Cargando…
A modular dCas9-SunTag DNMT3A epigenome editing system overcomes pervasive off-target activity of direct fusion dCas9-DNMT3A constructs
Detection of DNA methylation in the genome has been possible for decades; however, the ability to deliberately and specifically manipulate local DNA methylation states in the genome has been extremely limited. Consequently, this has impeded our understanding of the direct effect of DNA methylation o...
Autores principales: | , , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Cold Spring Harbor Laboratory Press
2018
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6071642/ https://www.ncbi.nlm.nih.gov/pubmed/29907613 http://dx.doi.org/10.1101/gr.233049.117 |
_version_ | 1783343907855138816 |
---|---|
author | Pflueger, Christian Tan, Dennis Swain, Tessa Nguyen, Trung Pflueger, Jahnvi Nefzger, Christian Polo, Jose M. Ford, Ethan Lister, Ryan |
author_facet | Pflueger, Christian Tan, Dennis Swain, Tessa Nguyen, Trung Pflueger, Jahnvi Nefzger, Christian Polo, Jose M. Ford, Ethan Lister, Ryan |
author_sort | Pflueger, Christian |
collection | PubMed |
description | Detection of DNA methylation in the genome has been possible for decades; however, the ability to deliberately and specifically manipulate local DNA methylation states in the genome has been extremely limited. Consequently, this has impeded our understanding of the direct effect of DNA methylation on transcriptional regulation and transcription factor binding in the native chromatin context. Thus, highly specific targeted epigenome editing tools are needed to address this. Recent adaptations of genome editing technologies, including fusion of the DNMT3A DNA methyltransferase catalytic domain to catalytically inactive Cas9 (dC9-D3A), have aimed to alter DNA methylation at desired loci. Here, we show that these tools exhibit consistent off-target DNA methylation deposition in the genome, limiting their capabilities to unambiguously assess the functional consequences of DNA methylation. To address this, we developed a modular dCas9-SunTag (dC9Sun-D3A) system that can recruit multiple DNMT3A catalytic domains to a target site for editing DNA methylation. dC9Sun-D3A is tunable, specific, and exhibits much higher induction of DNA methylation at target sites than the dC9-D3A direct fusion protein. Importantly, genome-wide characterization of dC9Sun-D3A binding sites and DNA methylation revealed minimal off-target protein binding and induction of DNA methylation with dC9Sun-D3A, compared to pervasive off-target methylation by dC9-D3A. Furthermore, we used dC9Sun-D3A to demonstrate the binding sensitivity to DNA methylation for CTCF and NRF1 in situ. Overall, this modular dC9Sun-D3A system enables precise DNA methylation deposition with the lowest off-target DNA methylation levels reported to date, allowing accurate functional determination of the role of DNA methylation at single loci. |
format | Online Article Text |
id | pubmed-6071642 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | Cold Spring Harbor Laboratory Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-60716422019-02-01 A modular dCas9-SunTag DNMT3A epigenome editing system overcomes pervasive off-target activity of direct fusion dCas9-DNMT3A constructs Pflueger, Christian Tan, Dennis Swain, Tessa Nguyen, Trung Pflueger, Jahnvi Nefzger, Christian Polo, Jose M. Ford, Ethan Lister, Ryan Genome Res Method Detection of DNA methylation in the genome has been possible for decades; however, the ability to deliberately and specifically manipulate local DNA methylation states in the genome has been extremely limited. Consequently, this has impeded our understanding of the direct effect of DNA methylation on transcriptional regulation and transcription factor binding in the native chromatin context. Thus, highly specific targeted epigenome editing tools are needed to address this. Recent adaptations of genome editing technologies, including fusion of the DNMT3A DNA methyltransferase catalytic domain to catalytically inactive Cas9 (dC9-D3A), have aimed to alter DNA methylation at desired loci. Here, we show that these tools exhibit consistent off-target DNA methylation deposition in the genome, limiting their capabilities to unambiguously assess the functional consequences of DNA methylation. To address this, we developed a modular dCas9-SunTag (dC9Sun-D3A) system that can recruit multiple DNMT3A catalytic domains to a target site for editing DNA methylation. dC9Sun-D3A is tunable, specific, and exhibits much higher induction of DNA methylation at target sites than the dC9-D3A direct fusion protein. Importantly, genome-wide characterization of dC9Sun-D3A binding