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

Lawrenson T, Shorinola O, Stacey N, Li C, Østergaard L, Patron N, Uauy C, Harwood W - Genome Biol. (2015)

Bottom Line: In B. oleracea, targeting of BolC.GA4.a leads to Cas9-induced mutations in 10 % of first generation plants screened.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.

View Article: PubMed Central - PubMed

Affiliation: John Innes Centre, Norwich Research Park, Colney, NR4 7UH, UK. tom.lawrenson@jic.ac.uk.

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.

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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
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Fig6: 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

Mentions: We also hypothesised that plants with homozygous mutations in BolC.GA4.a would show a dwarf phenotype similar to that observed in Arabidopsis ga4 mutants. Therefore, we performed a phenotypic screen of the 80 T0B. oleracea plants. All 80 T0 lines were grown to maturity, and at flowering two lines not previously characterised by the restriction digest/PCR assay were observed to be dwarf in stature (L2F1_A and L2F1_E; Fig. 6a). The BolC.GA4.a sequences from both dwarf plants were found to contain a series of mutant alleles in Target 1 and Target 2, in two independent leaf samples from each plant (Fig. 5b, Additional file 2). In addition, the mutation was restricted to BolC.GA4.a, as we were unable to detect any mutation in BolC.GA4.b. The identification of T0 plants with a visible knockout phenotype has also been reported in rice, tomato, and Arabidopsis [8, 10, 24].Fig. 6


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

Lawrenson T, Shorinola O, Stacey N, Li C, Østergaard L, Patron N, Uauy C, Harwood W - Genome Biol. (2015)

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
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4663725&req=5

Fig6: 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
Mentions: We also hypothesised that plants with homozygous mutations in BolC.GA4.a would show a dwarf phenotype similar to that observed in Arabidopsis ga4 mutants. Therefore, we performed a phenotypic screen of the 80 T0B. oleracea plants. All 80 T0 lines were grown to maturity, and at flowering two lines not previously characterised by the restriction digest/PCR assay were observed to be dwarf in stature (L2F1_A and L2F1_E; Fig. 6a). The BolC.GA4.a sequences from both dwarf plants were found to contain a series of mutant alleles in Target 1 and Target 2, in two independent leaf samples from each plant (Fig. 5b, Additional file 2). In addition, the mutation was restricted to BolC.GA4.a, as we were unable to detect any mutation in BolC.GA4.b. The identification of T0 plants with a visible knockout phenotype has also been reported in rice, tomato, and Arabidopsis [8, 10, 24].Fig. 6

Bottom Line: In B. oleracea, targeting of BolC.GA4.a leads to Cas9-induced mutations in 10 % of first generation plants screened.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.

View Article: PubMed Central - PubMed

Affiliation: John Innes Centre, Norwich Research Park, Colney, NR4 7UH, UK. tom.lawrenson@jic.ac.uk.

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.

Show MeSH
Related in: MedlinePlus