Fig 3. Inheritance of targeted mutations and Cas9 in progeny of primary CRISPR/Cas events.

Three primary events with cloned targeted mutations (lanes 1, 8 and 12; underlined) were used to generate genetic populations to assess inheritance of targeted mutations. The diploid event (lane 1; X46-3) was crossed to an inbred diploid line, M6 (lane 18) as the female parent while tetraploid events (lanes 8, 12; D46-44, D46-9) were selfed. Six progeny from the X46-3 population (lanes 2–7) and three progeny from the D46-44 (lanes 9–11) and D46-9 (lanes 13–15) populations were assessed for A) targeted mutations using a restriction digestion assay (top gel) and inheritance of Cas9 (bottom gel) and used for B) cloning targeted mutations using previously described methods (Fig 2 and S5 Fig). The PCR assay used for detecting Cas9 (A; bottom gel) produced a 1144 bp amplicon with each lane corresponding to the top gel and is further described in S6 Fig Wild-type Désirée and M6 were used as negative controls (lanes 16 and 18, respectively) and a 1:1 template mixture with wild-type and mutated DNA was used as a positive control (lane 17). The lengths of deletions (-) or insertions (+) of the targeted mutations in progeny (B) are in parenthesis to the left of each cloned mutation and the number of reads generated in the primary event (F₀) or individual progeny (F₁) are in brackets on the right. All targeted mutations were aligned to wild-type sequence and cloned from StALS1 unless indicated on the right. PAM sequences are in gray.