S01 - Session P1 - Targeted mutagenesis of CENTRORADIALIS using the CRISPR/Cas9 system in highbush blueberry

Authors: Masafumi Omori *, Hisayo Yamane, Keishi Osakabe, Yuriko Osakabe, Ryutaro Tao

Genome editing technology such as CRISPR/Cas9 enables us to modify specific genomic loci and may have the potential to accelerate molecular breeding of 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.). We intended to generate mutations in the flowering repressor gene, CENTRORADIALIS ( CEN ), however, plants with mutations in all alleles have yet to be obtained. For polyploid plants, mutations in all alleles are challenging, but may be necessary for the future practical use of this system as an efficient breeding technique. The aim of this study was to improve the CRISPR/Cas9-mediated gene editing efficiency of the mutation generation in tetraploid highbush blueberry. We first evaluated the performance of different promoters for driving gRNA and Cas9 expression by transient expression assay using blueberry leaves. The blueberry U6-7 promoter generated 17 times higher gRNA expression than the Arabidopsis U6 promoter, while the blueberry POLYUBIQUITIN ( UBQ3b ) promoter generated 2 times higher Cas9 transcript accumulations than the CaMV 35S promoter. In addition, the transient assay showed that CRISPR/Cas9 harboring blueberry endogenous promoters increased the mutation efficiency. We also evaluated the mutation efficiency of five different gRNAs targeting CEN and found that each gRNA yielded different mutation efficiency. Next, CRISPR/Cas9 vectors harboring different promoters and gRNAs were introduced into the blueberry genome by Agrobacterium -mediated transformation. Of the 56 stable transformants, 14 lines contained mutated alleles. Sequence analysis revealed 1- to 10-bp insertions/deletions in CEN alleles, with the highest mutated allele ratio of 100%. The new vector with blueberry promoters increased the average mutated frequency compared with the vector with Arabidopsis U6 and 35S promoters, which was consistent with the transient assay results. Our study suggested that selection of appropriate promoters and target sequences was critical for efficient genome editing in highbush blueberry.