Efficient Gene Editing in Tomato in the First Generation Using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-Associated9 System

Abstract

During the past 12 years, there has been rapid development of genome-editing strategies that make it possible to directly target regions of genes in a DNA sequence-specific manner. Two of these strategies, zinc finger nucleases (Urnov et al., 2010) and transcription activator-like nucleases (TALENs; Joung and Sander, 2013), are based on protein-DNA interactions, whereas a third technology, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated9 (Cas9) endonuclease (Liu and Fan, 2014), is an RNA-guided DNA endonuclease system. Each technology has advantages and disadvantages with regard to cost, ease of construction, efficiency of targeting, and specificity (Chen and Gao, 2014). Of the three genome-editing approaches, CRISPR/Cas9 has seen a meteoric rise during the past 2 years, with applications to bacterial, animal, human, and, most recently, plant systems (Pennisi, 2013). This is due, in part, to a greater number of advantages of CRISPR/Cas9, including the straightforward construct design and assembly, as compared with zinc finger nucleases and TALENs. The first reports of CRISPR/Cas9 editing in plants appeared in 2013, with successful application for both transient expression and recovery of stable transgenic lines. In addition to demonstration of the efficacy in the models Arabidopsis (Arabidopsis thaliana; Li et al., 2013) and Nicotiana benthamiana (Nekrasov et al., 2013), there have also been reports for three crop species, rice (Oryza sativa; Zhang et al., 2014), sorghum (Sorghum bicolor; Jiang et al., 2013), and wheat (Triticum aestivum; Wang et al., 2014). For stable transgenic lines, modifications reported for genes in primary transformants of Arabidopsis and rice were shown to persist into the next generation (Feng et al., 2014; Zhang et al., 2014). Thus, CRISPR/Cas9 is rapidly becoming the tool of choice for gene editing in plants, but further testing is needed to determine whether efficacy will be universal. Tomato (Solanum lycopersicum) is an ideal candidate for testing CRISPR/Cas9 gene editing in a dicot crop, because of the availability of efficient transformation methodology (Van Eck et al., 2006), the diploidy of the genome, a high-quality genome sequence (Tomato Genome Consortium, 2012), and its economic importance (fresh and processed), which in the United States alone accounts for more than $2 billion (http://www.ers.usda.gov/topics/crops/vegetables-pulses/tomatoes.aspx#.U85tkoBdVSY). To test the efficacy of CRISPR/Cas9 in tomato, we chose to target a gene that, when function was disrupted, would result in a distinctive, immediately recognizable phenotype early in the plant tissue culture phase of Agrobacterium tumefaciens-mediated transformation. A CRISPR/Cas9 construct was designed to target neighboring sequences in the second exon of the tomato homolog of Arabidopsis ARGONAUTE7 (SlAGO7), because loss-of-function mutations are recessive and result in plants whose typical compound flat leaves become needle like or wiry (Fig. 1; Lesley, 1928; Yifhar et al., 2012). SlAGO7 is required for the biogenesis of a class of small RNAs known as transacting short interfering RNAs, which regulate organ polarity through posttranscriptional silencing of AUXIN RESPONSE FACTOR (ARF) genes (Husbands et al., 2009). Strong alleles of slago7 thus produce lower levels of transacting short interfering RNAs and reduced ARF mRNA degradation, resulting in the first leaves of mutant plants having leaflets without petioles and later-formed leaves lacking laminae (Fig. 1C). These distinctive phenotypes allowed us to immediately identify first-generation transformed (T0) plants in which both alleles of SlAGO7 might be mutated. CRISPR/Cas9-mediated gene editing in stable transgenic tomato plants. A, Schematic illustrating two sgRNAs (red arrows) targeting the SlAGO7 coding sequence. Cas9/sgRNA1/sgRNA2 were expressed from the same plasmid (Belhaj et al., 2013). Black arrows indicate PCR primers used to evaluate mutation type and efficiency. B, A wild-type (WT) tomato plant at 9 weeks of age, and the fourth-produced leaf from the primary shoot (inset). C, The strong wiry2-1 allele of slago7. First formed leaves have leaflets lacking petioles (blue arrows), and later formed leaves are radialized (white arrows). D and E, A representative CRISPR/Cas9-slago7 (CR-slago7) plant (D) and its first five leaves showing the distinctive loss-of-function recessive wiry syndrome (E). Bars = 1 cm. F and G, PCR genotyping of eight representative CR-slago7 plants showing the type of DNA lesions generated, including homozygosity for the expected 90-bp deletion (plant 1). PCR for the presence of Cas9 is also shown along with wiry phenotypic evaluations. A range of indels was found in the other T0 plants. The PCR products from a subset of plants were cloned and sequenced to validate the deletion in plant 1 and to characterize additional DNA lesions (G). Note that the two deletions in plant 3 are smaller (3S)