DNA epigenome editing using CRISPR-Cas SunTag-directed DNMT3A

Abstract

Background: DNA methylation has widespread effects on gene expression during development. However, our ability to assign specific function to regions of DNA methylation is limited by the poor correlation between global patterns of DNA methylation and gene expression.

Results: Here, we utilize nuclease-deactivated Cas9 protein fused to repetitive peptide epitopes (SunTag) recruiting multiple copies of antibody-fused de novo DNA methyltransferase 3A (DNMT3A) (dCas9-SunTag-DNMT3A) to amplify the local DNMT3A concentration to methylate genomic sites of interest. We demonstrate that dCas9-SunTag-DNMT3A dramatically increases CpG methylation at the HOXA5 locus in human embryonic kidney (HEK293T) cells. Furthermore, using a single guide RNA, dCas9-SunTag-DNMT3A is able to methylate a 4.5-kb genomic region and repress HOXA5 gene expression. Reduced representation bisulfite sequencing and RNA-seq show that dCas9-SunTag-DNMT3A methylates regions of interest with minimal impact on the global DNA methylome and transcriptome.

Conclusions: This effective and precise tool enables site-specific manipulation of DNA methylation and may be used to address the relationship between DNA methylation and gene expression.

Fig. 1

dCas9-SunTag-DNMT3A methylates the HOXA5 promoter and suppresses gene expression. a Schematic of dCas9-SunTag-DNMT3A. Deactivated Cas9 (dCas9) was fused to SunTag epitopes and the single-chain variable fragment (scFv) was fused to green fluorescent protein (GFP) and DNMT3A1. With guide RNAs, multiple copies of ^scFvDNMT3A1 can be directed to specific loci and methylated regions of interest. b Lentiviral vectors of dCas9-SunTag-DNMT3A. The dCas9^SunTag vector contains the p2A self-cleaving peptide to separate dCas9^SunTag and blue fluorescent protein (BFP). Guide RNA vector contains the RFP657 fluorescence marker. c The guide RNA design at the HOXA5 locus. Guide RNA HOXA5 (sgHOXA5) was located in exon 1 of HOXA5 gene. d The percent of methylation across individual CpGs as determined by deep sequencing of amplicons across the locus. e The fold change relative to dCas9^SunTag + ^scFvDNMT3A1 as analyzed by quantitative real-time PCR for HOXA5 expression, measured 30 days after transduction of indicated constructs. ***P < 0.001 by Student t test

Fig. 2

dCas9-SunTag-DNMT3A methylates CpGs and CpHs within 4.5 kb of HOXA5 locus. a The percent of methylation across individual CpGs as determined by deep sequencing of amplicons within a 4.5-kb window of HOXA5 locus. b The percent of methylation across individual CpHs as determined by deep sequencing of amplicons within a 4.5-kb window of HOXA5 locus

Fig. 3

dCas9-SunTag-DNMT3A methylates the intragenic regions of KLF4 without affecting gene expression. a The percent of methylation across individual CpGs as determined using deep sequencing of amplicons in a 2.5-kb window of the KLF4 locus. The gray bar represents the guide RNA binding site. b The fold change relative to dCas9^SunTag + ^scFvDNMT3A1 as measured by quantitative PCR for KLF4 expression

Fig. 4

dCas9-SunTag-DNMT3A is capable of methylating 4.5-kb regions of HOXA5 locus with minimal effects on global methylome and transcriptome. a The methylation level of detected CpGs across the genome as determined by reduced representation bisulfite sequencing (RRBS). b The correlation between dCas9-SunTag-DNMT3A1-treated samples by Pearson’s correlation method. c The methylation status of detected CpGs at HOXA5 locus by whole genome bisulfite sequencing. The proportional methylation at each analyzed CpG is plotted from 0 to 1 (representing from 0 to 100% methylation). d The genome-wide gene expression analysis (log2 fold changes of FPKM) of dCas9-SunTag-DNMT3A1-treated samples compared to non-transduced cells (Neg) using RNA-seq. Red dot represents relative DNMT3A expression, and blue dot represents relative HOXA5 expression compared to Neg cells