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Reprogrammable CRISPR/Cas9-based system for inducing site-specific DNA methylation

Advances in sequencing technology allow researchers to map genome-wide changes in DNA methylation in development and disease. However, there is a lack of experimental tools to site-specifically manipulate DNA methylation to discern the functional consequences. We developed a CRISPR/Cas9 DNA methyltr...

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Autores principales: McDonald, James I., Celik, Hamza, Rois, Lisa E., Fishberger, Gregory, Fowler, Tolison, Rees, Ryan, Kramer, Ashley, Martens, Andrew, Edwards, John R., Challen, Grant A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The Company of Biologists Ltd 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4920199/
https://www.ncbi.nlm.nih.gov/pubmed/27170255
http://dx.doi.org/10.1242/bio.019067
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author McDonald, James I.
Celik, Hamza
Rois, Lisa E.
Fishberger, Gregory
Fowler, Tolison
Rees, Ryan
Kramer, Ashley
Martens, Andrew
Edwards, John R.
Challen, Grant A.
author_facet McDonald, James I.
Celik, Hamza
Rois, Lisa E.
Fishberger, Gregory
Fowler, Tolison
Rees, Ryan
Kramer, Ashley
Martens, Andrew
Edwards, John R.
Challen, Grant A.
author_sort McDonald, James I.
collection PubMed
description Advances in sequencing technology allow researchers to map genome-wide changes in DNA methylation in development and disease. However, there is a lack of experimental tools to site-specifically manipulate DNA methylation to discern the functional consequences. We developed a CRISPR/Cas9 DNA methyltransferase 3A (DNMT3A) fusion to induce DNA methylation at specific loci in the genome. We induced DNA methylation at up to 50% of alleles for targeted CpG dinucleotides. DNA methylation levels peaked within 50 bp of the short guide RNA (sgRNA) binding site and between pairs of sgRNAs. We used our approach to target methylation across the entire CpG island at the CDKN2A promoter, three CpG dinucleotides at the ARF promoter, and the CpG island within the Cdkn1a promoter to decrease expression of the target gene. These tools permit mechanistic studies of DNA methylation and its role in guiding molecular processes that determine cellular fate.
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spelling pubmed-49201992016-07-07 Reprogrammable CRISPR/Cas9-based system for inducing site-specific DNA methylation McDonald, James I. Celik, Hamza Rois, Lisa E. Fishberger, Gregory Fowler, Tolison Rees, Ryan Kramer, Ashley Martens, Andrew Edwards, John R. Challen, Grant A. Biol Open Methods & Techniques Advances in sequencing technology allow researchers to map genome-wide changes in DNA methylation in development and disease. However, there is a lack of experimental tools to site-specifically manipulate DNA methylation to discern the functional consequences. We developed a CRISPR/Cas9 DNA methyltransferase 3A (DNMT3A) fusion to induce DNA methylation at specific loci in the genome. We induced DNA methylation at up to 50% of alleles for targeted CpG dinucleotides. DNA methylation levels peaked within 50 bp of the short guide RNA (sgRNA) binding site and between pairs of sgRNAs. We used our approach to target methylation across the entire CpG island at the CDKN2A promoter, three CpG dinucleotides at the ARF promoter, and the CpG island within the Cdkn1a promoter to decrease expression of the target gene. These tools permit mechanistic studies of DNA methylation and its role in guiding molecular processes that determine cellular fate. The Company of Biologists Ltd 2016-05-11 /pmc/articles/PMC4920199/ /pubmed/27170255 http://dx.doi.org/10.1242/bio.019067 Text en © 2016. Published by The Company of Biologists Ltd http://creativecommons.org/licenses/by/3.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.
spellingShingle Methods & Techniques
McDonald, James I.
Celik, Hamza
Rois, Lisa E.
Fishberger, Gregory
Fowler, Tolison
Rees, Ryan
Kramer, Ashley
Martens, Andrew
Edwards, John R.
Challen, Grant A.
Reprogrammable CRISPR/Cas9-based system for inducing site-specific DNA methylation
title Reprogrammable CRISPR/Cas9-based system for inducing site-specific DNA methylation
title_full Reprogrammable CRISPR/Cas9-based system for inducing site-specific DNA methylation
title_fullStr Reprogrammable CRISPR/Cas9-based system for inducing site-specific DNA methylation
title_full_unstemmed Reprogrammable CRISPR/Cas9-based system for inducing site-specific DNA methylation
title_short Reprogrammable CRISPR/Cas9-based system for inducing site-specific DNA methylation
title_sort reprogrammable crispr/cas9-based system for inducing site-specific dna methylation
topic Methods & Techniques
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4920199/
https://www.ncbi.nlm.nih.gov/pubmed/27170255
http://dx.doi.org/10.1242/bio.019067
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