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

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.

Precision gynecologic oncology: circulating cell free DNA epigenomic analysis, artificial intelligence and the accurate detection of ovarian cancer

“…This study demonstrates the potential value of Precision Oncology based on the combination of AI and epigenomic analysis for both the accurate detection of and for the elucidation of the pathogenesis of OC. The latter is critical for the development and deployment of novel targeted therapeutics such as CRISPR-based DNA methylation editing 48 . Larger confirmation studies are clearly warranted.…”

Targeted DNA Methylation Editing Using an All-in-One System Establishes Paradoxical Activation of EBF3

“…These cell lines were grown in filter-capped cell culture flasks under standard conditions, maintained at 37 °C in a humidified atmosphere with 5% CO 2 and 21% O 2 , as recommended. We have described the DNA methylomes of these cell lines in our previous works [ 26 , 30 ].…”

“…The samples for FACS analysis were prepared based on previously published protocols from our lab [ 26 , 30 ]. Briefly, post-72 h of transfection, the cells were treated with trypsin and suspended in 250 μL of sterile auto MACS buffer containing 1 × DPBS, 1% FCS, and 2 mM EDTA.…”

“…Elaborate methods for next-generation and specific DNA methylation analysis techniques have already been documented in our prior publications [ 26 , 32 ]. Consequently, this publication will not delve into further specifics regarding these methodologies.…”

“…Based on the findings from our previous work [ 26 ], attempting the concurrent transient transfection of three substantial plasmids is a challenging and time-consuming task. The primary objective of these transfection experiments was to generate enough cells for subsequent gene expression investigations and more extensive chromatin analysis.…”