Targeted DNA Methylation Analysis Methods

Cheishvili, David; Petropoulos, Sophie; Christiansen, Steffan Noe; Szyf, Moshe

doi:10.1007/978-1-4939-6743-8_3

Cited by 4 publications

(1 citation statement)

References 46 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…For samples with cell numbers <1.0 × 10 6 , we recommend the use of EZ DNA Methylation-Direct Kit (Zymo Research), which allows for cell lysis and bisulfite conversion without an intermediate DNA purification step. Comprehensive methodologies for next-generation [ 31 ] and other targeted DNA methylation analysis methods [ 32 ] have been published previously and, therefore, will not be detailed further in this publication. For bisulfite-specific primer design, we recommend using the MethPrimer online design tool [ 33 ].…”

Section: Methodsmentioning

confidence: 99%

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

Smith

Banerjee

Waly

et al. 2021

Cancers

View full text Add to dashboard Cite

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.

show abstract

Section: Methodsmentioning

confidence: 99%

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

Smith

Banerjee

Waly

et al. 2021

Cancers

View full text Add to dashboard Cite

show abstract

Understanding the role of DNA methylation in successful biological invasions: a review

et al. 2018

View full text Add to dashboard Cite

Locus-Specific DNA Methylation Editing in Mammalian Cells using a CRISPR-Based System

Smith

Banerjee

Waly

et al. 2021

Preprint

View full text Add to dashboard Cite

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 a 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, a putative epigenetic driver of melanoma metastasis, achieving up to 304.00% gain of methylation and 99.99% relative demethylation, respectively. Further, we employ a novel, targeted screening approach to confirm minimal off-target activity and high on-target specificity of our editing sys-tem within our target locus.

show abstract

Targeted DNA Methylation Analysis Methods

Cited by 4 publications

References 46 publications

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

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

Understanding the role of DNA methylation in successful biological invasions: a review

Locus-Specific DNA Methylation Editing in Mammalian Cells using a CRISPR-Based System

Contact Info

Product

Resources

About