CRISPR/Cas9-mediated mutagenesis of phytoene desaturase in diploid and octoploid strawberry

Gene editing using CRISPR/Cas9 is a simple and powerful tool for elucidating genetic controls and for crop improvement. We demonstrate use of CRISPR/Cas methodology in diploid Fragaria vesca ssp. vesca Hawaii 4 and octoploid F. x ananassa Calypso by targeting the visible endogenous marker gene PDS (phytoene desaturase). Agrobacterium-mediated transformation of leaf and petiole explants was used for efficient stable integration of constructs expressing plant codon-optimised Cas9 and single guide sequences under control of either the Arabidopsis U6-26 consensus promoter and terminator or Fragaria vesca U6III regulatory sequences. More than 80% ( Hawaii 4) and 50% ( Calypso) putative transgenic shoot lines exhibited mutant phenotypes. Of mutant shoot lines selected for molecular analysis, approximately 55% ( Calypso) and 75% ( Hawaii 4) included albino regenerants with bi-allelic target sequence variants. Our results indicate the PDS gene is functionally diploid in Calypso and clearly demonstrate that CRISPR/Cas9 can be used to edit single copy genes at high frequency within the genome of the diploid and the same target in octoploid strawberry.