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

Lee, Jun Hyung; Mazarei, Mitra; Pfotenhauer, Alexander; Dorrough, Aubrey; Poindexter, Magen; Hewezi, Tarek; Lenaghan, Scott C.; Graham, David E.; Stewart, Neal C.

doi:10.3389/fpls.2019.01720

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

Journal Article · Fri Jan 31 00:00:00 EST 2020 · Frontiers in Plant Science

DOI:https://doi.org/10.3389/fpls.2019.01720· OSTI ID:1606824

Lee, Jun Hyung ^[1]; Mazarei, Mitra ^[1]; Pfotenhauer, Alexander ^[2]; Dorrough, Aubrey ^[1]; Poindexter, Magen ^[1]; Hewezi, Tarek ^[1]; Lenaghan, Scott C. ^[2];

^[3]; Stewart, Neal C. ^[1]

Univ. of Tennessee, Knoxville, TN (United States). Dept. of Plant Sciences
Univ. of Tennessee, Knoxville, TN (United States). Dept. of Plant Sciences, and Dept. of Food Science
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)

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.

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Research Organization:: Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)

Sponsoring Organization:: USDOE

Grant/Contract Number:: AC05-00OR22725

OSTI ID:: 1606824

Journal Information:: Frontiers in Plant Science, Vol. 10, Issue NA; ISSN 1664-462X

Publisher:: Frontiers Research FoundationCopyright Statement

Country of Publication:: United States

Language:: English

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Cited by: 8 works

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