Abstract
CRISPR/Cas9 mediated genome editing technology has experienced rapid advances in recent years and has been applied to a wide variety of plant species, including soybean. Several platforms have been developed for designing and cloning of single CRISPR targets or multiple targets in a single destination vector. This chapter provides an updated working protocol for applying CRISPR/Cas9 technology to target a single gene or multiple genes simultaneously in soybean. We describe two platforms for cloning single CRISPR targets and multiplexing targets, respectively, and reagent delivery methodologies. The protocols address crucial limiting steps that can limit CRISPR editing in soybean hairy roots, composite plants, and tissue culture-based regenerated whole plants. To date, transgenic soybean plants with mutagenesis in up to three target genes have been obtained with this procedure.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Cho HJ, Farrand SK, Noel GR, Widholm JM (2000) High-efficiency induction of soybean hairy roots and propagation of the soybean cyst nematode. Planta 210:195–204
Kereszt A, Li D, Indrasumunar A, Nguyen CD, Nontachaiyapoom S, Kinkema M, Gresshoff PM (2007) Agrobacterium rhizogenes-mediated transformation of soybean to study root biology. Nat Protoc 2:948–952
Liang Z, Chen K, Zhang Y, Liu J, Yin K, Qiu J, Gao CX (2018) Genome editing of bread wheat using biolistic delivery of CRISPR/Cas9 in vitro transcripts or ribonucleoproteins. Nat Protoc 13:413–430
Marton I, Zuker A, Shklarman E, Zeevi V, Tovkach A, Roffe S, Ovadis M, Tzfira T, Vainstein A (2010) Nontransgenic genome modification in plant cells. Plant Physiol 154:1079–1087
Woo JW, Kim J, Kwon SI, Corvalán C, Cho SW, Kim H, Kim SG, Kim ST, Choe S, Kim JS (2015) DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins. Nat Biotechnol 33:1162–1164
Zhang Y, Liang Z, Zong Y, Wang Y, Liu J, Chen K, Qiu JL, Gao C (2016) Efficient and transgene-free genome editing in wheat through transient expression of CRISPR/Cas9 DNA or RNA. Nat Commun 7:12617
Curtin SJ, Zhang F, Sander JD, Haun WJ, Starker C, Baltes NJ, Reyon D, Dahlborg EJ, Goodwin MJ, Coffman AP, Dobbs D, Joung JK, Voytas DF, Stupar RM (2011) Targeted mutagenesis of duplicated genes in soybean with zinc-finger nucleases. Plant Physiol 156:466–473
Haun W, Coffman A, Clasen BM, Demorest ZL, Lowy A, Ray E, Retterath A, Stoddard T, Juillerat A, Cedrone F, Mathis L, Voytas DF, Zhang F (2014) Improved soybean oil quality by targeted mutagenesis of the fatty acid desaturase 2 gene family. Plant Biotechnol J 12:934–940
Li Z, Liu ZB, Xing A, Moon BP, Koellhoffer JP, Huang L, Ward RT, Clifton E, Falco SC, Cigan AM (2015) Cas9-guide RNA directed genome editing in soybean. Plant Physiol 169:960–970
Curtin SJ, Xiong Y, Michno JM, 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 16:1125. https://doi.org/10.1111/pbi.12857
Cai Y, Chen L, Liu X, Guo C, Sun S, Wu C, Jiang B, Han T, Hou W (2018) CRISPR/Cas9-mediated targeted mutagenesis of GmFT2a delays flowering time in soybean. Plant Biotechnol J 16:176–185
Kanazashi Y, Hirose A, Takahashi I, Mikami M, Endo M, Hirose S, Toki S, Kaga A, Naito K, Ishimoto M, Abe J, Yamada T (2018) Simultaneous site-directed mutagenesis of duplicated loci in soybean using a single guide RNA. Plant Cell Rep 37:553–563
Michno JM, Wang XB, Liu J, Curtin SJ, Kono TJ, Stupar RM (2015) CRISPR/Cas mutagenesis of soybean and Medicago truncatula using a new web-tool and a modified Cas9 enzyme. GM Crops Food 6:243–253
Cermak T, Curtin SJ, Gil-Humanes H, Cegan R, Kono TJ, Konecna E, Belanto J, Starker C, Wathre JW, Greenstein RL, Voytas DF (2017) A multipurpose toolkit to enable advanced genome engineering in plants. Plant Cell 29:1196–1217
Zhu X, Xu Y, Yu S, Lu L, Ding M, Cheng J, Song G, Gao X, Yao L, Fan D, Meng S, Zhang X, Hu S, Tian Y (2014) An efficient genotyping method for genome-modified animals and human cells generated with CRISPR/Cas9 system. Sci Rep 4:6420–6428
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plantarum 15:473–497
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
Paz MM, Shou H, Guo Z, Zhang Z, Banerjee AK, Wang K (2004) Assessment of conditions affecting Agrobacterium-mediated soybean transformation using the cotyledonary node explant. Euphytica 136:167–179
Mankin SL, Hill DS, Olhoft PM, Toren E, Wenck AR, Nea L, Xing L, Brown J, Fu H, Ireland L, Jia H, Hillebrand H, Jones T, Song HS (2007) Disarming and sequencing of Agrobacterium rhizogenes strain K599 (NCPPB2659) plasmid pRi2659. In Vitro Cell Dev Biol 43:521–535
Konieczny A, Ausubel FM (1993) A procedure for mapping Arabidopsis mutations using co-dominant ecotype-specific PCR-based markers. Plant J 4:403–410
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Liu, J., Gunapati, S., Mihelich, N.T., Stec, A.O., Michno, JM., Stupar, R.M. (2019). Genome Editing in Soybean with CRISPR/Cas9. In: Qi, Y. (eds) Plant Genome Editing with CRISPR Systems. Methods in Molecular Biology, vol 1917. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-8991-1_16
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8991-1_16
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-8990-4
Online ISBN: 978-1-4939-8991-1
eBook Packages: Springer Protocols