Figure 3.

(A) Absence of transgene in plants from DsRED-negative selected seeds was confirmed by PCR using Cas9-specific primers. Examples are shown for tomato, Arabidopsis and rice, selecting different number of seeds and number of independent lines. In all cases, fluorescence and Cas9 PCR band did correlate perfectly (an image indicates the presence/absence of DsRED fluorescence in the original selected seed). (B) Sequence alignment of mutations detected in homozygosis in transgene-free T2 individual plants. CRISPR target sequence in red and PAM sequence in green. Nucleotide insertions are indicated in blue; deletions are also indicated with blue dashes. Deletion “del193” starts in target 2 and ends around 150 nts downstream target 3. It also includes an insertion of six nts that interestingly match with six nucleotides upstream target 2 (Supplementary Figure 5B).