Tomato fruit as a model for tissue-specific gene silencing in crop plants

Abstract

Use of CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated 9)-mediated genome editing has proliferated for use in numerous plant species to modify gene function and expression, usually in the context of either transient or stably inherited genetic alternations. While extremely useful in many applications, modification of some loci yields outcomes detrimental to further experimental evaluation or viability of the target organism. Expression of Cas9 under a promoter conferring gene knockouts in a tissue-specific subset of genomes has been demonstrated in insect and animal models, and recently in Arabidopsis. We developed an in planta GFP (green fluorescent protein) assay system to demonstrate fruit-specific gene editing in tomato using a phosphoenolpyruvate carboxylase 2 gene promoter. We then targeted a SET-domain containing polycomb protein, SlEZ2, previously shown to yield pleiotropic phenotypes when targeted via ³⁵S-driven RNA interference and we were able to characterize fruit phenotypes absent additional developmental perturbations. Tissue-specific gene editing will have applications in assessing function of essential genes otherwise difficult to study via germline modifications and will provide routes to edited genomes in tissues that could not otherwise be recovered when their germline modification perturbs their normal development.