Cas9-Guide RNA Directed Genome Editing in Soybean^{<a href="#fn1" rid="fn1" class=" fn">[OPEN]</a>}

Abstract

Plant transformation is most commonly achieved by Agrobacterium tumefaciens infection or particle bombardment, both of which have inherent challenges, such as random gene integration, endogenous gene interruption, multiple gene copies, and often unpredictable gene expression. Hundreds of events must be screened to identify a single copy-integrated gene that does not interrupt any endogenous gene. Site-specific integration (SSI) approach has been developed to place genes at previously screened genomic sites through recombinase-mediated cassette exchange (RMCE) using a recombinase, such as Causes Recombination (CRE) or flippase (FLP; Nanto et al., 2005; Chawla et al., 2006; Louwerse et al., 2007; Li et al., 2009). However, the SSI target sites are still generated by random insertions and must be maintained as unique lines to accept new genes by a second round of transformation.

DNA homology-directed recombination (HDR), commonly used to transform yeast (Saccharomyces cerevisiae) and some model animal species, is rarely successful in plant transformation. There are only a few reported attempts to change introduced genes or endogenous genes through HDR in model plants, such as Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum; Halfter et al., 1992; Offringa et al., 1993; Miao and Lam, 1995; Kempin et al., 1997; Hanin et al., 2001). In one example using a positive-negative selection scheme to enrich HDR events, gene targeting was estimated at a frequency below 5.3 × 10 ^{to endogenous targets of} Lotus japonicas, although no HDR events were ever obtained (Thykjaer et al., 1997). Helped by an effective positive-negative selection and efficient A. tumefaciens transformation, Terada et al. (2002, 2007) successfully modified a rice (Oryza sativa) endogenous gene Waxy and later, a gene family member Alcohol dehydrogenase2 by HDR.

DNA double-strand breaks (DSBs) are naturally repaired by nonhomologous end joining (NHEJ), HDR, or microhomology-mediated end joining (Bleuyard et al., 2006). Homing endonucleases, such as I-SceI and I-CreI, have been used to generate artificial DSBs to stimulate HDR. HDR frequency at an artificial I-SceI recognition site previously placed in tobacco was increased by up to 100-fold when an I-SceI expression cassette was introduced together with a donor DNA by A. tumefaciens transformation (Puchta et al., 1996; Siebert and Puchta, 2002). Mutations of artificially introduced I-SceI recognition site in maize (Zea mays) were detected in 1% of analyzed F1 plants when I-SceI was introduced by crossing and activated by gene excision (Yang et al., 2009). Through the codelivery of a donor and an I-SceI expression DNA by either A. tumefaciens or biolistic transformation, the 35S promoter of the donor DNA was precisely inserted at previously introduced I-SceI sites at practical frequencies (D’Halluin et al., 2008).

Because homing endonuclease recognition sites do not normally exist in animal or plant genomes, unique agents are developed to specifically recognize a given genomic sequence. Taking advantage of the natural degeneracy of I-CreI recognition sequence, both rational design and experimental screening approaches have been used to create I-CreI derivatives that can recognize various DNA sequences (Seligman et al., 2002; Smith et al., 2006). An engineered I-CreI derivative capable of recognizing a sequence at the maize liguleless locus was successfully used to produce mutations with 2- to 220-bp deletions or short insertions at the expected cleavage site (Gao et al., 2010).

Zinc finger nucleases (ZFNs) are a group of engineered endonucleases that uses custom-designed zinc fingers to bind a specified DNA sequence, allowing the linked FokI endonuclease domain to generate a DSB in the recognized sequence (Durai et al., 2005). ZFNs work in pairs, because FokI nuclease subunits have to form dimers to cleave DNA. Mutations, small deletions and insertions, or targeted gene integrations at introduced ZFNs recognition sites were achieved in Arabidopsis and tobacco (Lloyd et al., 2005; Wright et al., 2005; de Pater et al., 2009). ZFN-mediated gene targeting was also successfully used to introduce a Phosphinothricin acetyltransferase herbicide resistance gene into a tobacco endochitinase gene and a maize inositol-1,3,4,5,6-petakisphosphate2-kinase gene or to introduce specific mutations in an acetolactate synthase (ALS) gene in tobacco to confer resistance to sulfonylurea herbicide (Cai et al., 2009; Shukla et al., 2009; Townsend et al., 2009).

Transcription activator-like effector nucleases (TALENs) are another group of engineered endonucleases that can be designed to bind practically any DNA sequence. (Cermak et al., 2011; Chen and Gao, 2013; Sun and Zhao, 2013). Various frequencies of mutations were obtained when five Arabidopsis endogenous genes were targeted with multiple TALENs and some of the mutations were transmitted to the next generation (Christian et al., 2013). Two fatty acid desaturase2 genes were successfully modified using TALENs to obtain mutant soybean (Glycine max) with desired fatty acid profiles of 80% (w/w) oleic acid and 4% (w/w) linoleic acid (Haun et al., 2014). Targeted gene editing through TALEN-mediated HDR was also achieved to edit 6 bp of the ALS gene in tobacco (Zhang et al., 2013).

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated9 (Cas9)-guide RNA (gRNA) is the latest DSB technology developed based on the Streptococcus pyogenesCRISPR immune system (Barrangou et al., 2007; Jinek et al., 2012; Cong et al., 2013; Mali et al., 2013; for review, see Hsu et al., 2014; Sander and Joung, 2014). Although all homing endonucleases, ZFNs, and TALENs rely on protein domains to recognize specific DNA sequences, the Cas9-gRNA system utilizes a simple gRNA to target a specific DNA sequence. The 20-bp target sequence has to be followed by a protospacer adjacent motif (PAM) nucleotides NGG and is cleaved between the third and fourth nucleotides upstream of the PAM. The recognition of PAM by Cas9-gRNA initiates DNA strands separation and RNA-DNA heteroduplex formation that proceeds directionally toward the 5′ end of the target sequence (Sternberg et al., 2014; for review, see Doudna and Charpentier, 2014).

We applied a Cas9-gRNA system suitable for soybean genome editing and acquired up to approximately 76% targeted mutagenesis through NHEJ and targeted gene integration through HDR. The integrated genes transmitted to T1 generation and segregated according to Mendelian laws. The Cas9-gRNA system was also successfully used to edit soybean ALS1 gene to obtain a chlorsulfuron-resistant soybean.

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