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. 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.
| Original language | English |
|---|---|
| Article number | 1720 |
| Journal | Frontiers in Plant Science |
| Volume | 10 |
| DOIs | |
| State | Published - Jan 31 2020 |
Funding
We would like to thank Ralph Laurel for his help with plant care and Sarbottam Piya for useful discussion. This research was sponsored in part by the Laboratory Directed Research and Development Program of Oak Ridge National Laboratory, managed by UT-Battelle, LLC, for the US Department of Energy (DOE) under contract DE-AC05-00OR22725. This work was partially funded by the Center for Bioenergy Innovation supported by the Office of Biological and Environmental Research in the DOE Office of Science. This manuscript has been co-authored by UT-Battelle, LLC, under contract DE-AC05-00OR22725 with the DOE. The US government retains and the publisher, by accepting the article for publication, acknowledges that the US government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this manuscript, or allow others to do so, for US government purposes. DOE will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan (https://energy.gov/downloads/doe-public-access-plan). We also acknowledge funding from Hatch grants to CNS, SCL, and TH, as well as funding from UT AgResearch to support the Center for Agricultural Synthetic Biology.
| Funders | Funder number |
|---|---|
| Center for Agricultural Synthetic Biology | |
| DOE Office of Science | |
| DOE Public Access Plan | |
| Office of Biological and Environmental Research | |
| US Department of Energy | |
| U.S. Department of Energy | DE-AC05-00OR22725 |
| Oak Ridge National Laboratory |
Keywords
- CRISPR/Cas9
- DNA methylation
- bisulfite sequencing
- epigenetic change
- genome editing