Cas9 targeting of a GFP gene in soybean hairy roots. (A) Schematic showing the targeted GFP sequences. The targets were designed to the negative strand of GFP. Black arrows are all possible GN20GG target motifs. GFP imaging and amplicon sequencing of representative (B) C9 + GFP 5′ target events and (C) C9 + GFP 3′ target events. Each panel is an independent event and blue-light images were overlaid onto white-light images of roots. The same magnification was used for all images. Wild-type sequences are in green, deletions are shown as dashes, and SNPs are shown in orange. The targeted sequences are highlighted in grey and the PAM is highlighted in red. Percentages next to sequences indicate the number of reads with sequence over the number of total reads sequenced. On average, there were 4,282 and 8,409 reads per event from the 5′-target and 3′-target events, respectively.