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Title: Epigenetic Footprints of CRISPR/Cas9-Mediated Genome Editing in Plants

Abstract

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 T2 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.more » 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.« less

Authors:
 [1];  [1];  [2];  [1];  [1];  [1];  [2]; ORCiD logo [3];  [1]
  1. Univ. of Tennessee, Knoxville, TN (United States). Dept. of Plant Sciences
  2. Univ. of Tennessee, Knoxville, TN (United States). Dept. of Plant Sciences, and Dept. of Food Science
  3. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Publication Date:
Research Org.:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1606824
Grant/Contract Number:  
AC05-00OR22725
Resource Type:
Accepted Manuscript
Journal Name:
Frontiers in Plant Science
Additional Journal Information:
Journal Volume: 10; Journal Issue: NA; Journal ID: ISSN 1664-462X
Publisher:
Frontiers Research Foundation
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; CRISPR/Cas9; genome editing; bisulfite sequencing; DNA methylation; epigenetic change

Citation Formats

Lee, Jun Hyung, Mazarei, Mitra, Pfotenhauer, Alexander, Dorrough, Aubrey, Poindexter, Magen, Hewezi, Tarek, Lenaghan, Scott C., Graham, David E., and Stewart, Neal C. Epigenetic Footprints of CRISPR/Cas9-Mediated Genome Editing in Plants. United States: N. p., 2020. Web. https://doi.org/10.3389/fpls.2019.01720.
Lee, Jun Hyung, Mazarei, Mitra, Pfotenhauer, Alexander, Dorrough, Aubrey, Poindexter, Magen, Hewezi, Tarek, Lenaghan, Scott C., Graham, David E., & Stewart, Neal C. Epigenetic Footprints of CRISPR/Cas9-Mediated Genome Editing in Plants. United States. https://doi.org/10.3389/fpls.2019.01720
Lee, Jun Hyung, Mazarei, Mitra, Pfotenhauer, Alexander, Dorrough, Aubrey, Poindexter, Magen, Hewezi, Tarek, Lenaghan, Scott C., Graham, David E., and Stewart, Neal C. Fri . "Epigenetic Footprints of CRISPR/Cas9-Mediated Genome Editing in Plants". United States. https://doi.org/10.3389/fpls.2019.01720. https://www.osti.gov/servlets/purl/1606824.
@article{osti_1606824,
title = {Epigenetic Footprints of CRISPR/Cas9-Mediated Genome Editing in Plants},
author = {Lee, Jun Hyung and Mazarei, Mitra and Pfotenhauer, Alexander and Dorrough, Aubrey and Poindexter, Magen and Hewezi, Tarek and Lenaghan, Scott C. and Graham, David E. and Stewart, Neal C.},
abstractNote = {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 T2 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.},
doi = {10.3389/fpls.2019.01720},
journal = {Frontiers in Plant Science},
number = NA,
volume = 10,
place = {United States},
year = {2020},
month = {1}
}

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