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Data released on February 08, 2018

Supporting data for "Genome-wide determination of on-target and off-target characteristics for RNA-guided DNA Methylation by dCas9 methyltransferases"

Lin, L; Liu, Y; Xu, F; Huang, J; Daugaard , T, F; Petersen, T, S; Hansen, B; Ye, L; Zhou, Q; Fang, F; Yang, L; Li, S; Fløe, L; Jensen , K, T; Shrock , E; Chen, F; Yang, H; Wang, J; Liu, X; Xu, X; Bolund, L; Nielsen, A, L; Luo, Y (2018): Supporting data for "Genome-wide determination of on-target and off-target characteristics for RNA-guided DNA Methylation by dCas9 methyltransferases" GigaScience Database. http://dx.doi.org/10.5524/100406 RIS BibTeX Text

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.
We generated CRISPR-guided DNA methyltransferases by fusing the catalytic domain of DNMT3A or DNMT3B to the C terminus of the dCas9 protein from S. 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 (over 1000) of off-target differentially methylated regions (DMRs) were identified. The off-target DMRs, which were hypermethylated in cells expressing dCas9 methyltransferase and gRNAs, 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 qPCR, 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.

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Accessions (data included in GigaDB):

GEO : GSE74935
GEO : GSE92310
GEO : GSE92311
GEO : GSE92261

Keywords:

dna methylation crispr cas9 dnmt3a dnmt3b dcas9 specificity off-targets epigenome editing 

Epigenomic, Transcriptomic, Genomic

http://gigadb.org/images/data/cropped/100406.jpg

Funding:

  • Funding body - Danish Council for Independent Research
  • Award ID - DFF_1337-00128
  • Awardee - Y Luo
  • Funding body - Danish Council for Independent Research
  • Award ID - DFF-1335-00763A
  • Comment - Sapere Aude Young Research Talent Prize
  • Awardee - Y Luo
  • Funding body - Lundbeck Foundation
  • Award ID - R173-2014-1105
  • Awardee - Y Luo
  • Funding body - Lundbeck Foundation
  • Award ID - R151-2013-14439
  • Awardee - L Bolund
  • Funding body - Lundbeck Foundation
  • Award ID - R219-2016-1375
  • Awardee - L Lin

Samples: Table Settings

Columns:

Common Name
Scienfic Name
Sample Attributes
Taxonomic ID
Genbank Name

Sample IDTaxonomic IDCommon NameGenbank NameScientific NameSample Attributes
ChIP dCas9-BFP-DNMT3A (500 ng) + uPA gRNA (500 ng)9606HumanhumanHomo sapiens Description:Cells were transfected with ChIP dCas9...
Analyte type:DNA
Alternative accession-BioSample:SAMN06134707
...
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ChIP input9606HumanhumanHomo sapiens Description:Cells were transfected with ChIP input...
Analyte type:DNA
Alternative accession-BioSample:SAMN06134706
...
+
control9606HumanhumanHomo sapiens Description:Cells were transfected with a total of...
Analyte type:RNA
Alternative accession-BioSample:SAMN04261752
...
+
dCas9-BFP-DNMT19606HumanhumanHomo sapiens Description:Cells were transfected with a total of...
Analyte type:RNA
Alternative accession-BioSample:SAMN04261748
...
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dCas9-BFP-DNMT3A9606HumanhumanHomo sapiens Description:Cells were transfected with a total of...
Analyte type:RNA
Alternative accession-BioSample:SAMN04261749
...
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dCas9-BFP-DNMT3A (500 ng)9606HumanhumanHomo sapiens Description:Cells were transfected with dCas9-BFP-...
Analyte type:DNA
Alternative accession-BioSample:SAMN06134719
...
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dCas9-BFP-DNMT3A (500 ng) + TGFBR3 gRNA (500 ng)9606HumanhumanHomo sapiens Description:Cells were transfected with dCas9-BFP-...
Analyte type:DNA
Alternative accession-BioSample:SAMN06134721
...
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dCas9-BFP-DNMT3A (500 ng) + uPA gRNA (500 ng)9606HumanhumanHomo sapiens Description:Cells were transfected with dCas9-BFP-...
Analyte type:DNA
Alternative accession-BioSample:SAMN06134714
...
+
dCas9-BFP-DNMT3A (500 ng) + uPA gRNA (500 ng) -19606HumanhumanHomo sapiens Description:Cells were transfected with dCas9-BFP-...
Analyte type:DNA
Alternative accession-BioSample:SAMN08272689
...
+
dCas9-BFP-DNMT3A (500 ng) + uPA gRNA (500 ng) -29606HumanhumanHomo sapiens Description:Cells were transfected with dCas9-BFP-...
Analyte type:DNA
Alternative accession-BioSample:SAMN08272694
...
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Displaying 1-10 of 22 Sample(s).

Files: (FTP site) Table Settings

Columns:

File Description
Sample ID
Data Type
File Format
Size
Release Date
Download Link
File Attributes

File NameSample IDData TypeFile FormatSizeRelease Date 
ReadmeTEXT23.54 KB2018-02-08
ImageTIF236.69 KB2018-02-08
ImageUNKNOWN589.48 KB2018-02-08
ImageUNKNOWN853.65 KB2018-02-08
ImageUNKNOWN885.47 KB2018-02-08
ImageUNKNOWN1020.39 KB2018-02-08
ImageUNKNOWN1.13 MB2018-02-08
ImageUNKNOWN1.14 MB2018-02-08
ImageUNKNOWN1.16 MB2018-02-08
ImageUNKNOWN1.18 MB2018-02-08
Displaying 1-10 of 43 File(s).

History:

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