Fig. 2.

NbRRAall1/NbPDS2-gRNA generated indels. a gRNA targets of the N. benthamiana loci, REDUCED RESIDUAL ARABINOSE arabinosyl transferase (NbRRA) and PHYTOENE DESATURASE (NbPDS), were a BtgI and a AvrII site is situated 2 and 0 bp upstream of the protospacer adjacent motif (PAM), respectively. Given the predicted cut site of SpCas9, 3 bp upstream of the PAM sequence [44], all of the NbPDS2-gRNA derived mutation combinations will destroy the AvrII site in the NbPDS target site and only insertions starting with ‘G’ at the cut site, i.e. less than one fourth the insertions possible, will restore the BtgI site in the NbRRAall1 target site. Primers, flanking the gRNA targets, are indicated by arrows. b Western blot analysis of day 4 post infiltration leaves using anti Flag and anti GFP mAbs, cross-reacting to SpCas9 (154 kDa) and to a faint protein band corresponding to the un-cleaved fusion protein (SpCas9-2A-GFP, ca 180 kDa), respectively. c, d DNA from 4 days post infiltration leaf samples of NbRRAall1- and NbPDS2-gRNA/Cas9 infiltrations were isolated, PCR amplified and subjected to restriction enzyme digestions using BtgI (NbRRAall1) and AvrII (NbPDS2), respectively, with the resistant bands (indicated by arrow) isolated, cloned into pJet and 12 clones of each target sequenced revealing the resulting indels depicted