Targeted mutagenesis of <i>CENTRORADIALIS</i> 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 (<i>Vaccinium</i> 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 <i>CENTRORADIALIS</i> (<i>CEN</i>) region. A sequence analysis revealed 1- to 2-bp insertions/deletions in <i>CEN</i> 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 <i>CEN</i>-mutated lines imply a non-functional <i>CEN</i> 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.

能够对特定基因组位点进行精准修饰的基因组编辑技术，有望加速多种果树作物的育种进程。本研究旨在评估CRISPR/Cas9介导的蓝莓（*Vaccinium* spp.）基因组编辑效果。本研究首先针对‘Blue Muffin’与‘O’Neal’两个蓝莓品种优化了植株再生体系，以提升其遗传转化效率；同时验证了腋芽转化技术在蓝莓基因修饰中的应用潜力。结果显示，腋芽转化技术可缩短蓝莓遗传转化周期并提升转化效率。在两个品种获得的47个转基因株系中，有6个株系的*CENTRORADIALIS*（*CEN*）基因区域发生了突变。序列分析表明，*CEN*等位基因存在1至2个碱基的插入或缺失；向导RNA（gRNA）1与gRNA2对应的平均突变等位基因比例分别为19%与22%。4条gRNA中有2条（gRNA3、gRNA4）未诱导产生突变，这提示筛选合适的gRNA序列是基因组编辑成功的关键。*CEN*基因突变株系的生长表型表明，蓝莓基因组中功能失活的*CEN*等位基因可能会抑制营养生长。本研究结果证实，该CRISPR/Cas9基因组编辑方案可用于蓝莓基因的功能鉴定。