Fig. 4

Comparison of target edited gene mutation efficiency for transgenic lines of ‘Chardonnay’ expressing each construction. (a). Efficiency of target edited gene for each construction considering all sgRNAs. Types of mutation are divided in three groups (selected plantlets without mutation in white, selected plantlets with monoallelic mutation in grey, selected plantlets with biallelic mutation in black). All plantlets previously selected by Cas9 genotyping allowed to amplify a PCR product corresponding to the target gene. PCR products were then sequenced and aligned to identify mutation rate. 85, 97 and 71 plantlets were selected for construct 1, 2 and 3, respectively (Pairwise comparison of proportions; ***, P < 0.001). (b). Comparison of target edited mutation rate for each sgRNA and each construction. All sequences analyzed in Fig. 3a have been ordered according to the nature of the mutation. 4 types of mutation are considered: biallelic WT/WT in white, monoallelic WT/in or WT/Del in light grey, biallelic In/Del or InA/InB or DelA/DelB or DelA/DelB/SubsA in dark grey (where A and B are random mutation), Biallelic In/In or Del/Del in black. In = insertion of one or several nucleotides, Del = Deletion of one or several nucleotides, subs = substitution. (c). Alignment of the different edited sequences against wild-type sequence for the three sgRNAs of each construction. Wild-type sequence is highlighted in grey and all insertions or deletions are highlighted in red. Two alleles are represented for each plantlet to highlight monoallelic and biallelic mutations. The protein length and the ratio of plantlets showing the corresponding sequence are mentioned on the right and on the left, respectively