sites and DNA methylation revealed minimal off-target protein binding and induction of DNA methylation with dC9Sun-D3A, compared to pervasive off-target methylation by dC9-D3A. Furthermore, we used dC9Sun-D3A to demonstrate the binding sensitivity to DNA methylation for CTCF and NRF1 in situ. Overall, this modular dC9Sun-D3A system enables precise DNA methylation deposition with the lowest off-target DNA methylation levels reported to date, allowing accurate functional determination of the role of DNA methylation at single loci. Cold Spring Harbor Laboratory Press 2018-08 /pmc/articles/PMC6071642/ /pubmed/29907613 http://dx.doi.org/10.1101/gr.233049.117 Text en © 2018 Pflueger et al.; Published by Cold Spring Harbor Laboratory Press http://creativecommons.org/licenses/by-nc/4.0/ This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genome.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/. |
spellingShingle | Method Pflueger, Christian Tan, Dennis Swain, Tessa Nguyen, Trung Pflueger, Jahnvi Nefzger, Christian Polo, Jose M. Ford, Ethan Lister, Ryan A modular dCas9-SunTag DNMT3A epigenome editing system overcomes pervasive off-target activity of direct fusion dCas9-DNMT3A constructs |
title | A modular dCas9-SunTag DNMT3A epigenome editing system overcomes pervasive off-target activity of direct fusion dCas9-DNMT3A constructs |
title_full | A modular dCas9-SunTag DNMT3A epigenome editing system overcomes pervasive off-target activity of direct fusion dCas9-DNMT3A constructs |
title_fullStr | A modular dCas9-SunTag DNMT3A epigenome editing system overcomes pervasive off-target activity of direct fusion dCas9-DNMT3A constructs |
title_full_unstemmed | A modular dCas9-SunTag DNMT3A epigenome editing system overcomes pervasive off-target activity of direct fusion dCas9-DNMT3A constructs |
title_short | A modular dCas9-SunTag DNMT3A epigenome editing system overcomes pervasive off-target activity of direct fusion dCas9-DNMT3A constructs |
title_sort | modular dcas9-suntag dnmt3a epigenome editing system overcomes pervasive off-target activity of direct fusion dcas9-dnmt3a constructs |
topic | Method |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6071642/ https://www.ncbi.nlm.nih.gov/pubmed/29907613 http://dx.doi.org/10.1101/gr.233049.117 |
work_keys_str_mv | AT pfluegerchristian amodulardcas9suntagdnmt3aepigenomeeditingsystemovercomespervasiveofftargetactivityofdirectfusiondcas9dnmt3aconstructs AT tandennis amodulardcas9suntagdnmt3aepigenomeeditingsystemovercomespervasiveofftargetactivityofdirectfusiondcas9dnmt3aconstructs AT swaintessa amodulardcas9suntagdnmt3aepigenomeeditingsystemovercomespervasiveofftargetactivityofdirectfusiondcas9dnmt3aconstructs AT nguyentrung amodulardcas9suntagdnmt3aepigenomeeditingsystemovercomespervasiveofftargetactivityofdirectfusiondcas9dnmt3aconstructs AT pfluegerjahnvi amodulardcas9suntagdnmt3aepigenomeeditingsystemovercomespervasiveofftargetactivityofdirectfusiondcas9dnmt3aconstructs AT nefzgerchristian amodulardcas9suntagdnmt3aepigenomeeditingsystemovercomespervasiveofftargetactivityofdirectfusiondcas9dnmt3aconstructs AT polojosem amodulardcas9suntagdnmt3aepigenomeeditingsystemovercomespervasiveofftargetactivityofdirectfusiondcas9dnmt3aconstructs AT fordethan amodulardcas9suntagdnmt3aepigenomeeditingsystemovercomespervasiveofftargetactivityofdirectfusiondcas9dnmt3aconstructs AT listerryan amodulardcas9suntagdnmt3aepigenomeeditingsystemovercomespervasiveofftargetactivityofdirectfusiondcas9dnmt3aconstructs AT pfluegerchristian modulardcas9suntagdnmt3aepigenomeeditingsystemovercomespervasiveofftargetactivityofdirectfusiondcas9dnmt3aconstructs AT tandennis modulardcas9suntagdnmt3aepigenomeeditingsystemovercomespervasiveofftargetactivityofdirectfusiondcas9dnmt3aconstructs AT swaintessa modulardcas9suntagdnmt3aepigenomeeditingsystemovercomespervasiveofftargetactivityofdirectfusiondcas9dnmt3aconstructs AT nguyentrung modulardcas9suntagdnmt3aepigenomeeditingsystemovercomespervasiveofftargetactivityofdirectfusiondcas9dnmt3aconstructs AT pfluegerjahnvi modulardcas9suntagdnmt3aepigenomeeditingsystemovercomespervasiveofftargetactivityofdirectfusiondcas9dnmt3aconstructs AT nefzgerchristian modulardcas9suntagdnmt3aepigenomeeditingsystemovercomespervasiveofftargetactivityofdirectfusiondcas9dnmt3aconstructs AT polojosem modulardcas9suntagdnmt3aepigenomeeditingsystemovercomespervasiveofftargetactivityofdirectfusiondcas9dnmt3aconstructs AT fordethan modulardcas9suntagdnmt3aepigenomeeditingsystemovercomespervasiveofftargetactivityofdirectfusiondcas9dnmt3aconstructs AT listerryan modulardcas9suntagdnmt3aepigenomeeditingsystemovercomespervasiveofftargetactivityofdirectfusiondcas9dnmt3aconstructs |