Fig. 4.

TvDAO can be used to eliminate Cas9-carrying mutant progeny. A Progeny of the transgenic Cas9 #S1 plant exhibit resistance segregation on 1/2 MS medium containing 15 mM d-valine. Scale bar = 1 cm. B PCR-based genotyping confirms transgene elimination in survival T₂ plants of Cas9 #S1 under d-valine-conditioned negative selection. A1-A3 and D1-D3 are randomly selected alive or dying seedlings, respectively. C Sanger sequencing of target amplicons validates inherited mutations at the three endogenous target sites in the T₂ plant Cas9 #S1-A3. Black bold letters mark PAMs and target sequences of gRNAs are underlined. Insertion and deletions are in red. D The Cas9 #S1-A3 plant displays highly clustered trichomes as indicated by red arrows. Scale bar = 1 cm