Induction of targeted, heritable mutations in barley and Brassica oleracea using RNA-guided Cas9 nuclease

Abstract

Background: The RNA-guided Cas9 system represents a flexible approach for genome editing in plants. This method can create specific mutations that knock-out or alter target gene function. It provides a valuable tool for plant research and offers opportunities for crop improvement.

Results: We investigate the use and target specificity requirements of RNA-guided Cas9 genome editing in barley (Hordeum vulgare) and Brassica oleracea by targeting multicopy genes. In barley, we target two copies of HvPM19 and observe Cas9-induced mutations in the first generation of 23 % and 10 % of the lines, respectively. In B. oleracea, targeting of BolC.GA4.a leads to Cas9-induced mutations in 10 % of first generation plants screened. In addition, a phenotypic screen identifies T0 plants with the expected dwarf phenotype associated with knock-out of the target gene. In both barley and B. oleracea stable Cas9-induced mutations are transmitted to T2 plants independently of the T-DNA construct. We observe off-target activity in both species, despite the presence of at least one mismatch between the single guide RNA and the non-target gene sequences. In barley, a transgene-free plant has concurrent mutations in the target and non-target copies of HvPM19.

Conclusions: We demonstrate the use of RNA-guided Cas9 to generate mutations in target genes of both barley and B. oleracea and show stable transmission of these mutations thus establishing the potential for rapid characterisation of gene function in these species. In addition, the off-target effects reported offer both potential difficulties and specific opportunities to target members of multigene families in crops.

Fig. 1

Barley HvPM19 and B. oleracea BolC.GA4.a gene models and target sequences. a Morex HVVMRXALLmA0022M08_scaffold7 sequence contains the four barley HvPM19 gene copies (filled arrows). The target sequences for sgRNAHvPM19-1 and sgRNAHvPM19-3 (grey highlight) are shown below their respective gene models, with the protospacer-adjacent motif (PAM) highlighted in red. Recognition sequences for the restriction endonucleases SapI and MaeIII are underlined. b The B. oleracea BolC.GA4.a gene model includes two exons (filled boxes) separated by an intron (represented by a solid line). The B. oleracea BolC.GA4.a sequences for sgRNA1BolC.GA4.a (Target 1) and sgRNA2BolC.GA4.a (Target 2) are shown below the target regions in grey highlight with the PAM highlighted in red. Recognition sequences for the restriction endonucleases AflII, HaeIII and HphI are underlined. Primers for mutant detection are shown in both panels and detailed in Additional file 3

Fig. 2

Schematic of binary plasmid vectors delivered to barley and B. oleracea. Transcription units were assembled into the binary plasmid backbone pAGM4723 or pAGM8031 using Golden Gate Modular Cloning. a The barley constructs, sgRNAHvPM19-1 and sgRNAHvPM19-3 house a hygromycin resistance cassette consisting of the hygromycin phosphotransferase coding sequence (hptII) driven and terminated by the 35 s promoter (P-CaMV35s) and terminator (T-CaMV35s) from Cauliflower mosaic virus; a Cas9 expression cassette consisting of sequence encoding Cas9 from Streptococcus pyogenies with a carboxy-terminal nuclear-localization signal from Simian vacuolating virus 40 (SpCas9:NLS) driven by a ubiquitin promoter from Zea mays (P-ZmUbi) and terminated by a nopaline synthase terminator from Agrobacterium tumefaciens (T-AtNos); and single guide RNA (sgRNAHvPM19-1 or sgRNAHvPM19-3) driven by a Triticum aestivum U6 promoter (P-TaU6). b The Brassica construct, sgRNABolC.GA4.a, houses a kanamycin resistance cassette consisting of the neomycin phosphotransferase coding sequence (nptII) driven and terminated by P-CaMV35S and T-AtNos; SpCas9:NLS driven by a constitutive promoter from Cassava Vein Mosaic Virus (P-CsVMV) and a tandem pair of single guide RNAs (sgRNA1BolC.GA4.a and sgRNA2BolC.GA4.a) driven by the U626 promoter from Arabidopsis (P-AtU626)

