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Gene targeting (GT) is a technique that alter the structure of the specific genes at their original loci in the genome by homologous recombination (HR). It plays an important role in functional genomics because it enables precise modification of the endogenous genes into desired forms such as knockout, knock-in, introduction of point mutations, as well as generation of fusion genes. Also, site-directed mutagenesis by GT can also be applied as an excellent technique for molecular breeding and gene therapy, because it can directly reflect the knowledge acquired from functional genomics. In this section, we introduce well-established GT procedure in rice in combination with positive-negative-selection (PNS) strategy.

The Target-AID system, consisting of a complex of cytidine deaminase and deficient CRISPR/Cas9, enables highly specific genomic nucleotide substitutions without the need for template DNA. The Cas9-fused cytidine deaminase is guided by sgRNAs and catalyzes the conversion of cytosine to uracil. The resulting U-G DNA mismatches trigger nucleotide substitutions (C to T or G to A) through DNA replication and repair pathways. Target-AID also retains the benefits of conventional CRISPR/Cas9 including robustness in various organisms, high targeting efficiency, and multiplex simultaneous gene editing. Our research group recently developed plant-optimized Target-AID system and demonstrated targeted base editing in tomato and rice. In this chapter, we introduce methods for Target-AID application in tomato.