The clustered regularly interspaced short palindrome repeat (CRISPR)/CRISPR-associated protein Cas) system is a powerful and highly precise gene-editing tool in basic and applied research for crop improvement programs. CRISPR/Cas tool is being extensively used in plants to improve crop yield, quality, and nutritional value and make them tolerant to environmental stresses. CRISPR/Cas system consists of a Cas protein with DNA endonuclease activity and one CRISPR RNA transcript that is processed to form one or several short guide RNAs that direct Cas9 to the target DNA sequence. The expression levels of Cas proteins and gRNAs significantly influence the editing efficiency of CRISPR/Cas-mediated genome editing. This review focuses on insights into RNA Pol III promoters and their types that govern the expression levels of sgRNA in the CRISPR/Cas system. We discussed Pol III promoters structural and functional characteristics and their comparison with Pol II promoters. Further, the use of synthetic promoters to increase the targeting efficiency and overcome the structural, functional, and expressional limitations of RNA Pol III promoters has been discussed. Our review reports various studies that illustrate the use of endogenous U6/U3 promoters for improving editing efficiency in plants and the applicative approach of species-specific RNA pol III promoters for genome editing in model crops like Arabidopsis and tobacco, cereals, legumes, oilseed, and horticultural crops. We further highlight the significance of optimizing these species-specific promoters’ systematic identification and validation for crop improvement and biotic and abiotic stress tolerance through CRISPR/Cas mediated genome editing.

Fusarium wilt is a major devastating fungal disease of tomato (Solanum lycopersicum L.) caused by Fusarium oxysporum f. sp. lycopersici (Fol) which reduces the yield and production. Xylem sap protein 10 (XSP10) and Salicylic acid methyl transferase (SlSAMT) are two putative negative regulatory genes associated with Fusarium wilt of tomato. Fusarium wilt tolerance in tomato can be developed by targeting these susceptible (S) genes. Due to its efficiency, high target specificity, and versatility, CRISPR/Cas9 has emerged as one of the most promising techniques for knocking out disease susceptibility genes in a variety of model and agricultural plants to increase tolerance/resistance to various plant diseases in recent years. Though alternative methods, like RNAi, have been attempted to knock down these two S genes in order to confer resistance in tomato against Fusarium wilt, there has been no report of employing the CRISPR/Cas9 system for this specific intent. In this study, we provide a comprehensive downstream analysis of the two S genes via CRISPR/Cas9-mediated editing of single (XSP10 and SlSAMT individually) and dual-gene (XSP10 and SlSAMT simultaneously). Prior to directly advancing on to the generation of stable lines, the editing efficacy of the sgRNA-Cas9 complex was first validated using single cell (protoplast) transformation. In the transient leaf disc assay, the dual-gene editing showed strong phenotypic tolerance to Fusarium wilt disease with INDEL mutations than single-gene editing. In stable genetic transformation of tomato at the GE1 generation, dual-gene CRISPR transformants of XSP10 and SlSAMT primarily exhibited INDEL mutations than single-gene-edited lines. The dual-gene CRISPR-edited lines (CRELs) of XSP10 and SlSAMT at GE1 generation conferred a strong phenotypic tolerance to Fusarium wilt disease compared to single-gene-edited lines. Taken together, the reverse genetic studies in transient and stable lines of tomato revealed that, XSP10 and SlSAMT function together as negative regulators in conferring genetic tolerance to Fusarium wilt disease.