The evaluation of CRISPR-Cas9-mediated editing efficiency using endogenous promoters in tetraploid blueberry

Genome editing technology that enables researchers to modify specific genomic loci may accelerate the breeding of many fruit crops. We previously reported that the CRISPR-Cas9 genome editing system is a viable option for mutating specific gene targets in commercial tetraploid highbush blueberry (Vaccinium corymbosum L.) cultivars. However, plants with mutations in all alleles have yet to be obtained. Genome editing efficiency in polyploids is typically lower than that in diploids because of the greater number of target sequences. The aim of this study was to improve the efficiency of the CRISPR-Cas9-mediated editing of the tetraploid blueberry genome by using endogenous promoters driving gRNA and Cas9 expression. We first isolated the blueberry U6 and POLYUBIQUITIN (UBQ3b) promoters. A transient expression assay using blueberry leaves was established to evaluate the different promoters in terms of target gene expression and editing efficiency. The gRNA and Cas9 transcript levels were 18- and 2-fold higher for the blueberry U6-7 and UBQ3b promoters, respectively, than for the Arabidopsis thaliana U6 and CaMV 35S promoters. Our transient expression system successfully introduced mutations. Moreover, the mutated allele frequency was higher for the vector harboring blueberry promoters than for the conventional vector harboring A. thaliana U6 and 35S promoters. Thus, our findings suggest that the use of endogenous promoters may lead to the efficient editing of the tetraploid blueberry genome.