Fig. 2

Multiplex genome editing of LW1 in H99 and ILP1 inbred lines. (A) Selfed ears of ILP1 harvested 12–14 days after pollination, with excised immature embryo in inset. (B) Putative transgenic calli on selective medium. (C) CRISPR/Cas9-events on selective regeneration medium. (D, E) Putative transgenic shoots on selective regeneration medium after 2–3 weeks post-transfer to a growth chamber with 16 h/8 h light/dark photoperiod. (F) Sequence confirmation of genome edits in LW1. The top half of the gel shows amplification of the 5’ end of the gene spanning the LW1-gRNA4 target site, bottom half shows amplicons spanning target sites for LW1-gRNA1, LW1-gRNA2 and LW1-gRNA3 at 3’ end of gene. Primer locations are indicated in Fig. 1A. Lane 3 (ILP1) is the non-edited control plant producing a band of 900 bp. Lanes 4 (ILP-E1), 5 (ILP-E2), 6 (ILP-E3) and 7 (H99-E1) are the edited lines. Lane 1 is the NEB 1 kb + ladder. (G, H, I) Sanger sequencing results of PCR products shown in panel F (guide RNA targets are depicted with gray line above WT sequence, PAM sequences are depicted in orange boxes; red boxes with dashes and letters represent deletions and insertion, respectively