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CRISPR/Cas9-Based Gene Editing Using Egg Cell-Specific Promoters in Arabidopsis and Soybean

Zheng, Na and Li, Ting and Dittman, Jaime D. and Su, Jianbin and Li, Riqing and Gassmann, Walter and Peng, Deliang and Whitham, Steven A. and Liu, Shiming and Yang, Bing (2020) CRISPR/Cas9-Based Gene Editing Using Egg Cell-Specific Promoters in Arabidopsis and Soybean. Frontiers in Plant Science, 11 . Art. No. 800. ISSN 1664-462X. PMCID PMC7309964. https://resolver.caltech.edu/CaltechAUTHORS:20200706-131708863

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Abstract

CRISPR/Cas9-based systems are efficient genome editing tools in a variety of plant species including soybean. Most of the gene edits in soybean plants are somatic and non-transmissible when Cas9 is expressed under control of constitutive promoters. Tremendous effort, therefore, must be spent to identify the inheritable edits occurring at lower frequencies in plants of successive generations. Here, we report the development and validation of genome editing systems in soybean and Arabidopsis based on Cas9 driven under four different egg-cell specific promoters. A soybean ubiquitin gene promoter driving expression of green fluorescent protein (GFP) is incorporated in the CRISPR/Cas9 constructs for visually selecting transgenic plants and transgene-evicted edited lines. In Arabidopsis, the four systems all produced a collection of mutations in the T2 generation at frequencies ranging from 8.3 to 42.9%, with egg cell-specific promoter AtEC1.2e1.1p being the highest. In soybean, function of the gRNAs and Cas9 expressed under control of the CaMV double 35S promoter (2x35S) in soybean hairy roots was tested prior to making stable transgenic plants. The 2x35S:Cas9 constructs yielded a high somatic mutation frequency in soybean hairy roots. In stable transgenic soybean T1 plants, AtEC1.2e1.1p:Cas9 yielded a mutation rate of 26.8%, while Cas9 expression driven by the other three egg cell-specific promoters did not produce any detected mutations. Furthermore, the mutations were inheritable in the T2 generation. Our study provides CRISPR gene-editing platforms to generate inheritable mutants of Arabidopsis and soybean without the complication of somatic mutagenesis, which can be used to characterize genes of interest in Arabidopsis and soybean.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.3389/fpls.2020.00800DOIArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7309964PubMed CentralArticle
ORCID:
AuthorORCID
Li, Ting0000-0001-8273-5593
Additional Information:© 2020 Zheng, Li, Dittman, Su, Li, Gassmann, Peng, Whitham, Liu and Yang. 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. Received: 11 April 2020; Accepted: 19 May 2020; Published: 16 June 2020. We thank Diane Luth at the Iowa State University Plant Transformation Facility for soybean transformation. We thank the Iowa State University Crop Bioengineering Center for publication subvention. Data Availability Statement: All datasets generated for this study are included in the article/Supplementary Material. Author Contributions: BY, SW, and SL designed the research. NZ, TL, JD, JS, and RL performed the experiments. DP and WG analyzed the data. NZ and BY wrote the manuscript with input from all other authors. Funding was provided by the Iowa Soybean Association (SW and BY), the National Natural Science Foundation of China (31972248 to SL), and the China Scholarship Council (NZ). The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Funders:
Funding AgencyGrant Number
Iowa Soybean AssociationUNSPECIFIED
National Natural Science Foundation of China31972248
China Scholarship CouncilUNSPECIFIED
Subject Keywords:CRISPR/Cas9, gene editing, egg cell-specific promoter, Arabidopsis, Glycine max, soybean
PubMed Central ID:PMC7309964
Record Number:CaltechAUTHORS:20200706-131708863
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20200706-131708863
Official Citation:Zheng N, Li T, Dittman JD, Su J, Li R, Gassmann W, Peng D, Whitham SA, Liu S and Yang B (2020) CRISPR/Cas9-Based Gene Editing Using Egg Cell-Specific Promoters in Arabidopsis and Soybean. Front. Plant Sci. 11:800. doi: 10.3389/fpls.2020.00800
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:104226
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:06 Jul 2020 20:34
Last Modified:06 Jul 2020 20:34

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