Figure 1.

Schematic overview of the approach for targeted deletion of the hom2 gene. Two single guide RNAs (sgRNAs) were designed that target the 5′ region of the hom2 gene. A circular deletion vector was used as a repair template, comprised of homology arms of approximately 1200 bp flanking a nourseothricin resistance cassette. It also contains a phleomycin resistance cassette that was used to distinguish between the desired gene deletion by homologous recombination (nourseothricin resistant and phleomycin sensitive transformants) and the undesired ectopic integration of the full construct (nourseothricin and phleomycin resistant transformants). Moreover, linear PCR products with shorter homology arms were used as repair template, as indicated. The Cas9 protein, sgRNAs and repair template were introduced into protoplasts by PEG-mediated transformation. All repair templates resulted in confirmed gene deletions.