Targeted Multi-allelic Mutagenesis in Tetraploid Potato using CRISPR-Cas9

The CRISPR-Cas9 system has received increased focus among the new breeding technologies since it can easily be adapted to different targets. Mariette Andersson of the Swedish University of Agricultural Sciences and colleagues applied the CRISPR-Cas9-mediated genome editing in protoplasts of a tetraploid potato (Solanum tuberosum).

CRISPR/Cas9 yielded mutations in all four target alleles in a single transfection, in 2 percent of regenerated lines. Three different regions of the granule-bound starch synthase (GBSS) gene were targeted under different experimental setups, resulting in mutations in at least one allele in up to 12 percent of regenerated shoots, with multiple alleles mutated in up to 67 percent of confirmed mutated lines. Most mutations resulted in small insertions/deletions.

Phenotypic analysis of starch found that full knockout of the GBSS enzyme activity was only confirmed in four-allele mutated lines. This shows that a single remaining wildtype allele is sufficient to maintain enough GBSS enzyme activity to produce significant amounts of amylose.

For more information, read the article in Plant Cell Reports.