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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

Frequency of on- and off-target Cas9 activity in L2F1_8.2 T1Brassica plants. a The alignment of sgRNA1BolC.GA4.a and sgRNA2BolC.GA4.a target sequences in BolC.GA4.a with their corresponding sequences in BolC.GA4.b. Hyphens represent alignment matches while mismatches are shown in black highlight and white font. The PAM is highlighted in red and numbering of nucleotides is relative to the PAM. b Percentage of the T1 plants with mutations in BolC.GA4.a and BolC.GA4.b. Dark and light grey bars represent the percentages of BolC.GA4.a and BolC.GA4.b editing, respectively. N = 90 plants
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Fig7: Frequency of on- and off-target Cas9 activity in L2F1_8.2 T1Brassica plants. a The alignment of sgRNA1BolC.GA4.a and sgRNA2BolC.GA4.a target sequences in BolC.GA4.a with their corresponding sequences in BolC.GA4.b. Hyphens represent alignment matches while mismatches are shown in black highlight and white font. The PAM is highlighted in red and numbering of nucleotides is relative to the PAM. b Percentage of the T1 plants with mutations in BolC.GA4.a and BolC.GA4.b. Dark and light grey bars represent the percentages of BolC.GA4.a and BolC.GA4.b editing, respectively. N = 90 plants

Mentions: To examine the mutation frequency of the target locus BolC.GA4.a, the T1 progenies of lines L2F1_8.2 and L2E1_17.1 were screened for Cas9-induced mutations in Target 1 and 2 by PCR amplification of BolC.GA4.a followed by direct sequencing. Using the sequencing chromatograms it was possible to identify homozygous and heterozygous mutations. We detected mutations in the T1 progenies of L2F1_8.2, but not in L2E1_17.1. Heterozygous in-dels were observed in 68 of 90 L2F1_8.2 T1 progenies; however, no homozygous mutations were identified. Of these 68 T1 plants, 35 had mutations in Target 1, whereas Target 2 was mutated in 67 lines, suggesting a higher efficiency of the Target 2 sgRNA (Fig. 7b). None of the 90 T1 progenies inherited the complete 282-bp deletion between the two BolC.GA4.a target regions that was observed in the T0 generation.Fig. 7


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- and off-target Cas9 activity in L2F1_8.2 T1Brassica plants. a The alignment of sgRNA1BolC.GA4.a and sgRNA2BolC.GA4.a target sequences in BolC.GA4.a with their corresponding sequences in BolC.GA4.b. Hyphens represent alignment matches while mismatches are shown in black highlight and white font. The PAM is highlighted in red and numbering of nucleotides is relative to the PAM. b Percentage of the T1 plants with mutations in BolC.GA4.a and BolC.GA4.b. Dark and light grey bars represent the percentages of BolC.GA4.a and BolC.GA4.b editing, respectively. N = 90 plants
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig7: Frequency of on- and off-target Cas9 activity in L2F1_8.2 T1Brassica plants. a The alignment of sgRNA1BolC.GA4.a and sgRNA2BolC.GA4.a target sequences in BolC.GA4.a with their corresponding sequences in BolC.GA4.b. Hyphens represent alignment matches while mismatches are shown in black highlight and white font. The PAM is highlighted in red and numbering of nucleotides is relative to the PAM. b Percentage of the T1 plants with mutations in BolC.GA4.a and BolC.GA4.b. Dark and light grey bars represent the percentages of BolC.GA4.a and BolC.GA4.b editing, respectively. N = 90 plants
Mentions: To examine the mutation frequency of the target locus BolC.GA4.a, the T1 progenies of lines L2F1_8.2 and L2E1_17.1 were screened for Cas9-induced mutations in Target 1 and 2 by PCR amplification of BolC.GA4.a followed by direct sequencing. Using the sequencing chromatograms it was possible to identify homozygous and heterozygous mutations. We detected mutations in the T1 progenies of L2F1_8.2, but not in L2E1_17.1. Heterozygous in-dels were observed in 68 of 90 L2F1_8.2 T1 progenies; however, no homozygous mutations were identified. Of these 68 T1 plants, 35 had mutations in Target 1, whereas Target 2 was mutated in 67 lines, suggesting a higher efficiency of the Target 2 sgRNA (Fig. 7b). None of the 90 T1 progenies inherited the complete 282-bp deletion between the two BolC.GA4.a target regions that was observed in the T0 generation.Fig. 7

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