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

Abstract

BackgroundGenome 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.ResultsWe 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.ConclusionsWe 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 materialThe online version of this article (10.1186/s13007-018-0382-8) contains supplementary material, which is available to authorized users.

Gene targeting in Arabidopsis via an all‐in‐one strategy that uses a translational enhancer to aid Cas9 expression

“…Transcriptional and translational enhancer sequences have been reported to improve Cas9 expression and, in turn, the efficiency of CRISPR/Cas9‐mediated targeted mutagenesis in plants (Gasparis et al ., ; Kusano et al ., ). We therefore investigated whether the omega translational enhancer from tobacco mosaic virus (TMV) would improve gene targeting by enhancing Cas9 translation (Gallie and Kado, ; Mitsuhara et al ., ; Figure a).…”

Targeted mutation of barley (1,3;1,4)-β-glucan synthases reveals complex relationships between the storage and cell wall polysaccharide content

“…The efficiency of CRISPR/Cas9-induced mutations in the primary transformants ranged from 5-35% in this study. Previous studies in barley reported CRISPR/Cas9-induced mutation frequencies of 10-23% (Lawrenson et al, 2015), 44% (Holme et al, 2017) and ~80% (Gasparis et al, 2018;Kapusi et al, 2017) of plants after Agrobacterium-mediated transformation. In other cereals such as wheat, mutant production efficiency was reported from 0.3-5% using biolistic bombardment of CRISPR/Cas9 components (Zhang et al, 2016;Gil-Humanes et al, 2017;Liang et al, 2017;Sánchez-León et al, 2018) and Agrobacterium-mediated transformation (Okada et al, 2019).…”

How to start your monocot CRISPR/Cas project: plasmid design, efficiency detection, and offspring analysis

“…The CRISPR/Cas9 system has been successfully used for genome editing in a variety of monocots, including rice (Oryza sativa), wheat (Triticum sp. ), barley (Hordeum vulgare), and maize (Zea mays) (Feng et al 2018;Gasparis et al 2018;Hu et al 2018;Kis et al 2019;Okamoto et al 2017;Wang et al 2017;Zhang et al 2016). To perform CRISPR/Cas9-mediated genome editing, the Cas9 endonuclease is guided by a single guide RNA (sgRNA) to recognize the complementary sequence and create double-strand breaks (DSBs), thereby generating a short deletion or insertion.…”