Abstract
Soybean has a palaeopolyploid genome with nearly 75% of the genes present in multiple copies. Although the CRISPR/Cas9 system has been employed in soybean to generate site-directed mutagenesis, a systematical assessment of mutation efficiency of the CRISPR/Cas9 system for the multiple-copy genes is still urgently needed. Here, we successfully optimize one sgRNA CRISPR/Cas9 system in soybean by testing the efficiency, pattern, specificity of the mutations at multiple loci of GmFAD2 and GmALS. The results showed that simultaneous site-directed mutagenesis of two homoeologous loci by one sgRNA, the mutation frequency in the T0 generation were 64.71% for GmPDS, 60.0% for GmFAD2 and 42.86% for GmALS, respectively. The chimeric and heterozygous mutations were dominant types. Moreover, association of phenotypes with mutation pattern at target loci of GmPDS11 and GmPDS18 could help us further demonstrate that the CRISPR/Cas9 system can efficiently generate target specific mutations at multiple loci using one sgRNA in soybean, albeit with a relatively low transformation efficiency.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Bao A, Burritt DJ, Chen H, Zhou X, Cao D, Tran LP (2019) The CRISPR/Cas9 system and its applications in crop genome editing. Crit Rev Biotechnol 39:321–336. doi:https://doi.org/10.1080/07388551.2018.1554621
Bhowmik P et al (2018) Targeted mutagenesis in wheat microspores using CRISPR/Cas9. Sci Rep 8:6502. doi:https://doi.org/10.1038/s41598-018-24690-8
Cai Y et al (2019) Mutagenesis of GmFT2a and GmFT5a mediated by CRISPR/Cas9 contributes for expanding the regional adaptability of soybean. Plant Biotechnol J. doi:https://doi.org/10.1111/pbi.13199
Char SN, Li R, Yang B (2019) CRISPR/Cas9 for mutagenesis in rice. Methods Mol Biol 1864:279–293. https://doi.org/10.1007/978-1-4939-8778-8_19
Christian M et al (2010) Targeting DNA double-strand breaks with TAL effector nucleases. Genetics 186:757–761. https://doi.org/10.1534/genetics.110.120717
Do PT et al (2019) Demonstration of highly efficient dual gRNA CRISPR/Cas9 editing of the homeologous GmFAD2-1A and GmFAD2-1B genes to yield a high oleic, low linoleic and alpha-linolenic acid phenotype in soybean. BMC Plant Biol 19:311. doi:https://doi.org/10.1186/s12870-019-1906-8
Han Y et al (2019) Generation of semi-dwarf rice (Oryza sativa L.) lines by CRISPR/Cas9-directed mutagenesis of OsGA20ox2 and proteomic analysis of unveiled changes caused by mutations. 3 Biotech 9:387. https://doi.org/10.1007/s13205-019-1919-x
Howells RM, Craze M, Bowden S, Wallington EJ (2018) Efficient generation of stable, heritable gene edits in wheat using CRISPR/Cas9. BMC Plant Biol 18:215. doi:https://doi.org/10.1186/s12870-018-1433-z
Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821. https://doi.org/10.1126/science.1225829
Kaul T et al (2019) Data mining by pluralistic approach on CRISPR gene editing in plants. Front Plant Sci 10:801. https://doi.org/10.3389/fpls.2019.00801
Kim D, Alptekin B, Budak H (2018) CRISPR/Cas9 genome editing in wheat. Funct Integr Genomics 18:31–41. doi:https://doi.org/10.1007/s10142-017-0572-x
Lei Y, Lu L, Liu HY, Li S, Xing F, Chen LL (2014) CRISPR-P: a web tool for synthetic single-guide RNA design of CRISPR-system in plants. Mol Plant 7:1494–1496. doi:https://doi.org/10.1093/mp/ssu044
Liang Z, Chen K, Zhang Y, Liu J, Yin K, Qiu JL, Gao C (2018) Genome editing of bread wheat using biolistic delivery of CRISPR/Cas9 in vitro transcripts or ribonucleoproteins. Nat Protoc 13:413–430. doi:https://doi.org/10.1038/nprot.2017.145
