Fig. 6

Testing of tRNA-sgRNA CRISPR/Cas constructs in wheat protoplasts. Three wheat genes (a) were targeted: Target gene 1 (homoeologues TraesCS1A02G338200, TraesCS1B02G350600 and TraesCS1D02G340400), Target gene 2 (homoeologues TraesCS3A02G289300, TraesCS3B02G323900 and TraesCS3D02G289100) and Target gene 3 (homoeologues TraesCS5A02G116500, TraesCS5B02G117800 and TraesCS5D02G129600). Red arrows illustrate positions of the 20 bp sgRNA target sites. Dashed lines illustrate expected CRISPR/Cas-induced deletions. (b) Wheat protoplasts were co-transformed with a level 1 construct carrying one of the three SpCas9 variants (pFH23, pFH66 or pFH67) and the level 1 construct (pFH94) carrying the six sgRNAs (a) in a tRNA-sgRNA array. Genotyping by PCR has revealed shifted DNA bands (marked by red asterisks) corresponding to amplicons carrying CRISPR/Cas-induced deletions of expected sizes. The three panels shown come from different parts of the same DNA gel (Additional file 4: Fig. S5). ‘M’ is the DNA marker; ‘NTC’ is the no template control