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Lycopene epsilon-cyclase ( LcyE ) is a key enzyme in the carotenoid biosynthetic pathway of higher plants. Using the CRSPR/Cas9 and the geminiviral replicon, we optimized a method for targeted mutagenesis and golden SNP replacement of the LcyE gene in rice. We have exploited the geminiviral replicon amplification as a means to provide a large amount of donor template for the repair of a CRISPR-Cas-induced DNA double-strand break (DSB) in the target gene via homology-directed repair (HDR). Mutagenesis experiments performed on the Donggin variety achieved precise modification of the LcyE loci with an efficiency of up to 90%. In HDR experiments, our target was the LcyE allele (LcyE-H523L) derived from anther culture containing a golden SNP replacement. The phenotype of the homologous recombination (HR) mutant obtained through the geminiviral replicon-based template delivery system was tangerine color, and the frequency was 1.32% of the transformed calli. In addition, the total carotenoid content of the LcyE sg2-HDR1 and LcyE sg2-HDR2 lines was 6.8-9.6 times higher than that of the wild-type (WT) calli, respectively. The reactive oxygen species content was lower in the LcyE sg2-HDR1 and LcyE sg2-HDR2 lines. These results indicate that efficient HDR can be achieved in the golden SNP replacement using a single and modular configuration applicable to different rice targets and other crops. This work demonstrates the potential to replace all genes with elite alleles within one generation and greatly expands our ability to improve agriculturally important traits.

Genome Editing of Golden SNP-Carrying Lycopene Epsilon-Cyclase (LcyE) Gene Using the CRSPR-Cas9/HDR and Geminiviral Replicon System in Rice