Simultaneous site-directed mutagenesis of duplicated loci in soybean using a single guide RNA
Using a gRNA and Agrobacterium-mediated transformation, we performed simultaneous site-directed mutagenesis of two GmPPD loci in soybean. Mutations in GmPPD loci were confirmed in at least 33% of T2 seeds.
The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system is a powerful tool for site-directed mutagenesis in crops. Using a single guide RNA (gRNA) and Agrobacterium-mediated transformation, we performed simultaneous site-directed mutagenesis of two homoeologous loci in soybean (Glycine max), GmPPD1 and GmPPD2, which encode the orthologs of Arabidopsis thaliana PEAPOD (PPD). Most of the T1 plants had heterozygous and/or chimeric mutations for the targeted loci. The sequencing analysis of T1 and T2 generations indicates that putative mutation induced in the T0 plant is transmitted to the T1 generation. The inheritable mutation induced in the T1 plant was also detected. This result indicates that continuous induction of mutations during T1 plant development increases the occurrence of mutations in germ cells, which ensures the transmission of mutations to the next generation. Simultaneous site-directed mutagenesis in both GmPPD loci was confirmed in at least 33% of T2 seeds examined. Approximately 19% of double mutants did not contain the Cas9/gRNA expression construct. Double mutants with frameshift mutations in both GmPPD1 and GmPPD2 had dome-shaped trifoliate leaves, extremely twisted pods, and produced few seeds. Taken together, our data indicate that continuous induction of mutations in the whole plant and advancing generations of transgenic plants enable efficient simultaneous site-directed mutagenesis in duplicated loci in soybean.
KeywordsCRISPR/Cas9 Generation Glycine max Heritable mutation Null-segregant PEAPOD
We thank Professor Holger Puchta (University of Karlsruhe) for permission to use plasmid DNAs of pEn-Chimera and pDe-CAS9, and M. Suzuki and Y. Kitsui for general technical assistance. This work was supported by the Cabinet Office, Government of Japan [the Cross-ministerial Strategic Innovation Promotion Program (SIP) for TY].
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
- Bilyeu KD (2008) Genetics and genomics of soybean. In: Stacey G (ed) Plant genetics/genomics, vol 2. Springer, USA, pp 135–139Google Scholar
- Curtin SJ, Xiong Y, Michno J-M, Campbell BW, Stec AO, Čermák T, Starker C, Voytas DF, Eamens AL, Stupar RM (2017) CRISPR/Cas9 and TALENs generate heritable mutations for genes involved in small RNA processing of Glycine max and Medicago truncatula. Plant Biotechnol J. https://doi.org/10.1111/pbi.12857 PubMedGoogle Scholar
- Feng ZY, Mao YF, Xu NF, Zhang BT, Wei PL, Yang DL, Wang Z, Zhang ZJ, Zheng R, Yang L, Zeng L, Liu XD, Zhu JK (2014) Multigeneration analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis. Proc Natl Acad Sci USA 111:4632–4637CrossRefPubMedPubMedCentralGoogle Scholar
- Gonzalez N, Pauwels L, Baekelandt A, De Milde L, Van Leene J, Besbrugge N, Heyndrickx KS, Perez AC, Durand AN, De Clercq R, Van De Slijke E, Bossche RV, Eeckhout D, Gevaert K, Vandepoele K, De Jaeger G, Goossens A, Inze D (2015) A repressor protein complex regulates leaf growth in Arabidopsis. Plant Cell 27:2273–2287CrossRefPubMedPubMedCentralGoogle Scholar
- Rahman SM, Takagi Y, Miyamoto K, Kawakita T (1994) Inheritance of low linolenic acid content in soybean mutant line M-5. Breed Sci 44:379–382Google Scholar
- Schmutz J, Cannon SB, Schlueter J, Ma JX, Mitros T, Nelson W, Hyten DL, Song QJ, Thelen JJ, Cheng JL, Xu D, Hellsten U, May GD, Yu Y, Sakurai T, Umezawa T, Bhattacharyya MK, Sandhu D, Valliyodan B, Lindquist E, Peto M, Grant D, Shu SQ, Goodstein D, Barry K, Futrell-Griggs M, Abernathy B, Du JC, Tian ZX, Zhu LC, Gill N, Joshi T, Libault M, Sethuraman A, Zhang XC, Shinozaki K, Nguyen HT, Wing RA, Cregan P, Specht J, Grimwood J, Rokhsar D, Stacey G, Shoemaker RC, Jackson SA (2010) Genome sequence of the palaeopolyploid soybean. Nature 463:178–183CrossRefPubMedGoogle Scholar