Abstract:The modifcation of crop genomes employing functional components of the microbial CRISPR/Cas immune system is a rapidly developing area of applied research. Site-directed plant genome modifcation by this technology involves the construction of Cas endonuclease- and guide-RNA-encoding vectors, delivery of the plasmid DNA into plant cells, processing of the chosen genomic target site by the corresponding gene products and regeneration of plants from modifed cells. The utilization of this technology in local breed… Show more
“…1 a). For these sites, guide RNAs (gRNAs) were designed (Additional file 1 , Table S1) and incorporated in the pSH121 generic vector [ 15 ]. Fig.…”
Section: Resultsmentioning
confidence: 99%
“…The non-parametric Kolmogorov-Smirnov test was used to compare the distributions of mutation types induced by selected gRNAs. Protoplast isolation, transformation, and amplicon sequencing were performed as described previously [15].…”
Section: Prevalidation Of Grna Activity By Protoplast Transfectionmentioning
Background
The naked caryopsis character in barley is a domestication-associated trait defined by loss-of-function of the NUD gene. The functional NUD gene encodes an Apetala 2/Ethylene-Response Factor (AP2/ERF) controlling the formation of a cementing layer between pericarp and both lemma and palea. The downstream genes regulated by the NUD transcription factor and molecular mechanism of a cementing layer formation are still not sufficiently described. A naturally occurring 17-kb deletion in the nud locus is associated with the emergence of naked barley. Naked barley has been traditionally used for food and nowadays is considered as a dietary component for functional nutrition.
Results
In the present study, we demonstrate that targeted knockout of the NUD gene using RNA-guided Cas9 endonuclease leads to the phenotype conversion from hulled to naked barley. Using in vivo pre-testing systems, highly effective guide RNAs targeting the first exon of the NUD gene were selected. Expression cassettes harboring the cas9 and guide RNA genes were used to transform barley cv. Golden Promise via Agrobacterium-mediated DNA transfer. The recessive naked grain phenotype was observed in 57% of primary transformants, which indicates a frequent occurrence of homozygous or biallelic mutations. T-DNA-free homozygous lines with independently generated mutations in the NUD gene were obtained in the T1 generation. At homozygous state, all obtained mutations including one- and two-amino acid losses with the translational reading frame being retained invariably caused the naked grain phenotype.
Conclusions
The hulled and naked barley isogenic lines generated are a perfect experimental model for further studies on pleiotropic consequences of nud mutations on overall plant performance under particular consideration of yield-determining traits. Due to the high β-glucan content of its grains, naked barley is considered as being of particular dietary value. The possibility to convert hulled into naked barley cultivars by targeted mutagenesis allows breeders to extend the potential utilization of barley by the provision of functional food.
“…1 a). For these sites, guide RNAs (gRNAs) were designed (Additional file 1 , Table S1) and incorporated in the pSH121 generic vector [ 15 ]. Fig.…”
Section: Resultsmentioning
confidence: 99%
“…The non-parametric Kolmogorov-Smirnov test was used to compare the distributions of mutation types induced by selected gRNAs. Protoplast isolation, transformation, and amplicon sequencing were performed as described previously [15].…”
Section: Prevalidation Of Grna Activity By Protoplast Transfectionmentioning
Background
The naked caryopsis character in barley is a domestication-associated trait defined by loss-of-function of the NUD gene. The functional NUD gene encodes an Apetala 2/Ethylene-Response Factor (AP2/ERF) controlling the formation of a cementing layer between pericarp and both lemma and palea. The downstream genes regulated by the NUD transcription factor and molecular mechanism of a cementing layer formation are still not sufficiently described. A naturally occurring 17-kb deletion in the nud locus is associated with the emergence of naked barley. Naked barley has been traditionally used for food and nowadays is considered as a dietary component for functional nutrition.
Results
In the present study, we demonstrate that targeted knockout of the NUD gene using RNA-guided Cas9 endonuclease leads to the phenotype conversion from hulled to naked barley. Using in vivo pre-testing systems, highly effective guide RNAs targeting the first exon of the NUD gene were selected. Expression cassettes harboring the cas9 and guide RNA genes were used to transform barley cv. Golden Promise via Agrobacterium-mediated DNA transfer. The recessive naked grain phenotype was observed in 57% of primary transformants, which indicates a frequent occurrence of homozygous or biallelic mutations. T-DNA-free homozygous lines with independently generated mutations in the NUD gene were obtained in the T1 generation. At homozygous state, all obtained mutations including one- and two-amino acid losses with the translational reading frame being retained invariably caused the naked grain phenotype.
Conclusions
The hulled and naked barley isogenic lines generated are a perfect experimental model for further studies on pleiotropic consequences of nud mutations on overall plant performance under particular consideration of yield-determining traits. Due to the high β-glucan content of its grains, naked barley is considered as being of particular dietary value. The possibility to convert hulled into naked barley cultivars by targeted mutagenesis allows breeders to extend the potential utilization of barley by the provision of functional food.
“…An alternative approach for creating transgene-free edited plants is transient expression of CRISPR/Cas DNA as have been reported in many crops, including wheat 116,117 , barley 118 , tetraploid potato 119,120 , tomato 121 , and cotton 122 . Compared to stable transformation of CRISPR/Cas DNA, transient expression is especially useful in certain horticultural crops that are vegetatively propagated, self-incompatible, polyploid, and/or have long juvenile stages 123 .…”
Directed breeding of horticultural crops is essential for increasing yield, nutritional content, and consumer-valued characteristics such as shape and color of the produce. However, limited genetic diversity restricts the amount of crop improvement that can be achieved through conventional breeding approaches. Natural genetic changes in cisregulatory regions of genes play important roles in shaping phenotypic diversity by altering their expression. Utilization of CRISPR/Cas editing in crop species can accelerate crop improvement through the introduction of genetic variation in a targeted manner. The advent of CRISPR/Cas-mediated cis-regulatory region engineering (cis-engineering) provides a more refined method for modulating gene expression and creating phenotypic diversity to benefit crop improvement. Here, we focus on the current applications of CRISPR/Cas-mediated cis-engineering in horticultural crops. We describe strategies and limitations for its use in crop improvement, including de novo cis-regulatory element (CRE) discovery, precise genome editing, and transgene-free genome editing. In addition, we discuss the challenges and prospects regarding current technologies and achievements. CRISPR/Cas-mediated cis-engineering is a critical tool for generating horticultural crops that are better able to adapt to climate change and providing food for an increasing world population.
“…Guide-RNA secondary structures were modeled using the RNAfold tool (Gruber et al, 2008). pSH121 harboring a maize codon-optimized cas9 coding sequence under control of the maize POLYUBIQUITIN 1 promoter and a guide-RNA scaffold preceded by the rice U3 (RNA polymerase III-processed) promoter was used as a generic vector (Gerasimova et al, 2019). A synthetic double-stranded oligonucleotide carrying the target-specific part of the gRNA was annealed and integrated between the OsU3 promoter and the upstream gRNA scaffold using BsaI restriction and ligation.…”
Section: Preparation Of a Lox3 Knockout Constructmentioning
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