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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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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
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Fig3: 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

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)

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
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig3: 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
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