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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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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
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Fig1: 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

Mentions: We investigated the use and target specificity requirements of RNA-guided Cas9 genome editing in barley by focusing on a multi-copy gene. We selected HvPM19, which is present as four copies within a single barley BAC clone from the cultivar ‘Morex’ (HvPM19-1 to HvPM19-4; Fig. 1a). Relative to HvPM19-1, the HvPM19-2, HvPM19-3 and HvPM19-4 loci have sequence identities of 89.8 %, 89.5 % and 88.6 %, respectively, whereas HvPM19-3 and HvPM19-4 have greater sequence identity to HvPM19-2 (98.4 % and 99.6 %). This suggests that HvPM19-1 was involved in the more ancestral duplication event and that there was a series of very recent duplication events between HvPM19-2, HvPM19-3 and HvPM19-4.Fig. 1


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)

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
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4663725&req=5

Fig1: 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
Mentions: We investigated the use and target specificity requirements of RNA-guided Cas9 genome editing in barley by focusing on a multi-copy gene. We selected HvPM19, which is present as four copies within a single barley BAC clone from the cultivar ‘Morex’ (HvPM19-1 to HvPM19-4; Fig. 1a). Relative to HvPM19-1, the HvPM19-2, HvPM19-3 and HvPM19-4 loci have sequence identities of 89.8 %, 89.5 % and 88.6 %, respectively, whereas HvPM19-3 and HvPM19-4 have greater sequence identity to HvPM19-2 (98.4 % and 99.6 %). This suggests that HvPM19-1 was involved in the more ancestral duplication event and that there was a series of very recent duplication events between HvPM19-2, HvPM19-3 and HvPM19-4.Fig. 1

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