A weak 3’ regulatory sequence reduces the CRISPR-induced mutation rate.

a. to c. Each panel represents a terminator comparison in the same context. Terminators can be compared within each panel, not from one panel to another. The modules were assembled into pAGM4723 and transformed into Col-0 via Agrobacterium tumefaciens strain GV3101. LB: Left Border. SM: Selectable Marker (Glufosinate resistance gene). EC_enh.: 752 bp of the At2g21740 promoter fused to 548 bp of the At1g76750 promoter. UBI10: 1327 bp of the At4g05320 promoter. Cas9_2: Fauser et al., 2014 [13]. Cas9_3: Li et al., 2013 [25]. E9T: 631 bp of the Pisum sativum rbcS E9 terminator. OcsT: 714 bp of the Agrobacterium tumefaciens octopine synthase terminator. AgsT: 410 bp of the Agrobacterium tumefaciens agropine synthase terminator. U6-26p: 205 bp of the At3g13855 promoter. sgRNAEF: “extension-flip” sgRNA. U6-26T: 7, 67 or 192 bp of the At3g13855 terminator. RB: Right Border. For the comparison using the UBI10 promoter, the AgsT is in combination with U6-26192; OcsT is in combination with U6-2667. 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. Red dot: All the T2 lines from this family carry the same mutation, indicating a mutation more likely inherited from the T1 parent rather than being de novo from the T2 line. Bold and underlined: Most active construct(s) for each panel.