Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes

Abstract

Substantial efforts are being made to optimize the CRISPR/Cas9 system for precision crop breeding. The avoidance of transgene integration and reduction of off-target mutations are the most important targets for optimization. Here, we describe an efficient genome editing method for bread wheat using CRISPR/Cas9 ribonucleoproteins (RNPs). Starting from RNP preparation, the whole protocol takes only seven to nine weeks, with four to five independent mutants produced from 100 immature wheat embryos. Deep sequencing reveals that the chance of off-target mutations in wheat cells is much lower in RNP mediated genome editing than in editing with CRISPR/Cas9 DNA. Consistent with this finding, no off-target mutations are detected in the mutant plants. Because no foreign DNA is used in CRISPR/Cas9 RNP mediated genome editing, the mutants obtained are completely transgene free. This method may be widely applicable for producing genome edited crop plants and has a good prospect of being commercialized.

Figure 1. Development and validation of CRISPR/Cas9 RNP-mediated genome editing in wheat.

(a) The exons of TaGW2 and the target site of gw2-sgRNA in exon 8. The single nucleotide polymorphism in the targeted sequence of TaGW2-A1 and the PAM motif are highlighted in green and red, respectively. The XbaI restriction site is underlined. (b) Mutagenesis frequencies of TaGW2-A1, -B1 and -D1 (induced by gw2-RNPs or pGE-TaGW2) in wheat protoplasts analysed by PCR-RE assay. Mutation bands are indicated by red arrows. WT/D and WT/U indicate wild type PCR amplicons with or without restriction enzyme digestion. (c) Mutagenesis frequencies of TaGW2-A1, -B1 and -D1 in embryos treated with gw2-RNPs or pGE-TaGW2 revealed by deep amplicon sequencing.

Figure 2. Targeted knockout of TaGW2 by delivery of CRISPR/Cas9 ribonucleoproteins.

(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.

Figure 3. Flowchart of CRISPR/Cas9 RNP induced targeted mutagenesis in wheat.

RNP preparation takes 3–4 days (including Cas9 protein purification). RNP validation in vitro and in protoplasts takes 3–4 days (not including the time for plant preparation). RNP coating and delivery can be completed within 4 h. After this, the plantlets regenerate in 6–8 weeks. Identification of mutants by PCR-RE and DNA sequencing takes 3 days.