Key issues in editing elite-tomato lines by CRISPR-Cas9 system

Abstract Tomato ( Solanum lycopersicum ) is one of the most important vegetable crops worldwide. Conventional breeding methods played an important role in obtaining tomato varieties with desired traits, but selection is a time- and labor-intensive process. In recent years, CRISPR-Cas9 genome editing has emerged as a game-changing tool that offers a faster and more precise approach to improving elite tomatoes. This technique utilizes guide RNAs (gRNAs) and the Cas9 enzyme to introduce targeted modifications in the plant genome, enabling the enhancement of specific traits related to stress tolerance and fruit quality. However, CRISPR technology is not yet routinely used in the development of commercial lines. The selection of suitable targets using transcriptomic, literature, and multi-omics analyses could help to avoid adverse effects on plant growth and development. In addition, the design of efficient and specific gRNAs is crucial to escape undesired genetic alterations, which are generally referred to as off-target effects, as well as the choice of effective delivery systems for CRISPR components. In this review, the main challenges for choosing targets, designing gRNAs, and assembling constructs are illustrated. In addition, the crucial role of the appropriate delivery methods is discussed. Furthermore, screening protocols for identifying successfully edited crops and producing CRISPR-Cas9-free progeny were explored. Coupling CRISPR-Cas9 with advanced computational analyses and efficient transformation methods has enormous potential for tomato breeding in the future. However, it also faces limitations such as off-target effects, mosaicism, and delivery efficiency. Safety concerns also exist and should be considered. This constantly progressing technology is paving the way for more sustainable agricultural solutions, contributing to boosting crop productivity and quality.