English (United Kingdom)

https://curated-unify.zendy.io/wp-json/zendy-region/v1/featured_content/oa?rat=en

https://curated-unify.zendy.io/wp-json/zendy-region/v1/highlighted_journal/

Global: Zendy Plus annual

Presents the access of premium content as premium feature

Premium Content

Presents the keyphrase highlighting as premium feature

Keyphrase Highlighting

Presents the summarisation as premium feature

Summarisation

Insights

Presents the pdf analysis as premium feature

PDF Analysis

Presents the zaia usage as premium feature

ZAIA

Global: Zendy Tools annual

Global: Zendy Free Plan

Global: Zendy Tools monthly

Global: Zendy Plus monthly

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 T 0  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 T 1  lines revealed that mutations in the  HvCKX1  gene were transmitted to the next generation of plants. Among 220 screened T 1  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 T 1  plants. Moreover, the knockout mutations of the  Nud  gene generated phenotype mutants with naked grains, and the phenotypic changes were identifiable in T 0  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.    Electronic supplementary material  The online version of this article (10.1186/s13007-018-0382-8) contains supplementary material, which is available to authorized users.

A simple and efficient CRISPR/Cas9 platform for induction of single and multiple, heritable mutations in barley (Hordeum vulgare L.)