Welcome to the new version of CaltechAUTHORS. Login is currently restricted to library staff. If you notice any issues, please email coda@library.caltech.edu
Published June 16, 2020 | Published + Supplemental Material
Journal Article Open

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

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.

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.

Attached Files

Published - fpls-11-00800.pdf

Supplemental Material - Data_Sheet_1_CRISPR_Cas9-Based_Gene_Editing_Using_Egg_Cell-Specific_Promoters_in_Arabidopsis_and_Soybean.pdf

Files

Data_Sheet_1_CRISPR_Cas9-Based_Gene_Editing_Using_Egg_Cell-Specific_Promoters_in_Arabidopsis_and_Soybean.pdf

Additional details

Created:
August 19, 2023
Modified:
October 20, 2023