Validation of CRISPR construct activity and gene function in melon via a hairy root transformation system

Abstract

Melon (Cucumis melo L.), an important cash fruit crop with high nutritional value, is cultivated worldwide. To promote the application of gene editing technology and accelerate functional analysis of genes in melon, we developed an efficient protocol for inducing the formation of hairy roots. Using melon cotyledon as explants and Agrobacterium rhizogenes (A. rhizogenes) K599 as the engineering bacterium, a large number of hairy roots could be induced within a month and the transformed hairy roots accounted for 68.61% of the total hairy roots. On average, 2.61 positive hairy roots were formed on each explant. By transforming hairy roots with a CRISPR/Cas9 gene editing construct, the availability of target sites can be assessed in planta in a brief time. The gene editing targets are preliminarily divided into three types: full editing, partial editing, and no editing, and the efficacy of target sites was further validated by stable transformation. Then, we found that the efficiency of gene editing was promoted by the number of sgRNA expression cassettes. Finally, we used this system to analyze the function of melon CmRHL1 in root hair development and found that melon root hair development was significantly inhibited by the mutation of this gene. In summary, the hairy root editing method established in this study may be used to quickly validate the activity of CRISPR/Cas9 constructs and characterize gene function during root development, serving as a complementary tool for heritable genome editing in melon.