Genome-wide determination of on-target and off-target characteristics for RNA-guided DNA methylation by dCas9 methyltransferases

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  • Lin Lin
  • Yong Liu, Department of Biomedicine, Aarhus University, Aarhus, Denmark., Fengping Xu, Department of Biology, University of Copenhagen, Copenhagen N, Denmark, Jinrong Huang, Department of Biology, University of Copenhagen, Copenhagen N, Denmark,
  • Tina Fuglsang Daugaard
  • Trine Skov Petersen, Department of Biomedicine, Aarhus University, Aarhus, Denmark.,
  • Bettina Hansen
  • Lingfei Ye, BGI-Shenzhen, Shenzhen 518083, China., Qing Zhou, China National GeneBank-Shenzhen, BGI-Research, Shenzhen 518083, China., Fang Fang, BGI-Shenzhen, Shenzhen 518083, China., China National GeneBank-Shenzhen, BGI-Research, Shenzhen 518083, China., Ling Yang, China National GeneBank-Shenzhen, BGI-Research, Shenzhen 518083, China., Shengting Li, Department of Biomedicine, Aarhus University, Aarhus, Denmark., BGI-Shenzhen, Shenzhen 518083, China.,
  • Lasse Fløe
  • Kristopher Torp Jensen, Department of Biomedicine, Aarhus University, Aarhus, Denmark., Ellen Shrock, Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA, Fang Chen, Department of Biology, University of Copenhagen, Copenhagen N, Denmark, Huanming Yang, James D. Watson Institute of Genome Sciences, Hangzhou 310058, China., Jian Wang, China National GeneBank-Shenzhen, BGI-Research, Shenzhen 518083, China., Xin Liu, China National GeneBank-Shenzhen, BGI-Research, Shenzhen 518083, China., Xun Xu, China National GeneBank-Shenzhen, BGI-Research, Shenzhen 518083, China.,
  • Lars Bolund
  • Anders Lade Nielsen
  • Yonglun Luo

Background: Fusion of DNA methyltransferase domains to the nuclease-deficient clustered regularly interspaced short palindromic repeat (CRISPR) associated protein 9 (dCas9) has been used for epigenome editing, but the specificities of these dCas9 methyltransferases have not been fully investigated.

Findings: We generated CRISPR-guided DNA methyltransferases by fusing the catalytic domain of DNMT3A or DNMT3B to the C terminus of the dCas9 protein from Streptococcus pyogenes and validated its on-target and global off-target characteristics. Using targeted quantitative bisulfite pyrosequencing, we prove that dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B can efficiently methylate the CpG dinucleotides flanking its target sites at different genomic loci (uPA and TGFBR3) in human embryonic kidney cells (HEK293T). Furthermore, we conducted whole genome bisulfite sequencing (WGBS) to address the specificity of our dCas9 methyltransferases. WGBS revealed that although dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B did not cause global methylation changes, a substantial number (more than 1000) of the off-target differentially methylated regions (DMRs) were identified. The off-target DMRs, which were hypermethylated in cells expressing dCas9 methyltransferase and guide RNAs, were predominantly found in promoter regions, 5΄ untranslated regions, CpG islands, and DNase I hypersensitivity sites, whereas unexpected hypomethylated off-target DMRs were significantly enriched in repeated sequences. Through chromatin immunoprecipitation with massive parallel DNA sequencing analysis, we further revealed that these off-target DMRs were weakly correlated with dCas9 off-target binding sites. Using quantitative polymerase chain reaction, RNA sequencing, and fluorescence reporter cells, we also found that dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B can mediate transient inhibition of gene expression, which might be caused by dCas9-mediated de novo DNA methylation as well as interference with transcription.

Conclusion: Our results prove that dCas9 methyltransferases cause efficient RNA-guided methylation of specific endogenous CpGs. However, there is significant off-target methylation indicating that further improvements of the specificity of CRISPR-dCas9 based DNA methylation modifiers are required.

Original languageEnglish
JournalGigaScience
Volume7
Issue number3
Pages (from-to)1-19
Number of pages19
ISSN2047-217X
DOIs
StatePublished - 1 Mar 2018
Externally publishedYes

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