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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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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)
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Fig2: 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)

Mentions: We independently targeted two ancestral HvPM19 gene copies (HvPM19-1 and HvPM19-3) in the spring barley cultivar ‘Golden Promise’ which is amenable to Agrobacterium-mediated transformation. We were able to amplify HvPM19-4 from Golden Promise, but unable to amplify HvPM19-2 suggesting that this cultivar lacks this copy of HvPM19. Two binary constructs, sgRNAHvPM19-1, referred to as pPM19-1 and sgRNAHvPM19-3, referred to as pPM19-3 (Fig. 2a), were designed to independently target HvPM19-1 and HvPM19-3, respectively. The 20 base-pair target sequence in pPM19-1 has a single nucleotide mismatch with each of the corresponding sequences in HvPM19-3 and HvPM19-4, while the target sequence in pPM19-3 has three mismatches with HvPM19-1 and one mismatch with HvPM19-4 (Fig. 3a).Fig. 2


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)

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)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig2: 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)
Mentions: We independently targeted two ancestral HvPM19 gene copies (HvPM19-1 and HvPM19-3) in the spring barley cultivar ‘Golden Promise’ which is amenable to Agrobacterium-mediated transformation. We were able to amplify HvPM19-4 from Golden Promise, but unable to amplify HvPM19-2 suggesting that this cultivar lacks this copy of HvPM19. Two binary constructs, sgRNAHvPM19-1, referred to as pPM19-1 and sgRNAHvPM19-3, referred to as pPM19-3 (Fig. 2a), were designed to independently target HvPM19-1 and HvPM19-3, respectively. The 20 base-pair target sequence in pPM19-1 has a single nucleotide mismatch with each of the corresponding sequences in HvPM19-3 and HvPM19-4, while the target sequence in pPM19-3 has three mismatches with HvPM19-1 and one mismatch with HvPM19-4 (Fig. 3a).Fig. 2

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