Fig. 3

Frequency of on-target and off-target Cas9 activity on the HvPM19 gene copies at T₁. a Alignment of sgRNAHvPM19-1 and sgRNAHvPM19-3 target sequences (grey highlight) with the corresponding sequences of the other copies of HvPM19. Hyphens represent alignment matches while mismatches are shown in black highlight and white font. The PAM is highlighted in red and the numbering of nucleotides is relative to the PAM. b Percentage of T₁ plants with mutations in the corresponding copies of HvPM19 for sgRNAHvPM19-1 (T₀-181 and T₀-122) and sgRNAHvPM19-3 (T₀-211). Dark and light grey bars represent the percentages for HvPM19-1 and HvPM19-3 editing, respectively

Fig. 4

Germline transmission of Cas9 induced mutations from T₁ to T₂ plants in barley and B. oleracea in the absence of the T-DNA construct. a Sequence alignment of T₁-181_E1 and five homozygous T₂ progeny with homozygous 1-bp deletion in HvPM19_1. b Sequence alignment from representative clones of T₁ heterozygote mutants and direct Sanger sequencing of their T₂ progeny with homozygous mutations in the absence of the T-DNA. The numbers of clones supporting T₁ mutant alleles are indicated on the right. c Sequence alignments of BolC.GA4.a Target 2 in homozygous T₁ and T-DNA free T₂ plants. Across panels the target sequences for sgRNAHvPM19-1 and sgRNABolC.GA4.a (grey) and PAM (red) are highlighted and Cas9 induced insertions and deletions are indicated by red font or red hyphens, respectively. Names of homozygous T₂ plants that lack the presence of the T-DNA construct are indicated in blue; individual homozygous plants with the same allele are shown on the same row and are labelled with a ‘p’ prefix

Fig. 5

Mutant alleles detected in T₀ B. oleracea. Alignment of wild-type and mutant sequences surrounding the target sequences (grey highlight) and PAM (red highlight) in mutants identified by restriction digest/PCR screen (a) and by phenotypic screen (b). Insertions and deletions are indicated by red font or red hyphens, respectively. For large deletions, red arrows indicate the direction of the deletions. For each line in panel b (L2F1_A and L2F1_E), 16 clones were examined and the frequencies of each mutant allele (represented as clones with mutant allele/total number of clones examined) are indicated at the right side of the panel

Fig. 6

Mutations in BolC.GA4.a result in dwarf stature and affect the pod valve margin. a Wild-type B. oleracea DH1012 (left) and L2F1_A with a mutation in BolC.GA4.a showing a severe dwarf phenotype. Scale bar 10 cm. b Height of homozygous T₁ plants with wild type (n = 11) or bolC.ga4.a mutant (n = 16) alleles. c Schematic cross section of B. oleracea pod with replum/valve margin region indicated by dashed square. Lignified tissue is indicated in red, unlignified cells are indicated in blue, and developing seeds are in green. d, e Cross-section of replum valve margin region of B. oleracea wild-type pod (d) and L2F1_A mutant pod (e); scale bars 200 μm

Fig. 7

Frequency of on- and off-target Cas9 activity in L2F1_8.2 T₁ Brassica plants. a The alignment of sgRNA1BolC.GA4.a and sgRNA2BolC.GA4.a target sequences in BolC.GA4.a with their corresponding sequences in BolC.GA4.b. Hyphens represent alignment matches while mismatches are shown in black highlight and white font. The PAM is highlighted in red and numbering of nucleotides is relative to the PAM. b Percentage of the T₁ plants with mutations in BolC.GA4.a and BolC.GA4.b. Dark and light grey bars represent the percentages of BolC.GA4.a and BolC.GA4.b editing, respectively. N = 90 plants