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Targeted genome modifications in soybean with CRISPR/Cas9.

ABSTRACT: The ability to selectively alter genomic DNA sequences in vivo is a powerful tool for basic and applied research. The CRISPR/Cas9 system precisely mutates DNA sequences in a number of organisms. Here, the CRISPR/Cas9 system is shown to be effective in soybean by knocking-out a green fluorescent protein (GFP) transgene and modifying nine endogenous loci.Targeted DNA mutations were detected in 95% of 88 hairy-root transgenic events analyzed. Bi-allelic mutations were detected in events transformed with eight of the nine targeting vectors. Small deletions were the most common type of mutation produced, although SNPs and short insertions were also observed. Homoeologous genes were successfully targeted singly and together, demonstrating that CRISPR/Cas9 can both selectively, and generally, target members of gene families. Somatic embryo cultures were also modified to enable the production of plants with heritable mutations, with the frequency of DNA modifications increasing with culture time. A novel cloning strategy and vector system based on In-Fusion® cloning was developed to simplify the production of CRISPR/Cas9 targeting vectors, which should be applicable for targeting any gene in any organism.The CRISPR/Cas9 is a simple, efficient, and highly specific genome editing tool in soybean. Although some vectors are more efficient than others, it is possible to edit duplicated genes relatively easily. The vectors and methods developed here will be useful for the application of CRISPR/Cas9 to soybean and other plant species.

SUBMITTER: Jacobs TB

PROVIDER: S-EPMC4365529 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Targeted genome modifications in soybean with CRISPR/Cas9.

Jacobs Thomas B TB LaFayette Peter R PR Schmitz Robert J RJ Parrott Wayne A WA

BMC biotechnology 20150312

<h4>Background</h4>The ability to selectively alter genomic DNA sequences in vivo is a powerful tool for basic and applied research. The CRISPR/Cas9 system precisely mutates DNA sequences in a number of organisms. Here, the CRISPR/Cas9 system is shown to be effective in soybean by knocking-out a green fluorescent protein (GFP) transgene and modifying nine endogenous loci.<h4>Results</h4>Targeted DNA mutations were detected in 95% of 88 hairy-root transgenic events analyzed. Bi-allelic mutations ...[more]

PMID: 25879861

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Project description:BackgroundThe plant architecture has significant effects on grain yield of various crops, including soybean (Glycine max), but the knowledge on optimization of plant architecture in order to increase yield potential is still limited. Recently, CRISPR/Cas9 system has revolutionized genome editing, and has been widely utilized to edit the genomes of a diverse range of crop plants.ResultsIn the present study, we employed the CRISPR/Cas9 system to mutate four genes encoding SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors of the SPL9 family in soybean. These four GmSPL9 genes are negatively regulated by GmmiR156b, a target for the improvement of soybean plant architecture and yields. The soybean Williams 82 was transformed with the binary CRISPR/Cas9 plasmid, assembled with four sgRNA expression cassettes driven by the Arabidopsis thaliana U3 or U6 promoter, targeting different sites of these four SPL9 genes via Agrobacterium tumefaciens-mediated transformation. A 1-bp deletion was detected in one target site of the GmSPL9a and one target site of the GmSPL9b, respectively, by DNA sequencing analysis of two T0-generation plants. T2-generation spl9a and spl9b homozygous single mutants exhibited no obvious phenotype changes; but the T2 double homozygous mutant spl9a/spl9b possessed shorter plastochron length. In T4 generation, higher-order mutant plants carrying various combinations of mutations showed increased node number on the main stem and branch number, consequently increased total node number per plants at different levels. In addition, the expression levels of the examined GmSPL9 genes were higher in the spl9b-1 single mutant than wild-type plants, which might suggest a feedback regulation on the expression of the investigated GmSPL9 genes in soybean.ConclusionsOur results showed that CRISPR/Cas9-mediated targeted mutagenesis of four GmSPL9 genes in different combinations altered plant architecture in soybean. The findings demonstrated that GmSPL9a, GmSPL9b, GmSPL9c and GmSPL9 function as redundant transcription factors in regulating plant architecture in soybean.

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Targeted genome modifications in soybean with CRISPR/Cas9.

Publications

Targeted genome modifications in soybean with CRISPR/Cas9.

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