Benoit Danilo, Laura Perrot, Kostlend Mara
Jan 16, 2019
Plant Cell Reports
Precise genome editing technologies are rapidly emerging as prime tools for improving crop characteristics. The CRISPR/ Cas9 system has proved highly versatile and efficient for inducing targeted DNA double-strand breaks (DSBs) in genomes. DNA DSBs in eukaryotes can be repaired using two different pathways: non-homologous end joining (NHEJ) or homologous recombination (HR). Repair of a DSB through NHEJ can be error prone and result in partially predicted deletions or insertions in the target sequence that can lead to gene knock-out (KO). In contrast, homologydirected repair (HDR) of a DSB through HR can be predicted and requires a DNA donor template with homologous flanking sequences that allows gene editing or knock-in (KI). The NHEJ-repair pathway is predominant in plants and has been widely used to obtain targeted KO of genes of interest. Although HDR gene editing (or KI) holds promise for crop breeding, this approach remains more difficult to master. It has a low success rate (few percent or less) in eukaryotic cells and is associated with the technical hurdle of delivering the donor template at the right moment and in the vicinity of the DSB in plant cells. Point mutations in the acetolactate synthase gene (ALS) (also known as the acetohydroxy acid synthase gene (AHAS)) can lead to sulfonylurea herbicide resistance. ALS genes are involved in the biosynthetic pathway for branchedchain amino acid synthesis, for which they encode the first enzyme involved in the production of valine, leucine or isoleucine (McCourt and Duggleby 2006). CRISPR/Cas9 modifications targeting ALS genes have been performed in many plant species including maize (Svitashev et al. 2015), soybean (Li et al. 2015), rice (Endo et al. 2016; Sun et al. 2016) and potato (Butler et al. 2015). In addition to its use in agriculture, herbicide resistance can be used in vitro to select for and recover rare events of HDR-mediated editing of the ALS gene. In this respect, ALS targeting can be an efficient tool for the improvement of HDR-mediated strategies. In tomato (Solanum lycopersicum), three distinct ALS genes, ALS1 (Solyc03g044330), ALS2 (Solyc07g061940) and ALS3 (Solyc06g059880) are localized on chromosomes 3, 7 and 6, respectively. Although ALS1 and ALS2 show high similarity (94% identity at the amino acid level), ALS3 is more distinct with 75% and 78% identity with ALS1 and ALS2, respectively. In vegetatively propagated plants or for woody perennials with long juvenile phases, CRISPR/Cas9-mediated genome modifications must be free of any other DNA insertions. In tomato, after transformation via Agrobacterium tumefaciens, the transgene carrying the single guide RNA (sgRNA) and the CAS9 gene are eliminated in the next T1 generation upon segregation (Nekrasov et al. 2017). The delivery of the CRISPR/Cas9 ribonucleoprotein (RNP) by biolistic or by PEG fusion in protoplasts has in a few cases produced DNAfree modifications (Svitashev et al. 2016). Among species for which the Agrobacterium tumefaciens transformation system is routinely used, only in tobacco has transgene-free CRISPR/Cas9-mediated KO been reported, and only after labour-intensive screening using next-generation sequencing coupled with high-resolution melting (Chen et al. 2018). Moreover, Agrobacterium-mediated delivery is generally reputed to be less efficient for HDR-mediated genome editing (Svitashev et al. 2015; Endo et al. 2016; Sun et al. 2016). In the present study, we developed a selection protocol based on the CRISPR/Cas9 system and Agrobacteriummediated transformation for efficient gene editing of the Communicated by Neal Stewart.