Locus-Specific DNA Methylation Editing in Melanoma Cell Lines Using a CRISPR-Based System

SIMPLE SUMMARY: DNA methylation is an important modification of the genome that is implicated in the pathogenesis of numerous human diseases, including cancer. DNA methylation changes can alter the expression of critical genes, predisposing to disease progression. Existing techniques that can modify DNA methylation to investigate disease etiology are severely limited with regard to specificity, which means that establishing a causal link between DNA methylation changes and disease progression is difficult. The advent of CRISPR-based technologies has provided a powerful tool for more specific editing of DNA methylation. Here, we describe a comprehensive protocol for the design and application of a CRISPR-dCas9-based tool for editing DNA methylation at a target locus in human melanoma cell lines alongside protocols for downstream techniques used to evaluate subsequent methylation and gene expression changes in methylation-edited cells. Furthermore, we demonstrate highly efficacious methylation and demethylation of the EBF3 promoter across a panel of melanoma cell lines. ABSTRACT: DNA methylation is a key epigenetic modification implicated in the pathogenesis of numerous human diseases, including cancer development and metastasis. Gene promoter methylation changes are widely associated with transcriptional deregulation and disease progression. The advent of CRISPR-based technologies has provided a powerful toolkit for locus-specific manipulation of the epigenome. Here, we describe a comprehensive global workflow for the design and application of a dCas9-SunTag-based tool for editing the DNA methylation locus in human melanoma cells alongside protocols for downstream techniques used to evaluate subsequent methylation and gene expression changes in methylation-edited cells. Using transient system delivery, we demonstrate both highly efficacious methylation and demethylation of the EBF3 promoter, which is a putative epigenetic driver of melanoma metastasis, achieving up to a 304.00% gain of methylation and 99.99% relative demethylation, respectively. Furthermore, we employ a novel, targeted screening approach to confirm the minimal off-target activity and high on-target specificity of our designed guide RNA within our target locus.