dCas9) with TET1 can efficiently demethylate genomic DNA of silkworm Bombyx mori, in a programmable target region specific manner. We first developed an all-in-one vector to maximize the targeting efficiency of dCas9-TET1. Then we selected 3 endogenous genes that were previously found to harbor methylated DNA, and designed gRNAs within the methylated region. Co-transfection of dCas9-TET1 and gRNA successfully erased methylation marks near the targeting region, with efficiencies from about 17.50% to 40.00%. Furthermore, targeted demethylation on gene body resulted in increased mRNA transcription level. Unlike the previously widely used decitabine, a methylation inhibitor, dCas9-TET1 is more effective and specific, and has no unwanted impact on whole-genome methylation. DCas9-TET1 provides a powerful tool for investigating the functional significance of DNA methylation in a locus-specific manner, and for exploring the unknown links between methylation and development in insects.

Keywords: Bombyx mori; CRISPR/dCas9; Decitabine; Demethylation; Epigenetic.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Bombyx / genetics*
CRISPR-Cas Systems*
DNA Methylation*
Epigenesis, Genetic*
Gene Editing / methods*