A modified sgRNA is slightly more efficient to trigger mutations.

a. Original sgRNA proposed by Mali et al., 2013 [3]. Extension-Flip (EF) sgRNA proposed by Chen et al., 2013 [8]. b. and c. Each panel represents a sgRNA backbone comparison in the same context. sgRNA backbones can be compared within each panel but not from one panel to another. The modules were assembled into pICSL4723 (RB+OD, with an overdrive) or pAGM4723 (RB, without an overdrive) and transformed into Col-0 via Agrobacterium tumefaciens strain GV3101. LB: Left Border. SM: Selectable Marker (Glufosinate resistance gene). CsVMV: 517 bp of a promoter from Cassava Vein Mosaic virus. UBI10: 1327 bp of the At4g05320 promoter. Cas9_2: Fauser et al., 2014 [13]. OcsT: 714 bp of the Agrobacterium tumefaciens octopine synthase terminator. U6-26p: 205 bp of the At3g13855 promoter. U6-26T: 7 bp of the At3g13855 terminator. RB: Right Border. c. Five lines were tested for sgRNAEF and four lines for sgRNA instead of six. The sgRNA targets ADH1. CRISPR activity measured in % of homozygous or biallelic stable mutants in the second generation after transformation (T2). Each dot represents an independent T2 family. Bold and underlined: Most active construct(s) for each panel.