Targeted mutagenesis of CENTRORADIALIS using CRISPR/Cas9 system through the improvement of genetic transformation efficiency of tetraploid highbush blueberry

Genome editing technology, which enables researchers to modify specific genomic loci, may be useful for accelerating the breeding of many fruit crops. The aim of this study was to evaluate the CRISPR/Cas9-mediated editing of the blueberry (Vaccinium spp.) genome. We first optimised the plant regeneration system to increase the genetic transformation efficiency for ‘Blue Muffin’ and ‘O’Neal’. We also tested the utility of the axillary bud transformation technique for modifying blueberry genes. We revealed that the axillary bud transformation method accelerated the blueberry transformation process and increased the transformation rate. Of the 47 transgenic lines obtained for two cultivars, six lines contained a mutated CENTRORADIALIS (CEN) region. A sequence analysis revealed 1- to 2-bp insertions/deletions in CEN alleles, with an average mutated allele ratio of 19% and 22% for gRNA1 and gRNA2, respectively. Two of four gRNAs (gRNA 3, 4) did not produce mutations, suggesting that selecting appropriate gRNA sequences is critical for genome editing. The growth phenotypes of the CEN-mutated lines imply a non-functional CEN allele in the blueberry genome may restrict vegetative growth. The results described herein confirm the utility of the CRISPR/Cas9 genome editing protocol for functionally characterising blueberry genes.