BackgroundThe 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.ResultsTargeted 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.ConclusionsThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-015-0131-2) contains supplementary material, which is available to authorized users.

CRISPR (clustered regularly interspaced short palindromic repeats), first discovered as an immune system of prokaryotes, has become a powerful tool for genome editing in eukaryotes (Gaj et al., 2013). Co-expression of the CRISPR-associated endonuclease (Cas9) with a chimeric guide-RNA (gRNA) targeting a GN 19 NGG motif results in a double-strand DNA break near NGG, the protospacer adjacent motif (PAM) (Jinek et al., 2012). Processing by the endogenous DNA repair machinery generates small indels that, when located within a coding sequence, can disrupt the reading frame and render the gene nonfunctional. The CRISPR/ Cas9 system has been successfully applied in several herbaceous systems (Belhaj et al., 2013;Harrison et al., 2014). Here we report its application in the woody perennial Populus using the 4-coumarate:CoA ligase (4CL) gene family as a case study. We achieved 100% mutational efficiency for two 4CL genes targeted, with every transformant examined carrying biallelic modifications. The CRISPR/Cas9 system is highly sensitive to single nucleotide polymorphisms (SNPs), as cleavage for a third 4CL gene was abolished due to SNPs in the target sequence. For outcrossing species with a highly heterozygous genome, gRNA design must take into account the frequent occurrence of SNPs to achieve efficient genome editing.Two 4CL genes, 4CL1 and 4CL2, associated with lignin and flavonoid biosynthesis, respectively (Hu et al., 1998;Harding et al., 2002), were targeted for CRISPR/Cas9 editing. The Populus tremula 9 alba clone 717-1B4 (717) routinely used for transformation is divergent from the genome-sequenced Populus trichocarpa (Hamzeh & Dayanandan, 2004). Therefore, the 4CL1 and 4CL2 gRNAs designed from the reference genome were interrogated with in-house 717 RNA-Seq data to ensure the absence of SNPs which could limit Cas9 efficiency (Supporting Information Fig. S1). A third gRNA designed for 4CL5, a genome duplicate of 4CL1, was also included. The corresponding 717 sequence harbors one SNP in each allele near/within the PAM, both of which are expected to abolish targeting by the 4CL5-gRNA (Fig. S1). All three gRNA target sites are located within the first exon (Fig. S1a).