Figure 2: Targeted knockout of TaGW2 by delivery of CRISPR/Cas9 ribonucleoproteins.
From: Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes
(a) PCR-RE assay results for 12 representative tagw2 mutants induced by gw2-RNPs. Lanes T0-1 to T0-12 show the PCR products of the 12 mutants after XbaI digestion. Lanes labelled WT/D and WT/U are the PCR products amplified from wild type (WT) plants with and without XbaI digestion, respectively. The sizes (bp) of the undigested WT amplicons for TaGW2-A1, -B1 or -D1 are indicated on the right side. (b) Indels caused by gw2-RNPs in the TaGW2-B1 and/or D1 homoeologs in 12 representative mutants. Hyphens denote deleted nucleotides. Nucleotides inserted are labelled green. The PAM motif (CCT) is shown in red. (c) Comparison of on-target (TaGW2-B1 and TaGW2-D1) and off-target (TaGW2-A1) mutation efficiencies induced by CRISPR/Cas9 RNPs (with gw2-RNPs) and TECCDNA (with pGE-TaGW2). N.D., not detected. (d) Distribution of the individuals carrying 1–6 mutated alleles among the 28 mutants generated by gw2-RNPs and the 30 mutants induced by pGE-TaGW2. N.D., not detected.
