Dataset Information

Induction of targeted, heritable mutations in barley and Brassica oleracea using RNA-guided Cas9 nuclease.

ABSTRACT: 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.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.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.

SUBMITTER: Lawrenson T

PROVIDER: S-EPMC4663725 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

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Induction of targeted, heritable mutations in barley and Brassica oleracea using RNA-guided Cas9 nuclease.

Lawrenson Tom T Shorinola Oluwaseyi O Stacey Nicola N Li Chengdao C Østergaard Lars L Patron Nicola N Uauy Cristobal C Harwood Wendy W

Genome biology 20151130

<h4>Background</h4>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.<h4>Results</h4>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 tw ...[more]

PMID: 26616834

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Project description:Background:Genome editing of monocot plants can be accomplished by using the components of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR associated Cas9) technology specifically optimized for these types of plants. Here, we present the development of RNA-guided Cas9 system for simplex and multiplex genome editing in barley. Results:We developed a set of customizable RNA-guided Cas9 binary vectors and sgRNA modules for simplex and multiplex editing in barley. To facilitate the design of RNA-guided Cas9 constructs, the pBract derived binary vectors were adapted to Gateway cloning and only one restriction enzyme was required for construction of the sgRNA. We designed a synthetic, codon optimized Cas9 gene containing the N terminal SV40 nuclear localization signal and the UBQ10 Arabidopsis 1st intron. Two different sgRNAs were constructed for simplex editing and one polycistronic tRNA-gRNA construct (PTG) for multiplex editing using an endogenous tRNA processing system. The RNA-guided Cas9 constructs were validated in transgenic barley plants produced by Agrobacterium-mediated transformation. The highest mutation rate was observed in simplex editing of the cytokinin oxidase/dehydrogenase HvCKX1 gene, where mutations at the hvckx1 locus were detected in 88% of the screened T0 plants. We also proved the efficacy of the PTG construct in the multiplex editing of two CKX genes by obtaining 9 plants (21% of all edited plants) with mutations induced in both HvCKX1 and HvCKX3. Analysis of the T1 lines revealed that mutations in the HvCKX1 gene were transmitted to the next generation of plants. Among 220 screened T1 plants we identified 85 heterozygous and 28 homozygous mutants, most of them bearing frameshift mutations in the HvCKX1 gene. We also observed independent segregation of mutations and the Cas9-sgRNA T-DNA insert in several T1 plants. Moreover, the knockout mutations of the Nud gene generated phenotype mutants with naked grains, and the phenotypic changes were identifiable in T0 plants. Conclusions:We demonstrated the effectiveness of an optimized RNA-guided Cas9 system that can be used for generating homozygous knockout mutants in the progeny of transgenic barely plants. This is also the first report of successful multiplex editing in barley using a tRNA processing system.

Dataset Information

Induction of targeted, heritable mutations in barley and Brassica oleracea using RNA-guided Cas9 nuclease.

Publications

Induction of targeted, heritable mutations in barley and Brassica oleracea using RNA-guided Cas9 nuclease.

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