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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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Related in: MedlinePlus

Mutant alleles detected in T0B. 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
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Fig5: Mutant alleles detected in T0B. 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

Mentions: Eighty independent transgenic lines were generated by Agrobacterium-mediated transformation, and 20 of these T0 plantlets were screened by the restriction digest/PCR assay to detect mutations in the target sequences. We identified in-dels at the target sites in BolC.GA4.a in two out of 20 T0 lines (L2F1_8.2 and L2E1_17.1). Mutations in L2E1_17.1 were confirmed by TA cloning and Sanger sequencing of the PCR products (Fig. 5a). Line L2F1_8.2 showed a 282-bp deletion that corresponds to re-joining of the DNA at exactly 3 bp from the PAM in both target regions. As in barley, the detection of the mutations required an enrichment of the target by restriction digest prior to PCR.Fig. 5


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)

Mutant alleles detected in T0B. 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
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig5: Mutant alleles detected in T0B. 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
Mentions: Eighty independent transgenic lines were generated by Agrobacterium-mediated transformation, and 20 of these T0 plantlets were screened by the restriction digest/PCR assay to detect mutations in the target sequences. We identified in-dels at the target sites in BolC.GA4.a in two out of 20 T0 lines (L2F1_8.2 and L2E1_17.1). Mutations in L2E1_17.1 were confirmed by TA cloning and Sanger sequencing of the PCR products (Fig. 5a). Line L2F1_8.2 showed a 282-bp deletion that corresponds to re-joining of the DNA at exactly 3 bp from the PAM in both target regions. As in barley, the detection of the mutations required an enrichment of the target by restriction digest prior to PCR.Fig. 5

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