Researchers demonstrate targeted epigenome editing in the promoter region of several genes using sgRNA/dCas9 complexes researchsquare Uni_Stuttgart CRISPR geneediting genetics epigenome epigenetics
By Susha Cheriyedath, M.Sc.Jul 17 2023Reviewed by Benedette Cuffari, M.Sc. In a recent study posted to the preprint server Research Square* while under review for publication in Epigenetics & Chromatin, researchers develop and characterize a highly specific EpiEditing system using catalytically inactivated Cas9 to achieve allele-specific deoxyribonucleic acid methylation .
Background Epigenomic editing involves the precise reprogramming of specific genomic regions using a tool called an EpiEditor. EpiEditor typically consists of components such as dCas9, DNMT3A/3L, and single guide ribonucleic acid . By introducing methylation to a particular locus, gene expression can be modulated.
For targets with single nucleotide polymorphisms in the protospacer adjacent motif region, a 20 nucleotide genomic sequence upstream of the PAM was selected as the sgRNA binding site. Comparatively, for targets with SNPs in the sgRNA seed region, a 20 nt sequence upstream of the nearest PAM containing the SNP was used.
DNA methylation analysis was also performed using Trim Galore!, PEAR, bwameth, and MethylDackel. RNA isolation and complementary DNA synthesis were followed by expression analysis using gene-specific primers and library generation with two-step polymerase chain reaction . Allelic expression ratios were determined for genes with SNPs in exons.
The experiments were categorized based on the location of SNP in sgRNA, including the seed region, position two of the PAM sequence, or PAM position three. SNPs in the PAM region leading to guanine to Y change, where Y represents cysteine or taurine , were preferred, as these changes exhibited the best discrimination of scFv-DNMT3A/3L-sfGFP, dCas9-10x SunTag-BFP, and sgRNA-DsRed constructs into HEK293 cells.
Heterozygous SNPs within the seed region of the sgRNA or PAM region of dCas9 were targeted to achieve allelic discrimination. The efficiency of targeted DNA methylation varied among the targets, with most successful targets reaching average DNA methylation of more than 50% at selected CpG sites.
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