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

Dataset type: Epigenomic, Transcriptomic, Genomic
Data released on February 08, 2018

Lin L; Liu Y; Xu F; Huang J; Daugaard TF; Petersen TS; Hansen B; Ye L; Zhou Q; Fang F; Yang L; Li S; Fløe L; Jensen KT; Shrock E; Chen F; Yang H; Wang J; Liu X; Xu X; Bolund L; Nielsen AL; 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

DOI10.5524/100406

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.

Additional details

Read the peer-reviewed publication(s):

Lin, L., Liu, Y., Xu, F., Huang, J., Daugaard, T. F., Petersen, T. S., … Luo, Y. (2018). Genome-wide determination of on-target and off-target characteristics for RNA-guided DNA methylation by dCas9 methyltransferases. GigaScience, 7(3). doi:10.1093/gigascience/giy011

Accessions (data included in GigaDB):

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





Sample IDTaxonomic IDCommon NameGenbank NameScientific NameSample Attributes
dCas9-BFP-DNMT3A (500 ng) + uPA gRNA (500 ng) -39606HumanhumanHomo sapiens Description:Cells were transfected with dCas9-BFP-...
Analyte type:DNA
Alternative accession-BioSample:SAMN08272693
...
+
dCas9-BFP-DNMT3A (50 ng) + TGFBR3 gRNA (50 ng)9606HumanhumanHomo sapiens Description:Cells were transfected with dCas9-BFP-...
Analyte type:DNA
Alternative accession-BioSample:SAMN06134716
...
+
dCas9-BFP-DNMT3A (50 ng) + uPA gRNA (50 ng)9606HumanhumanHomo sapiens Description:Cells were transfected with dCas9-BFP-...
Analyte type:DNA
Alternative accession-BioSample:SAMN06134717
...
+
dCas9-BFP-DNMT3B9606HumanhumanHomo sapiens Description:Cells were transfected with a total of...
Analyte type:RNA
Alternative accession-BioSample:SAMN04261750
...
+
dCas9-BFP-DNMT3B (500 ng)9606HumanhumanHomo sapiens Description:Cells were transfected with dCas9-BFP-...
Analyte type:DNA
Alternative accession-BioSample:SAMN06134718
...
+
dCas9-BFP-DNMT3B (500 ng) + TGFBR3 gRNA (500 ng)9606HumanhumanHomo sapiens Description:Cells were transfected with dCas9-BFP-...
Analyte type:DNA
Alternative accession-BioSample:SAMN06134720
...
+
dCas9-BFP-DNMT3B (500 ng) + uPA gRNA (500 ng)9606HumanhumanHomo sapiens Description:Cells were transfected with dCas9-BFP-...
Analyte type:DNA
Alternative accession-BioSample:SAMN06134722
...
+
dCas9-BFP-EGFP9606HumanhumanHomo sapiens Description:Cells were transfected with a total of...
Analyte type:RNA
Alternative accession-BioSample:SAMN04261751
...
+
pUC199606HumanhumanHomo sapiens Description:Cells were transfected with pUC19 in s...
Analyte type:DNA
Alternative accession-BioSample:SAMN06134715
...
+
pUC19-49606HumanhumanHomo sapiens Description:Cells were transfected with pUC19-4 in...
Analyte type:DNA
Alternative accession-BioSample:SAMN08272692
...
+
Displaying 11-20 of 22 Sample(s).




File NameSample IDData TypeFile FormatSizeRelease Date 
ImageTIF236.69 KB2018-02-08
Tabular dataUNKNOWN4 GB2018-02-08
Tabular dataUNKNOWN4.1 GB2018-02-08
Tabular dataUNKNOWN3.9 GB2018-02-08
ImageUNKNOWN1.61 MB2018-02-08
ImageUNKNOWN589.48 KB2018-02-08
ImageUNKNOWN1.74 MB2018-02-08
ImageUNKNOWN1.13 MB2018-02-08
ImageUNKNOWN1.16 MB2018-02-08
ImageUNKNOWN1.63 MB2018-02-08
Displaying 1-10 of 43 File(s).
Funding body Awardee Award ID Comments
Danish Council for Independent Research Y Luo DFF_1337-00128
Danish Council for Independent Research Y Luo DFF-1335-00763A Sapere Aude Young Research Talent Prize
Lundbeck Foundation Y Luo R173-2014-1105
Lundbeck Foundation L Bolund R151-2013-14439
Lundbeck Foundation L Lin R219-2016-1375
Date Action
February 8, 2018 Dataset publish
February 8, 2018 Publisher name updated from : GigaScience
July 3, 2018 Manuscript Link added : 10.1093/gigascience/giy011