Two efficient CRISPR/Cas9 systems for gene editing in soybean

Carrijo, Jéssica; Illa-Berenguer, Eudald; LaFayette, Peter R.; Torres, Nathália Miranda Feitosa; Aragão, F. J. L.; Parrott, W. A.; Vianna, Giovanni R.

doi:10.1007/s11248-021-00246-x

Cited by 23 publications

(11 citation statements)

References 51 publications

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“…In soybean multiplex gene editing, previous work reported the application of four sgRNAs with two different soybean Pol III-dependent promoters ( GmU3 and GmU6 ) driving the expression of the individual sgRNAs, and this system was used to knock out flavanone-3-hydroxylase (F3H) and flavone synthase II (FNS II) gene to increase the isoflavone content ( Zhang et al., 2020 ). The TCTU and STU systems with three targets were evaluated in soybean hairy roots ( Carrijo et al., 2021 ). They found that the TCTU was more efficient than the STU system.…”

Section: Discussionmentioning

confidence: 99%

“…An individual expression unit in the whole cassette is separated by Csy4 hairpins or tRNA Gly genes, generating functional individual units after processing ( Xie et al., 2015 ; Čermák et al., 2017 ). In soybean, the TCTU system was used to increase isoflavone content ( Zhang et al., 2020 ), and STU was used in soybean gene editing ( Carrijo et al., 2021 ). However, the CRISPR/Cas9-mediated multiplex gene editing system for soybean still needs to be evaluated and modified.…”

Section: Introductionmentioning

confidence: 99%

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Multiplex CRISPR/Cas9-mediated raffinose synthase gene editing reduces raffinose family oligosaccharides in soybean

Cao

Wang

et al. 2022

Front. Plant Sci.

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Soybean [Glycine max (L.) Merr.] is an important world economic crop. It is rich in oil, protein, and starch, and soluble carbohydrates in soybean seeds are also important for human and livestock consumption. The predominant soluble carbohydrate in soybean seed is composed of sucrose and raffinose family oligosaccharides (RFOs). Among these carbohydrates, only sucrose can be digested by humans and monogastric animals and is beneficial for metabolizable energy, while RFOs are anti-nutritional factors in diets, usually leading to flatulence and indigestion, ultimately reducing energy efficiency. Hence, breeding efforts to remove RFOs from soybean seeds can increase metabolizable energy and improve nutritional quality. The objective of this research is to use the multiplex Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9‐mediated gene editing system to induce the knockout of soybean raffinose synthase (RS) genes RS2 and RS3 simultaneously to reduce RFOs in mature seeds. First, we constructed five types of multiplex gene editing systems and compared their editing efficiency in soybean hairy roots. We confirmed that the two-component transcriptional unit (TCTU) and single transcriptional unit (STU) systems with transfer RNA (tRNA) as the cleavage site performed better than other systems. The average editing efficiency at the four targets with TCTU-tRNA and STU-tRNA was 50.5% and 46.7%, respectively. Then, we designed four single-guide RNA (sgRNA) targets to induce mutations at RS2 and RS3 by using the TCTU-tRNA system. After the soybean transformation, we obtained several RS2 and RS3 mutation plants, and a subset of alleles was successfully transferred to the progeny. We identified null single and double mutants at the T2 generation and analyzed the seed carbohydrate content of their progeny. The RS2 and RS3 double mutants and the RS2 single mutant exhibited dramatically reduced levels of raffinose and stachyose in mature seeds. Further analysis of the growth and development of these mutants showed that there were no penalties on these phenotypes. Our results indicate that knocking out RS genes by multiplex CRISPR/Cas9-mediated gene editing is an efficient way to reduce RFOs in soybean. This research demonstrates the potential of using elite soybean cultivars to improve the soybean meal trait by multiplex CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9-mediated gene editing.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Multiplex CRISPR/Cas9-mediated raffinose synthase gene editing reduces raffinose family oligosaccharides in soybean

Cao

Wang

et al. 2022

Front. Plant Sci.

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“…Two transcription systems were also tested in soybean including the single transcriptional unit (STU), SpCas9 and sgRNA are driven by only one promoter, and in the conventional system, the two-component transcriptional unit (TCTU), SpCas9, is under the control of a pol II promoter, and sgRNAs are under the control of a pol III promoter. The results showed that the STU is more efficient (Carrijo et al, 2021). Cpf1 (Cas12a) systems have also been established in soybean for GE (Duan et al, 2021;Kim and Choi, 2021).…”

Section: Development Of Transformation Methods For New Breeding Techn...mentioning

confidence: 99%

Progress in Soybean Genetic Transformation Over the Last Decade

Guo

Qiu

et al. 2022

Front. Plant Sci.

