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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

Germline transmission of Cas9 induced mutations from T1 to T2 plants in barley and B. oleracea in the absence of the T-DNA construct. a Sequence alignment of T1-181_E1 and five homozygous T2 progeny with homozygous 1-bp deletion in HvPM19_1. b Sequence alignment from representative clones of T1 heterozygote mutants and direct Sanger sequencing of their T2 progeny with homozygous mutations in the absence of the T-DNA. The numbers of clones supporting T1 mutant alleles are indicated on the right. c Sequence alignments of BolC.GA4.a Target 2 in homozygous T1 and T-DNA free T2 plants. Across panels the target sequences for sgRNAHvPM19-1 and sgRNABolC.GA4.a (grey) and PAM (red) are highlighted and Cas9 induced insertions and deletions are indicated by red font or red hyphens, respectively. Names of homozygous T2 plants that lack the presence of the T-DNA construct are indicated in blue; individual homozygous plants with the same allele are shown on the same row and are labelled with a ‘p’ prefix
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Fig4: Germline transmission of Cas9 induced mutations from T1 to T2 plants in barley and B. oleracea in the absence of the T-DNA construct. a Sequence alignment of T1-181_E1 and five homozygous T2 progeny with homozygous 1-bp deletion in HvPM19_1. b Sequence alignment from representative clones of T1 heterozygote mutants and direct Sanger sequencing of their T2 progeny with homozygous mutations in the absence of the T-DNA. The numbers of clones supporting T1 mutant alleles are indicated on the right. c Sequence alignments of BolC.GA4.a Target 2 in homozygous T1 and T-DNA free T2 plants. Across panels the target sequences for sgRNAHvPM19-1 and sgRNABolC.GA4.a (grey) and PAM (red) are highlighted and Cas9 induced insertions and deletions are indicated by red font or red hyphens, respectively. Names of homozygous T2 plants that lack the presence of the T-DNA construct are indicated in blue; individual homozygous plants with the same allele are shown on the same row and are labelled with a ‘p’ prefix

Mentions: We found that the mutations detected in the T2 progenies matched those observed in the corresponding T1 parent in all cases examined. For instance, the homozygous 1-bp deletion observed in line T1-181_E1 was also present in all its T2 progenies that segregated away from the T-DNA construct (Table 1; Fig. 4a). Likewise, a range of mutations found in heterozygous T1 plants were also identified in homozygous T2 individuals in the absence of the T-DNA construct (Fig. 4b). Taken together, the T1 and T2 sequence data from six lines, originating from two independent T0 events (T0-181 and T0-122), provide strong evidence of stable germline transmission of Cas9-induced mutations in barley in the absence of the T-DNA.Fig. 4


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)

Germline transmission of Cas9 induced mutations from T1 to T2 plants in barley and B. oleracea in the absence of the T-DNA construct. a Sequence alignment of T1-181_E1 and five homozygous T2 progeny with homozygous 1-bp deletion in HvPM19_1. b Sequence alignment from representative clones of T1 heterozygote mutants and direct Sanger sequencing of their T2 progeny with homozygous mutations in the absence of the T-DNA. The numbers of clones supporting T1 mutant alleles are indicated on the right. c Sequence alignments of BolC.GA4.a Target 2 in homozygous T1 and T-DNA free T2 plants. Across panels the target sequences for sgRNAHvPM19-1 and sgRNABolC.GA4.a (grey) and PAM (red) are highlighted and Cas9 induced insertions and deletions are indicated by red font or red hyphens, respectively. Names of homozygous T2 plants that lack the presence of the T-DNA construct are indicated in blue; individual homozygous plants with the same allele are shown on the same row and are labelled with a ‘p’ prefix
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig4: Germline transmission of Cas9 induced mutations from T1 to T2 plants in barley and B. oleracea in the absence of the T-DNA construct. a Sequence alignment of T1-181_E1 and five homozygous T2 progeny with homozygous 1-bp deletion in HvPM19_1. b Sequence alignment from representative clones of T1 heterozygote mutants and direct Sanger sequencing of their T2 progeny with homozygous mutations in the absence of the T-DNA. The numbers of clones supporting T1 mutant alleles are indicated on the right. c Sequence alignments of BolC.GA4.a Target 2 in homozygous T1 and T-DNA free T2 plants. Across panels the target sequences for sgRNAHvPM19-1 and sgRNABolC.GA4.a (grey) and PAM (red) are highlighted and Cas9 induced insertions and deletions are indicated by red font or red hyphens, respectively. Names of homozygous T2 plants that lack the presence of the T-DNA construct are indicated in blue; individual homozygous plants with the same allele are shown on the same row and are labelled with a ‘p’ prefix
Mentions: We found that the mutations detected in the T2 progenies matched those observed in the corresponding T1 parent in all cases examined. For instance, the homozygous 1-bp deletion observed in line T1-181_E1 was also present in all its T2 progenies that segregated away from the T-DNA construct (Table 1; Fig. 4a). Likewise, a range of mutations found in heterozygous T1 plants were also identified in homozygous T2 individuals in the absence of the T-DNA construct (Fig. 4b). Taken together, the T1 and T2 sequence data from six lines, originating from two independent T0 events (T0-181 and T0-122), provide strong evidence of stable germline transmission of Cas9-induced mutations in barley in the absence of the T-DNA.Fig. 4

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