CRISPR-Guided Functional Genomics of Anthocyanin Biosynthesis Pathways in Strawberry

Abstract

Anthocyanins are flavonoid-derived secondary metabolites responsible for the characteristic red pigmentation and antioxidant potential of strawberry fruits. In cultivated strawberry, Fragaria × ananassa, anthocyanin accumulation significantly influences fruit quality, nutritional value, postharvest shelf life, and consumer acceptance. Recent advances in genome editing technologies, particularly Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas systems, have revolutionized functional genomics and crop improvement strategies. This study presents a comprehensive investigation of anthocyanin biosynthesis pathways in strawberry using CRISPR-guided functional genomics approaches. Candidate structural and regulatory genes, including FaCHS, FaCHI, FaF3H, FaDFR, FaANS, FaUFGT, and transcription factors such as FaMYB10 and FabHLH3, were targeted through CRISPR/Cas9-mediated editing. Functional validation was performed using Agrobacterium-mediated transformation, targeted mutagenesis, transcriptome profiling, and metabolomic analyses. Gene-edited strawberry lines exhibited significant alterations in fruit pigmentation, anthocyanin concentration, and expression profiles of flavonoid pathway genes. Knockout of FaMYB10 resulted in white-fruited phenotypes with reduced pelargonidin accumulation, whereas activation or promoter editing enhanced anthocyanin biosynthesis and fruit coloration. Transcriptomic analyses revealed extensive regulatory networks involving light-responsive and stress-associated pathways. The study demonstrates the effectiveness of CRISPR-guided functional genomics for dissecting complex metabolic pathways in polyploid crops and highlights its potential for precision breeding of nutritionally enhanced strawberry cultivars. These findings contribute to the understanding of anthocyanin regulation and provide a molecular framework for future improvement of fruit quality traits in strawberry breeding programs.