In genome editing tools such as CRISPR, it is beneficial if no foreign DNA is integrated into the genome. Mariette Andersson and her colleagues from the Swedish University of Agricultural Sciences in Sweden used a DNA‐free genome editing method using CRISPR-Cas9ribonucleoproteins (RNP) to target the granule bound starch synthase gene (GBSS) in potato (Solanum tuberosum) protoplasts.
The RNP method was done using previously developed protoplast isolation, transfection and regeneration protocols. The Cas9 protein to be used was designed using RNA produced either synthetically or by in vitro transcription. RNP with synthetically produced RNA induced mutations at a 9% frequency, with all mutated lines being transgene‐free.
A mutagenesis frequency of 25% of all regenerated shoots was found when using RNP with in vitro transcriptionally produced RNA. However, shoots with confirmed mutations had unintended DNA inserts. The inserts originated both from DNA template remnants from the in vitro transcription and from chromosomal potato DNA.
In the regenerated shoots from the RNP‐experiments, mutations were induced in all four GBSSalleles resulting in a complete knockout of the enzyme’s function.
For more information on this study, read the article in Physiologia Plantarum.