CRISPR/Cas9-edited SPL-CNR quantitatively control tomato fruit ripening

The spontaneous Colorless non-ripening (Cnr) mutant fails to ripen and has an epiallele resulting in the transcriptional inhibition of the SQUAMOSA promoter binding protein-like (SPL)-CNR gene. Therefore, CNR has been suggested to function as a master regulator for fruit ripening. However, knockout mutants generated by CRISPR/Cas9 in ripening regulators, such as RIN and NOR, exhibited weak phenotypes of ripening inhibition compared to the corresponding spontaneous mutants. In this study, two guide RNAs were designed to target the SPL-CNR locus, resulting in 67.7 % and 12.8 % efficiency in the targeted regions among transgenic plants. Three CRISPR/Cas9-edited SPL-CNR lines (CR-CNRs) were used for further analysis, all disrupting the SPL-CNR promoter-binding domain. CR-CNR ripe fruit exhibited a light red color and displayed inhibited carotenoid accumulation, ethylene biosynthesis, and softening. In addition, volatiles in CR-CNRs were altered with an absence of cis-3-hexenal and 1-hexanol and an increased accumulation of trans-2-hexenal. The expression levels of key genes involved in carotenoid synthesis, cell wall modification, ethylene biosynthesis, and ripening regulators were downregulated in the ripening fruit of CR-CNRs. Our results suggest that SPL-CNR functions as a positive ripening regulator and is a potential target for modulating ethylene, fruit softening, and flavor-related volatiles in tomato.