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

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.

View Accepted Manuscript (DOE)

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

Publisher:: Frontiers Research FoundationCopyright Statement

Country of Publication:: United States

Language:: English

References (49)

Phytopathogen-induced changes to plant methylomes Hewezi, Tarek; Pantalone, Vince; Bennett, Morgan Plant Cell Reports, Vol. 37, Issue 1 https://doi.org/10.1007/s00299-017-2188-y	journal	July 2017
Characterization of oleosin genes from forage sorghum in Arabidopsis and yeast reveals their role in storage lipid stability Ojha, Rabishankar; Kaur, Simranjit; Sinha, Kshitija Planta, Vol. 254, Issue 5 https://doi.org/10.1007/s00425-021-03744-8	journal	October 2021
Role of DNA methylation in imprinting disorders: an updated review Elhamamsy, Amr Rafat Journal of Assisted Reproduction and Genetics, Vol. 34, Issue 5 https://doi.org/10.1007/s10815-017-0895-5	journal	March 2017
CRISPR/Cas9-mediated targeted mutagenesis in Nicotiana tabacum Gao, Junping; Wang, Genhong; Ma, Sanyuan Plant Molecular Biology, Vol. 87, Issue 1-2 https://doi.org/10.1007/s11103-014-0263-0	journal	October 2014
Walking a Fine Germline: Synthesizing Public Opinion and Legal Precedent to Develop Policy Recommendations for Heritable Gene-Editing Benston, Shawna Journal of Bioethical Inquiry, Vol. 19, Issue 3 https://doi.org/10.1007/s11673-022-10186-8	journal	April 2022
Putting DNA methylation in context: from genomes to gene expression in plants Niederhuth, Chad E.; Schmitz, Robert J. Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, Vol. 1860, Issue 1 https://doi.org/10.1016/j.bbagrm.2016.08.009	journal	January 2017
Genome-wide High-Resolution Mapping and Functional Analysis of DNA Methylation in Arabidopsis Zhang, Xiaoyu; Yazaki, Junshi; Sundaresan, Ambika Cell, Vol. 126, Issue 6 https://doi.org/10.1016/j.cell.2006.08.003	journal	September 2006
ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering Gaj, Thomas; Gersbach, Charles A.; Barbas, Carlos F. Trends in Biotechnology, Vol. 31, Issue 7, p. 397-405 https://doi.org/10.1016/j.tibtech.2013.04.004	journal	July 2013
Competition between DNA methylation and transcription factors determines binding of NRF1 Domcke, Silvia; Bardet, Anaïs Flore; Adrian Ginno, Paul Nature, Vol. 528, Issue 7583 https://doi.org/10.1038/nature16462	journal	December 2015
High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells Fu, Yanfang; Foden, Jennifer A.; Khayter, Cyd Nature Biotechnology, Vol. 31, Issue 9 https://doi.org/10.1038/nbt.2623	journal	June 2013
GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases Tsai, Shengdar Q.; Zheng, Zongli; Nguyen, Nhu T. Nature Biotechnology, Vol. 33, Issue 2 https://doi.org/10.1038/nbt.3117	journal	December 2014
Transcription factors as readers and effectors of DNA methylation Zhu, Heng; Wang, Guohua; Qian, Jiang Nature Reviews Genetics, Vol. 17, Issue 9 https://doi.org/10.1038/nrg.2016.83	journal	August 2016
Rapid improvement of domestication traits in an orphan crop by genome editing Lemmon, Zachary H.; Reem, Nathan T.; Dalrymple, Justin Nature Plants, Vol. 4, Issue 10 https://doi.org/10.1038/s41477-018-0259-x	journal	October 2018
Knockout of OsNramp5 using the CRISPR/Cas9 system produces low Cd-accumulating indica rice without compromising yield Tang, Li; Mao, Bigang; Li, Yaokui Scientific Reports, Vol. 7, Issue 1 https://doi.org/10.1038/s41598-017-14832-9	journal	October 2017
CRISPR/Cas9-mediated efficient and heritable targeted mutagenesis in tomato plants in the first and later generations Pan, Changtian; Ye, Lei; Qin, Li Scientific Reports, Vol. 6, Issue 1 https://doi.org/10.1038/srep24765	journal	April 2016
