Possible Reversion of CRISPR-Cas9-Edited Sequences in Octoploid Strawberry

Gene editing is more challenging in octoploids due to the presence of multiple copies of each gene. However, the ability to edit genes in these plants would allow editing in commercial varieties. Here, we delivered sequences targeting <i>FaMYB9</i> into octoploid strawberry "Honeoye" and identified several gene-edited lines. Among them, the heterozygous gene-edited line <i>FaMYB9</i>^CR-15 had curved and wrinkled leaves at 3 months, whereas leaves of 3-month-old wild-type (WT) strawberry seedlings were elliptical with a smooth surface. At that stage, <i>FaMYB9</i>^CR-15 leaves also had large patches of wax. We identified 11,402 differentially expressed genes, divided into four clusters, between WT and <i>FaMYB9</i>^CR-15 seedlings at 3 months. Notably, cluster 4 genes-related to nonhomologous end joining, microhomology-mediated end joining repairs, homologous recombination, nucleotide excision repair, and mismatch repair-were more highly expressed in the gene-edited line than in the WT. Surprisingly, by 6 months of age, <i>FaMYB9</i>^CR-15 leaves had become smooth with small patches of wax, and expression levels of cluster 4 genes were significantly lower than at 3 months. Over the same period, the percentage of <i>FaMYB9</i> loci harboring the mutant allele decreased from 70.2% to 43.7%. These findings lead us to conclude that there could be reversion of mutated sequences in octoploid strawberry, emphasizing the challenges of gene editing high-ploidy materials.