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Soybean is one of the important food, feed, and biofuel crops in the world. Soybean genome modification by genetic transformation has been carried out for trait improvement for more than 4 decades. However, compared to other major crops such as rice, soybean is still recalcitrant to genetic transformation, and transgenic soybean production has been hampered by limitations such as low transformation efficiency and genotype specificity, and prolonged and tedious protocols. The primary goal in soybean transformation over the last decade is to achieve high efficiency and genotype flexibility. Soybean transformation has been improved by modifying tissue culture conditions such as selection of explant types, adjustment of culture medium components and choice of selection reagents, as well as better understanding the transformation mechanisms of specific approaches such as Agrobacterium infection. Transgenesis-based breeding of soybean varieties with new traits is now possible by development of improved protocols. In this review, we summarize the developments in soybean genetic transformation to date, especially focusing on the progress made using Agrobacterium-mediated methods and biolistic methods over the past decade. We also discuss current challenges and future directions.

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“…To conduct gene editing research, an efficient transformation system is essential; however, no transformation system suitable for pyrethrum has yet been developed. Agrobacterium rhizogenes -mediated hairy root transformation is considered a rapid, simple, and highly efficient method that enables rapid characterization of improved CRISPR/Cas9 systems in hairy roots [ 26 – 29 ]. This report describes an A. rhizogenes -mediated hairy root transformation system that can quickly verify the effect of gene editing in pyrethrum, and tests the gene editing effect of a ribozyme-mediated CRISPR/Cas9 system using a pol II promoter in this context.…”

Section: Introductionmentioning

confidence: 99%

Ribozyme-mediated CRISPR/Cas9 gene editing in pyrethrum (Tanacetum cinerariifolium) hairy roots using a RNA polymerase II-dependent promoter

Zeng

et al. 2022

Plant Methods

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Background Traditional CRISPR/Cas9 systems that rely on U6 or U3 snRNA promoters (RNA polymerase III-dependent promoters) can only achieve constitutive gene editing in plants, hampering the functional analysis of specifically expressed genes. Ribozyme-mediated CRISPR/Cas9 systems increase the types of promoters which can be used to transcribe sgRNA. Therefore, such systems allow specific gene editing; for example, transcription of the artificial gene Ribozyme-sgRNA-Ribozyme (RGR) is initiated by an RNA polymerase II-dependent promoter. Genetic transformation is indispensable for editing plant genes. In certain plant species, including pyrethrum, genetic transformation remains challenging to do, limiting the functional verification of novel CRISPR/Cas9 systems. Thus, this study’s aim was to develop a simple Agrobacterium rhizogenes-mediated hairy root transformation system to analyze the function of a ribozyme-mediated CRISPR/Cas9 system in pyrethrum. Results A hairy root transformation system for pyrethrum is described, with a mean transformation frequency of 7%. Transgenic hairy roots transformed with the pBI121 vector exhibited significantly increased beta-glucuronidase staining as a visual marker of transgene expression. Further, a ribozyme-based CRISPR/Cas9 vector was constructed to edit the TcEbFS gene, which catalyzes synthesis of the defense-related compound (E)-β-farnesene in pyrethrum. The vector was transferred into the hairy roots of pyrethrum and two stably transformed hairy root transgenic lines obtained. Editing of the TcEbFS gene in the hairy roots was evaluated by gene sequencing, demonstrating that both hairy root transgenic lines had DNA base loss at the editing target site. Gas chromatography–mass spectrometry showed that the (E)-β-farnesene content was significantly decreased in both hairy root transgenic lines compared with the empty vector control group. Altogether, these results show that RGR can be driven by the CaMV35S promoter to realize TcEbFS gene editing in pyrethrum hairy roots. Conclusion An A. rhizogenes-mediated hairy root transformation and ribozyme-mediated CRISPR/Cas9 gene editing system in pyrethrum was established, thereby facilitating gene editing in specific organs or at a particular developmental stage in future pyrethrum research.

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Two efficient CRISPR/Cas9 systems for gene editing in soybean

Cited by 23 publications

References 51 publications

Multiplex CRISPR/Cas9-mediated raffinose synthase gene editing reduces raffinose family oligosaccharides in soybean

Multiplex CRISPR/Cas9-mediated raffinose synthase gene editing reduces raffinose family oligosaccharides in soybean

Progress in Soybean Genetic Transformation Over the Last Decade

Ribozyme-mediated CRISPR/Cas9 gene editing in pyrethrum (Tanacetum cinerariifolium) hairy roots using a RNA polymerase II-dependent promoter

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