Efficient and transgene-free genome editing in wheat through transient expression of CRISPR/Cas9 DNA or RNA

Abstract

Editing plant genomes is technically challenging in hard-to-transform plants and usually involves transgenic intermediates, which causes regulatory concerns. Here we report two simple and efficient genome-editing methods in which plants are regenerated from callus cells transiently expressing CRISPR/Cas9 introduced as DNA or RNA. This transient expression-based genome-editing system is highly efficient and specific for producing transgene-free and homozygous wheat mutants in the T0 generation. We demonstrate our protocol to edit genes in hexaploid bread wheat and tetraploid durum wheat, and show that we are able to generate mutants with no detectable transgenes. Our methods may be applicable to other plant species, thus offering the potential to accelerate basic and applied plant genome-engineering research.

Figure 1. Overview of the genome-editing methods based on TECCDNA or TECCRNA.

The CRISPR/Cas9 DNA (plasmid constructs) or RNA (in vitro synthesized transcripts) is delivered into immature wheat embryos by particle bombardment. After transient expression and function, CRISPR/Cas9 DNA or RNA becomes degraded, while the bombarded embryos produce callus cells from which seedlings are regenerated. The T0 seedlings are examined using PCR-RE assay and DNA sequencing to identify targeted mutants.

Figure 2. Development and validation of the TECCDNA method.

(a) Sequence of an sgRNA designed to target a site within a conserved region of exon 3 of TaGASR7 homoeologues. The protospacer-adjacent motif (PAM) sequence is highlighted in red and the BcnI restriction site is underlined. The outcome of PCR-RE assays analysing 12 representative tagasr7 mutants is shown. Lanes T0-1 to T0-12 show blots of PCR fragments amplified from independent wheat plants digested with BcnI. Lanes labelled WT/D and WT/U are PCR fragments amplified from WT plants with and without BcnI digestion, respectively. The bands marked by red arrowheads are caused by CRISPR/Cas9-induced mutations. (b) Genotypes of 12 representative mutant plants identified by sequencing. (c) Schematic of the structure of the pGE-sgRNA vector and five primer sets used for detecting transgene-free mutants. sgRNA refers to sgRNAs targeting TaGASR7, TaDEP1, TaNAC2, TaPIN1, TaLOX2, TdGASR7 and TaGW2, respectively. (d) Outcome of tests for transgene-free mutants using five primer sets in 12 representative tagasr7 mutant plants. Lanes without bands identify transgene-free mutants. Lanes labelled WT and plasmid show the PCR fragments amplified from a WT plant and the pGE-TaGASR7 vector, respectively.

Figure 3. Development and testing of TECCRNA method.

(a) Sites within a conserved region of exon 8 of wheat TaGW2 homoeologues targeted by CRISPR/Cas9 systems. The PAM sequence is highlighted in red, the XbaI restriction sites are underlined and the single-nucleotide polymorphism in the targeted sequences is highlighted in green. Outcome of PCR-RE assays analysing nine representative tagw2 mutants is shown. Lanes T0-1 to T0-9 show blots of PCR fragments amplified from independent wheat plants digested with XbaI. Lanes labelled WT/D and WT/U are PCR fragments amplified from WT plants with and without XbaI digestion, respectively. The bands marked by red arrowheads are caused by CRISPR/Cas9-induced mutations. (b) CRISPR/Cas9-induced mutant TaGW2 alleles identified by sequencing. (c) Comparison the mutagenesis frequencies for three homoeologues of TaGW2 of three genome-editing methods.

Figure 4. Phenotypic analysis of TaGASR7 and TaDEP1 mutants generated using TECCDNA genome-editing method.

(a) TKW of WT and tagasr7-aabbdd mutant Bobwhite and Kenong199 plants. Data are from eight replicates for Bobwhite and ten replicates for Kenong199. (b) Plant heights of tadep1-aabbdd mutant plants compared with WT. Data are from 15 replicates. Values in a,b are mean±s.d. **P<0.01 (t-tests). (c) The morphology of plant heights of Kenong199 for WT and tadep1-aabbdd mutant in the vegetative and reproductive growth stages. Scale bar, 6 cm.