Methylation profiling

Dataset Information

0

Assessment of on- and off-target effects of dSpCas9-DNMT3A and dSpCas9-TET1 fusion proteins and the impact of modulating promoter strength on the specificity of CRISPR/dCas9-based epigenetic editing tools


ABSTRACT: Inferring causal relationship between epigenetic marks and gene expression requires molecular tools which can precisely modify specific genomic regions in a living cell. In this work, we present a comprehensive, modular and extensible CRISPR/dCas9-based molecular toolbox for targeted epigenetic modulation and direct gene regulation. It features a system for expression of orthogonal dCas9 proteins fused to catalytic domains of various effectors, and includes a multi-gRNA system for targeting dCas9 orthologs to up to six loci. This part of the study aimed to determine the impact of promoter strength on modulating on- and off-target activity of dSpCas9-DNMT3A and dSpCas9-TET1 fusion proteins. The dSpCas9-DNMT3A N-terminal fusion was used to target IL6ST with four sgRNAs, and the dSpCas9-TET1 N-terminal fusion was used to target MGAT3 with five gRNAs. For each locus, two types of constructs were used: i) construct having both the fusion protein and the selection marker (PuroR) under the same strong, constitutive CAG promoter; ii) the fusion construct under the weak EFS promoter, while maintaining efficient selection of transfected cells with PuroR under the strong SV40 promoter. Epigenome-wide profiling was conducted using the Illumina Infinium Human MethylationEPIC Beadchip arrays to assess on- and off-target activity of the fusion proteins in transfected HEK293 cells.

ORGANISM(S): Homo sapiens

PROVIDER: GSE126528 | GEO | 2019/09/12

REPOSITORIES: GEO

Dataset's files

Source:
Action DRS
Other
Items per page:
1 - 1 of 1

Similar Datasets

2016-09-23 | GSE83890 | GEO
2019-10-11 | GSE134994 | GEO
2019-10-11 | GSE134995 | GEO
2015-10-26 | E-GEOD-72001 | biostudies-arrayexpress
| PRJNA420755 | ENA
2016-06-01 | E-GEOD-74935 | biostudies-arrayexpress
2019-05-07 | GSE123882 | GEO
2019-05-07 | GSE123830 | GEO
| PRJNA522270 | ENA
2017-03-10 | GSE95064 | GEO