 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Dec 14, 2016 |
| Title |
Genome-wide determination of on-target and off-target characteristics for RNA-guided DNA Methylation by dCas9 methyltransferases (CRISPRme) [WGBS] |
| Organism |
Homo sapiens |
| Experiment type |
Methylation profiling by high throughput sequencing
|
| Summary |
Fusion of active protein domains to the nuclease-deficient clustered regularly interspaced short palindromic repeat (CRISPR) associated protein 9 (dCas9) has been widely used for epigenome editing, but the specificities of these engineered proteins have still not been fully investigated. Targeted methylation of specific gene loci offers a direct approach to perturb DNA methylation-associated biological processes. In this study, we generated and validated the global off-target characteristics of CRISPR-guided DNA methyltransferases (CRISPRme) by fusing the catalytic domain of DNMT3A or DNMT3B to the C terminus of the dCas9 protein from S. pyogenes. Using targeted quantitative bisulfite pyrosequencing and whole genome bisulfite sequencing (WGBS), we prove that CRISPRme can efficiently methylate the CpG dinucleotides flanking its target sites in genomic loci (uPA and TGFBR3) in human cells (HEK293T) with CpG-methylation levels exceeding 70% for some target sites. Using qPCR, fluorescent reporter cells, and RNA sequencing, we found that CRISPRme can mediate transient inhibition of gene expression which appears to result from Cas9-mediated interference with transcription rather than de novo DNA methylation. Analyses of whole genome methylation did not identify global methylation changes, however a substantial number of CRISPRme off-target differentially methylated regions (DMR, over 6000) were still identified. The majority of these DMRs were hypermethylated both in cells expressing CRISPRme alone and cells expressing CRISPRme together with gRNAs. These off-target hypermethylated sites were enriched in gene bodies, introns, 5’UTR, CGI shores, Alu sequences, open chromatin and PAM rich regions, but not correlated with off-target binding sites predicted by ChIP-seq. Our results prove that CRISPRme allows for efficient RNA-guided methylation of endogenous CpGs, however with high frequencies of off-target methylation indicating that further improvements of the specificity of CRISPR-dCas9 based DNA methylation modifiers are still required.
|
| |
|
| Overall design |
Nine samples transfected with pUC19, or dCas9 methyltransferases with or without gRNAs were analyzed by WGBS.
|
| |
|
| Contributor(s) |
Lin L, Liu Y, Xu F, Huang J, Daugaard TF, Petersen TS, Hansen B, Ye L, Zhou Q, Fang F, Li S, Fløe L, Shrock E, Yang H, Wang J, Xu X, Bolund L, Nielse AL, Luo Y |
| Citation(s) |
29635374 |
| Submission date |
Dec 13, 2016 |
| Last update date |
Dec 25, 2019 |
| Contact name |
Yonglun Luo |
| E-mail(s) |
alun@biomed.au.dk
|
| Phone |
0045-22411944
|
| Organization name |
Aarhus University
|
| Street address |
Wilhelm Meyers Allé 4
|
| City |
aarhus |
| ZIP/Postal code |
8000 |
| Country |
Denmark |
| |
|
| Platforms (1) |
|
| Samples (15)
|
| GSM2425578 |
dCas9-BFP-DNMT3A (500 ng) + uPA gRNA (500 ng) |
| GSM2425579 |
dCas9-BFP-DNMT3B (500 ng) + uPA gRNA (500 ng) |
| GSM2425580 |
dCas9-BFP-DNMT3A (500 ng) + TGFBR3 gRNA (500 ng) |
|
| This SubSeries is part of SuperSeries: |
| GSE92311 |
Genome-wide determination of on-target and off-target characteristics for RNA-guided DNA Methylation by dCas9 methyltransferases (CRISPRme) |
|
| Relations |
| BioProject |
PRJNA357200 |
| SRA |
SRP095006 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE92310_RAW.tar |
122.0 Gb |
(http)(custom) |
TAR (of BEDGRAPH, TXT) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
Less...
More...