Lu Y, Zhu JK (2017) Precise editing of a target base in the rice genome using a modified CRISPR/Cas9 system. Mol Plant 10:523–525. https://doi.org/10.1016/j.molp.2016.11.013
Ma X et al (2015) A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol Plant 8:1274–1284. https://doi.org/10.1016/j.molp.2015.04.007
Paz MM, Martinez JC, Kalvig AB, Fonger TM, Wang K (2006) Improved cotyledonary node method using an alternative explant derived from mature seed for efficient Agrobacterium-mediated soybean transformation. Plant Cell Rep 25:206–213. https://doi.org/10.1007/s00299-005-0048-7
Perez L et al (2019) CRISPR/Cas9 mutations in the rice Waxy/GBSSI gene induce allele-specific and zygosity-dependent feedback effects on endosperm starch biosynthesis. Plant Cell Rep 38:417–433. doi:https://doi.org/10.1007/s00299-019-02388-z
Puchta H (2005) The repair of double-strand breaks in plants: mechanisms and consequences for genome evolution. J Exp Bot 56:1–14. doi:https://doi.org/10.1093/jxb/eri025
Qin G, Gu H, Ma L, Peng Y, Deng XW, Chen Z, Qu LJ (2007) Disruption of phytoene desaturase gene results in albino and dwarf phenotypes in Arabidopsis by impairing chlorophyll, carotenoid, and gibberellin biosynthesis. Cell Res 17:471–482. doi:https://doi.org/10.1038/cr.2007.40
Schaeffer SM, Nakata PA (2015) CRISPR/Cas9-mediated genome editing and gene replacement in plants: transitioning from lab to field. Plant Sci 240:130–142. https://doi.org/10.1016/j.plantsci.2015.09.011
Schmutz J et al (2010) Genome sequence of the palaeopolyploid soybean. Nature 463:178–183. doi:https://doi.org/10.1038/nature08670
Walter KL, Strachan SD, Ferry NM, Albert HH, Castle LA, Sebastian SA (2014) Molecular and phenotypic characterization of Als1 and Als2 mutations conferring tolerance to acetolactate synthase herbicides in soybean. Pest Manag Sci 70:1831–1839. doi:https://doi.org/10.1002/ps.3725
Zhang H et al (2014) The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation. Plant Biotechnol J 12:797–807. doi:https://doi.org/10.1111/pbi.12200
Zhang P et al (2019) Multiplex CRISPR/Cas9-mediated metabolic engineering increases soybean isoflavone content and resistance to soybean mosaic virus. Plant Biotechnol J. doi:https://doi.org/10.1111/pbi.13302
Acknowledgements
This study was supported by National Natural Science Foundation of China (31501327), the Jilin Scientific and Technological Development Program (20170101014JC) and the Agricultural Science and Technology Innovation Project of Jilin Province (CXGC2017ZY024 and CXGC2018ZY027) to Dr. Ling Zhang. The Ministry of Agriculture of China for Transgenic Research (2016ZX08004-004) to Dr. Yingshan Dong. We are grateful to Dr. Feng Liu (Nanjing Agricultural University) for kindly providing the CPRISP/Cas9 vector.
Author information
Authors and Affiliations
Contributions
LZ and YSD directed and designed the experiments. TL and HMQ contributed to phenotypic survey and photography. LZ performed the vector construct and soybean transformation experiment. LZ and TL performed mutation identification the data processing. YZW performed the bioinformatics analysis of genes. LZ and ZJX drafted and revised the manuscript.
Corresponding author
Ethics declarations
Conflict of interest
The authors have declared that no competing interests exist.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Zhang, L., Wang, Y., Li, T. et al. Target-specific mutations efficiency at multiple loci of CRISPR/Cas9 system using one sgRNA in soybean. Transgenic Res 30, 51–62 (2021). https://doi.org/10.1007/s11248-020-00228-5
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11248-020-00228-5