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

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 techn...

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Autores principales:	Jim Smith, Rakesh Banerjee, Reema Waly, Arthur Urbano, Gregory Gimenez, Robert Day, Michael R. Eccles, Robert J. Weeks, Aniruddha Chatterjee
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	CRISPR dCas9 SunTag DNA methylation epigenetic editing melanoma Neoplasms. Tumors. Oncology. Including cancer and carcinogens RC254-282
Acceso en línea:	https://doaj.org/article/66939659e9bd4b11a53e5d958255dd07
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spelling	oai:doaj.org-article:66939659e9bd4b11a53e5d958255dd072021-11-11T15:31:38ZLocus-Specific DNA Methylation Editing in Melanoma Cell Lines Using a CRISPR-Based System10.3390/cancers132154332072-6694https://doaj.org/article/66939659e9bd4b11a53e5d958255dd072021-10-01T00:00:00Zhttps://www.mdpi.com/2072-6694/13/21/5433https://doaj.org/toc/2072-6694DNA 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 <i>EBF3</i> 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.Jim SmithRakesh BanerjeeReema WalyArthur UrbanoGregory GimenezRobert DayMichael R. EcclesRobert J. WeeksAniruddha ChatterjeeMDPI AGarticleCRISPRdCas9SunTagDNA methylationepigenetic editingmelanomaNeoplasms. Tumors. Oncology. Including cancer and carcinogensRC254-282ENCancers, Vol 13, Iss 5433, p 5433 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	CRISPR dCas9 SunTag DNA methylation epigenetic editing melanoma Neoplasms. Tumors. Oncology. Including cancer and carcinogens RC254-282
spellingShingle	CRISPR dCas9 SunTag DNA methylation epigenetic editing melanoma Neoplasms. Tumors. Oncology. Including cancer and carcinogens RC254-282 Jim Smith Rakesh Banerjee Reema Waly Arthur Urbano Gregory Gimenez Robert Day Michael R. Eccles Robert J. Weeks Aniruddha Chatterjee Locus-Specific DNA Methylation Editing in Melanoma Cell Lines Using a CRISPR-Based System
description	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 <i>EBF3</i> 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.
format	article
author	Jim Smith Rakesh Banerjee Reema Waly Arthur Urbano Gregory Gimenez Robert Day Michael R. Eccles Robert J. Weeks Aniruddha Chatterjee
author_facet	Jim Smith Rakesh Banerjee Reema Waly Arthur Urbano Gregory Gimenez Robert Day Michael R. Eccles Robert J. Weeks Aniruddha Chatterjee
author_sort	Jim Smith
title	Locus-Specific DNA Methylation Editing in Melanoma Cell Lines Using a CRISPR-Based System
title_short	Locus-Specific DNA Methylation Editing in Melanoma Cell Lines Using a CRISPR-Based System
title_full	Locus-Specific DNA Methylation Editing in Melanoma Cell Lines Using a CRISPR-Based System
title_fullStr	Locus-Specific DNA Methylation Editing in Melanoma Cell Lines Using a CRISPR-Based System
title_full_unstemmed	Locus-Specific DNA Methylation Editing in Melanoma Cell Lines Using a CRISPR-Based System
title_sort	locus-specific dna methylation editing in melanoma cell lines using a crispr-based system
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/66939659e9bd4b11a53e5d958255dd07
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Locus-Specific DNA Methylation Editing in Melanoma Cell Lines Using a CRISPR-Based System

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