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Epigenetic Footprints of CRISPR/Cas9-Mediated Genome Editing in Plants
CRISPR/Cas9 has been widely applied to various plant species accelerating the pace of plant genome editing and precision breeding in crops. Unintended effects beyond off-target nucleotide mutations are still somewhat unexplored. We investigated the degree and patterns of epigenetic changes after gen...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026911/ https://www.ncbi.nlm.nih.gov/pubmed/32117329 http://dx.doi.org/10.3389/fpls.2019.01720 |
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author | Lee, Jun Hyung Mazarei, Mitra Pfotenhauer, Alexander C. Dorrough, Aubrey B. Poindexter, Magen R. Hewezi, Tarek Lenaghan, Scott C. Graham, David E. Stewart, C. Neal |
author_facet | Lee, Jun Hyung Mazarei, Mitra Pfotenhauer, Alexander C. Dorrough, Aubrey B. Poindexter, Magen R. Hewezi, Tarek Lenaghan, Scott C. Graham, David E. Stewart, C. Neal |
author_sort | Lee, Jun Hyung |
collection | PubMed |
description | CRISPR/Cas9 has been widely applied to various plant species accelerating the pace of plant genome editing and precision breeding in crops. Unintended effects beyond off-target nucleotide mutations are still somewhat unexplored. We investigated the degree and patterns of epigenetic changes after gene editing. We examined changes in DNA methylation in genome-edited promoters of naturally hypermethylated genes (AT1G72350 and AT1G09970) and hypomethylated genes (AT3G17320 and AT5G28770) from Arabidopsis. Transgenic plants were developed via Agrobacterium-mediated floral dip transformation. Homozygous edited lines were selected from segregated T(2) plants by an in vitro digestion assay using ribonucleoprotein complex. Bisulfite sequencing comparisons were made between paired groups of edited and non-edited plants to identify changes in DNA methylation of the targeted loci. We found that directed mutagenesis via CRISPR/Cas9 resulted in no unintended morphological or epigenetic alterations. Phenotypes of wild-type, transgenic empty vector, and transgenic edited plants were similar. Epigenetic profiles revealed that methylation patterns of promoter regions flanking target sequences were identical among wild-type, transgenic empty vector, and transgenic edited plants. There was no effect of mutation type on epigenetic status. We also evaluated off-target mutagenesis effects in the edited plants. Potential off-target sites containing up to 4-bp mismatch of each target were sequenced. No off-target mutations were detected in candidate sites. Our results showed that CRISPR/Cas9 did not leave an epigenetic footprint on either the immediate gene-edited DNA and flanking DNA or introduce off-target mutations. |
format | Online Article Text |
id | pubmed-7026911 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-70269112020-02-28 Epigenetic Footprints of CRISPR/Cas9-Mediated Genome Editing in Plants Lee, Jun Hyung Mazarei, Mitra Pfotenhauer, Alexander C. Dorrough, Aubrey B. Poindexter, Magen R. Hewezi, Tarek Lenaghan, Scott C. Graham, David E. Stewart, C. Neal Front Plant Sci Plant Science CRISPR/Cas9 has been widely applied to various plant species accelerating the pace of plant genome editing and precision breeding in crops. Unintended effects beyond off-target nucleotide mutations are still somewhat unexplored. We investigated the degree and patterns of epigenetic changes after gene editing. We examined changes in DNA methylation in genome-edited promoters of naturally hypermethylated genes (AT1G72350 and AT1G09970) and hypomethylated genes (AT3G17320 and AT5G28770) from Arabidopsis. Transgenic plants were developed via Agrobacterium-mediated floral dip transformation. Homozygous edited lines were selected from segregated T(2) plants by an in vitro digestion assay using ribonucleoprotein complex. Bisulfite sequencing comparisons were made between paired groups of edited and non-edited plants to identify changes in DNA methylation of the targeted loci. We found that directed mutagenesis via CRISPR/Cas9 resulted in no unintended morphological or epigenetic alterations. Phenotypes of wild-type, transgenic empty vector, and transgenic edited plants were similar. Epigenetic profiles revealed that methylation patterns of promoter regions flanking target sequences were identical among wild-type, transgenic empty vector, and transgenic edited plants. There was no effect of mutation type on epigenetic status. We also evaluated off-target mutagenesis effects in the edited plants. Potential off-target sites containing up to 4-bp mismatch of each target were sequenced. No off-target mutations were detected in candidate sites. Our results showed that CRISPR/Cas9 did not leave an epigenetic footprint on either the immediate gene-edited DNA and flanking DNA or introduce off-target mutations. Frontiers Media S.A. 2020-01-31 /pmc/articles/PMC7026911/ /pubmed/32117329 http://dx.doi.org/10.3389/fpls.2019.01720 Text en Copyright © 2020 Lee, Mazarei, Pfotenhauer, Dorrough, Poindexter, Hewezi, Lenaghan, Graham and Stewart http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Plant Science Lee, Jun Hyung Mazarei, Mitra Pfotenhauer, Alexander C. Dorrough, Aubrey B. Poindexter, Magen R. Hewezi, Tarek Lenaghan, Scott C. Graham, David E. Stewart, C. Neal Epigenetic Footprints of CRISPR/Cas9-Mediated Genome Editing in Plants |
title | Epigenetic Footprints of CRISPR/Cas9-Mediated Genome Editing in Plants |
title_full | Epigenetic Footprints of CRISPR/Cas9-Mediated Genome Editing in Plants |
title_fullStr | Epigenetic Footprints of CRISPR/Cas9-Mediated Genome Editing in Plants |
title_full_unstemmed | Epigenetic Footprints of CRISPR/Cas9-Mediated Genome Editing in Plants |
title_short | Epigenetic Footprints of CRISPR/Cas9-Mediated Genome Editing in Plants |
title_sort | epigenetic footprints of crispr/cas9-mediated genome editing in plants |
topic | Plant Science |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026911/ https://www.ncbi.nlm.nih.gov/pubmed/32117329 http://dx.doi.org/10.3389/fpls.2019.01720 |
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