Multigeneration analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis Feng, Z.; Mao, Y.; Xu, N. Proceedings of the National Academy of Sciences, Vol. 111, Issue 12 https://doi.org/10.1073/pnas.1400822111	journal	February 2014
Critical roles of DNA demethylation in the activation of ripening-induced genes and inhibition of ripening-repressed genes in tomato fruit Lang, Zhaobo; Wang, Yihai; Tang, Kai Proceedings of the National Academy of Sciences, Vol. 114, Issue 22 https://doi.org/10.1073/pnas.1705233114	journal	May 2017
CRISPR-Cas9 Targeted Mutagenesis Leads to Simultaneous Modification of Different Homoeologous Gene Copies in Polyploid Oilseed Rape ( Brassica napus ) Braatz, Janina; Harloff, Hans-Joachim; Mascher, Martin Plant Physiology, Vol. 174, Issue 2 https://doi.org/10.1104/pp.17.00426	journal	April 2017
Towards CRISPR/Cas crops - bringing together genomics and genome editing Scheben, Armin; Wolter, Felix; Batley, Jacqueline New Phytologist, Vol. 216, Issue 3 https://doi.org/10.1111/nph.14702	journal	August 2017
The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation Zhang, Hui; Zhang, Jinshan; Wei, Pengliang Plant Biotechnology Journal, Vol. 12, Issue 6 https://doi.org/10.1111/pbi.12200	journal	May 2014
Genome editing of the disease susceptibility gene CsLOB1 in citrus confers resistance to citrus canker Jia, Hongge; Zhang, Yunzeng; Orbović, Vladimir Plant Biotechnology Journal, Vol. 15, Issue 7 https://doi.org/10.1111/pbi.12677	journal	January 2017
CRISPR/Cas9-mediated targeted mutagenesis of GmFT2a delays flowering time in soya bean Cai, Yupeng; Chen, Li; Liu, Xiujie Plant Biotechnology Journal, Vol. 16, Issue 1 https://doi.org/10.1111/pbi.12758	journal	June 2017
Genotyping genome-edited mutations in plants using CRISPR ribonucleoprotein complexes Liang, Zhen; Chen, Kunling; Yan, Yan Plant Biotechnology Journal, Vol. 16, Issue 12 https://doi.org/10.1111/pbi.12938	journal	May 2018
Genetic Screens in Human Cells Using the CRISPR-Cas9 System Wang, Tim; Wei, Jenny J.; Sabatini, David M. Science, Vol. 343, Issue 6166 https://doi.org/10.1126/science.1246981	journal	December 2013
Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR Haeussler, Maximilian; Schönig, Kai; Eckert, Hélène Genome Biology, Vol. 17, Issue 1 https://doi.org/10.1186/s13059-016-1012-2	journal	July 2016
Advanced Methylome Analysis after Bisulfite Deep Sequencing: An Example in Arabidopsis Dinh, Huy Q.; Dubin, Manu; Sedlazeck, Fritz J. PLoS ONE, Vol. 7, Issue 7 https://doi.org/10.1371/journal.pone.0041528	journal	July 2012
Editorial: Epigenetic Modifications Associated with Abiotic and Biotic Stresses in Plants: An Implication for Understanding Plant Evolution Yaish, Mahmoud W. Frontiers in Plant Science, Vol. 8 https://doi.org/10.3389/fpls.2017.01983	journal	November 2017
CRISPR for Crop Improvement: An Update Review Jaganathan, Deepa; Ramasamy, Karthikeyan; Sellamuthu, Gothandapani Frontiers in Plant Science, Vol. 9 https://doi.org/10.3389/fpls.2018.00985	journal	July 2018
Epigenetic Regulation of Juvenile-to-Adult Transition in Plants Xu, Yunmin; Zhang, Lu; Wu, Gang Frontiers in Plant Science, Vol. 9 https://doi.org/10.3389/fpls.2018.01048	journal	July 2018
DNA Methylation and the Evolution of Developmental Complexity in Plants Bräutigam, Katharina; Cronk, Quentin Frontiers in Plant Science, Vol. 9 https://doi.org/10.3389/fpls.2018.01447	journal	October 2018
Dynamic DNA Methylation in Plant Growth and Development Bartels, Arthur; Han, Qiang; Nair, Pooja International Journal of Molecular Sciences, Vol. 19, Issue 7 https://doi.org/10.3390/ijms19072144	journal	July 2018
Targeted DNA Methylation Analysis by Next-generation Sequencing Masser, Dustin R.; Stanford, David R.; Freeman, Willard M. Journal of Visualized Experiments, Issue 96 https://doi.org/10.3791/52488	journal	January 2015
Accurate sodium bisulfite sequencing in plants Henderson, Ian R.; Chan, Simon R.; Cao, Xiaofeng Epigenetics, Vol. 5, Issue 1 https://doi.org/10.4161/epi.5.1.10560	journal	January 2010
