U6 promoter efficacy analysis at the protoplast level. StU6 promoters were here defined as 350 nt upstream of the transcription start site (Supplementary Fig. 1 and Supplementary Fig. 2D), and the 257 bp AtU6-1 promoter of Arabidopsis thaliana was obtained from the vector pHBT-pcoCas927. (A) Structure of the Granular Bound Starch Synthase (GBSS) gene3 with the guide RNA (gRNA1) targeting part of the conserved motif and proposed active site KTGGL23. gRNA1 includes a diagnostic BsrI restriction enzyme site spanning the SpCas9 cleavage site28, −3 bp upstream of the Photospacer Adjacent Motif (PAM) (red), which upon digestion yields the allele specific fragments: allele III (286, 140), allele I and II (289, 140) and allele IV (289, 144). The outer most primer set includes the SNPs (+SNPs) and gives rise to PCR amplicons of 426 (allele III), 429 (alleles I and II) and 433 bp (allele IV), and the innermost (-SNPs) to a PCR amplicon of 218 bp (all alleles). These length SNPs were conserved between the cultivars Desirée and Wotan. (B) Construct design and StU6-1-4 versus AtU6-1 promoter analysis at the cell pool (protoplast) level as evidenced by the presence of indel mediated destruction of the BsrI site (BsrI resistant band). (C) Indel Detection by Amplicon Analysis (IDAA) chromatograms for the + SNP and –SNP PCR amplicons of WT and AtU6-1 and StU6-1 derived indels. + SNP IDAA reveals allele complexity, while –SNP and BsrI digested + SNP amplicons permit estimation of editing efficacy. (D) Sequence analysis of 34 individual clones of the BsrI resistant band of StU6-1 (B) isolated and cloned into the pJet vector, confirmed the indel distribution of the IDAA (Fig. 1C, panel: StU6-1, -SNP) also peaking at −4 bp deletions. WT peak positions are indicated by dotted lines. PPDK and NOS designate Pyruvate phosphate dikinase promoter and nopaline synthase terminator, respectively. All experiments were done in Desirée, except for panel C (upper right side) and D, which were done in the cultivar Wotan.