Reactivation of a loss-of-function SlGLK2 allele by frame-restoring genome editing in tomato

Restoration of the crop improvement-related loss-of-function gene, Solanum lycopersicum GOLDEN2-LIKE, through an additional mutation by a conventional genome editing technique. Plant domestication and crop improvement processes have inadvertently led to the loss of gene functions that contribute to crop quality. In widely cultivated tomato (Solanum lycopersicum) varieties, the uniform ripening (u) mutation, a loss-of-function allele of the Solanum lycopersicum Golden2-like 2 (SlGLK2), has been selected to improve fruit appearance. However, this selection is associated with reduced fruit nutritional quality, lowering the levels of sugars and tocopherols due to impaired plastid development. In this study, the function of SlGLK2 was restored by introducing an additional mutation using the temperature-tolerant Lachnospiraceae bacterium ND 2006 Cas12a (LbCas12a) genome editing system. A 13-bp deletion in the linker region of SlGLK2 protein in the CR-Slglk2-4 line restored the reading frame and led to enhanced chlorophyll accumulation and plastid development, particularly in the stem end region of the fruit. The edited line also showed increased total soluble solids and tocopherol content in ripe fruit. Biochemical, transcriptomic, and transcriptional activity analyses confirmed the functional restoration of SlGLK2 in the CR-Slglk2-4 line. This study not only provides a useful Micro-Tom line for investigating plastid biogenesis in tomato fruit but also demonstrates the potential of genome editing to restore the latent functions of pseudogenized genes in modern crops, offering a new approach for recovering traits lost during plant domestication or crop improvement.