For centuries, combining useful traits into a single tomato plant has been done by selective crossbreeding that resulted in hundreds of extant modern cultivars. However, crossbreeding is a labor-intensive process that requires between 5 and 7 years to develop a new variety. More recently, genome editing with the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has been established as an efficient method to accelerate the breeding process by introducing targeted modifications to plant genomes via generation of targeted double-strand breaks (DSBs). CRISPR/Cas9 has been used to generate a variety of specific changes ranging from gene knockouts to gene replacements, and can also be easily multiplexed to modify several targets simultaneously. Given that (1) generating knockout mutations only requires a DSB that is frequently repaired by the error-prone nonhomologous end joining (NHEJ) pathway resulting in gene function inactivation, and (2) the genetic basis of many useful agronomic traits consists of loss of gene function, multiple traits can be created in a plant in one generation by simultaneously introducing DSBs into multiple genes of interest. On the other hand, more precise modifications, such as allele replacement, can be achieved by gene targeting-a less efficient process in which an external template is used to repair the DSB by homologous recombination (HR). These technical breakthroughs allow the design and customization of plant traits to achieve the ideal plant type ("ideotype"). Here, we describe protocols to assemble CRISPR/Cas9 constructs for both single and multiplex gene knockouts as well as gene targeting and to generate and identify genome-edited tomato plants via Agrobacterium-mediated transformation in tissue culture.
Keywords: CRISPR/Cas9; Gene replacement; Gene-editing; Solanum lycopersicum; Tomato.