CRISPR/Cas-mediated genome editing for efficient tomato breeding: past achievements and future directions

The limited genetic diversity in tomatoes presents significant hurdles for traditional breeding efforts. However, the emergence of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated protein 9 (CRISPR/Cas9) genome editing has revolutionized the rate and efficiency of tomato breeding. CRISPR/Cas9 technology plays a crucial role in editing and thoroughly characterizing various traits in tomatoes. CRISPR/Cas9 has shown efficacy in transferring novel domestication traits between cultivated tomatoes and their wild relatives and vice versa. A number of notable developments in CRISPR/Cas technology include the utilization of online resources for multiplexing and designing single-guide RNA (sgRNA), as well as utilizing cutting-edge cloning methods such as GoldenBraid, BioBrick, and Golden Gate cloning technology. Furthermore, improved transformation methods, such as Agrobacterium -mediated methods, the development of CRISPR/Cas constructs, and the use of DNA-free protoplasts for Cas9-guide RNA (gRNA) ribonucleoprotein (RNP) complexes, contribute to the advancement of gene editing capabilities. The toolkit for precise genome modification has also been expanded by the application of Cas9 variants, such as Cas9-NG/XNG-Cas9 and protospacer-adjacent motif (PAM) free Cas12a (Cpf1), together with techniques such as base/prime editing using Target- Activation-Induced Cytidine Deaminase (AID) technology and homologous recombination (HR)-based gene knock-in (HKI) mediated by geminivirus replicon. This review focuses on the significant advancements made in current research using CRISPR/Cas technology for fast and successful tomato breeding.