Strategies for analyzing bisulfite sequencing data Wreczycka, Katarzyna; Gosdschan, Alexander; Yusuf, Dilmurat Journal of Biotechnology, Vol. 261 https://doi.org/10.1016/j.jbiotec.2017.08.007	journal	November 2017
Gene body DNA methylation in plants Bewick, Adam J.; Schmitz, Robert J. Current Opinion in Plant Biology, Vol. 36 https://doi.org/10.1016/j.pbi.2016.12.007	journal	April 2017
Improving CRISPR Genome Editing by Engineering Guide RNAs Moon, Su Bin; Kim, Do Yon; Ko, Jeong-Heon Trends in Biotechnology, Vol. 37, Issue 8 https://doi.org/10.1016/j.tibtech.2019.01.009	journal	August 2019
CRISPR plants now subject to tough GM laws in European Union Callaway, Ewen Nature, Vol. 560, Issue 7716 https://doi.org/10.1038/d41586-018-05814-6	journal	July 2018
Off-target Effects in CRISPR/Cas9-mediated Genome Engineering Zhang, Xiao-Hui; Tee, Louis Y.; Wang, Xiao-Gang Molecular Therapy - Nucleic Acids, Vol. 4 https://doi.org/10.1038/mtna.2015.37	journal	January 2015
With a free pass, CRISPR-edited plants reach market in record time Waltz, Emily Nature Biotechnology, Vol. 36, Issue 1 https://doi.org/10.1038/nbt0118-6b	journal	January 2018
Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method Zhang, Xiuren; Henriques, Rossana; Lin, Shih-Shun Nature Protocols, Vol. 1, Issue 2 https://doi.org/10.1038/nprot.2006.97	journal	June 2006
Dynamics and function of DNA methylation in plants Zhang, Huiming; Lang, Zhaobo; Zhu, Jian-Kang Nature Reviews Molecular Cell Biology, Vol. 19, Issue 8 https://doi.org/10.1038/s41580-018-0016-z	journal	May 2018
Erratum: Corrigendum: CRISPR/Cas9-mediated efficient and heritable targeted mutagenesis in tomato plants in the first and later generations Pan, Changtian; Ye, Lei; Qin, Li Scientific Reports, Vol. 7, Issue 1 https://doi.org/10.1038/srep46916	journal	December 2017
Risk associated with off-target plant genome editing and methods for its limitation Zhao, Hui; Wolt, Jeffrey D. Emerging Topics in Life Sciences, Vol. 1, Issue 2 https://doi.org/10.1042/ETLS20170037	journal	November 2017
The CRISPR/Cas9 system and its applications in crop genome editing Bao, Aili; Burritt, David J.; Chen, Haifeng Critical Reviews in Biotechnology, Vol. 39, Issue 3 https://doi.org/10.1080/07388551.2018.1554621	journal	January 2019
Analysis of the Arabidopsis superman allelic series and the interactions with other genes demonstrate developmental robustness and joint specification of male–female boundary, flower meristem termination and carpel compartmentalization Breuil-Broyer, Stéphanie; Trehin, Christophe; Morel, Patrice Annals of Botany, Vol. 117, Issue 5 https://doi.org/10.1093/aob/mcw023	journal	April 2016
Genome-Wide Analysis of DNA Methylation in Soybean Song, Qing-Xin; Lu, Xiang; Li, Qing-Tian Molecular Plant, Vol. 6, Issue 6 https://doi.org/10.1093/mp/sst123	journal	November 2013
Cyst Nematode Parasitism Induces Dynamic Changes in the Root Epigenome Hewezi, Tarek; Lane, Thomas; Piya, Sarbottam Plant Physiology, Vol. 174, Issue 1 https://doi.org/10.1104/pp.16.01948	journal	March 2017
A CRISPR/Cas9 toolkit for multiplex genome editing in plants Xing, Hui-Li; Dong, Li; Wang, Zhi-Ping BMC Plant Biology, Vol. 14, Issue 1 https://doi.org/10.1186/s12870-014-0327-y	journal	November 2014
Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR Haeussler, Maximilian; Schönig, Kai; Eckert, Hélène Figshare https://doi.org/10.6084/m9.figshare.c.3643583	collection	January 2016

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59 BASIC BIOLOGICAL SCIENCES
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DNA methylation
bisulfite sequencing
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genome editing

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

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