Outlook for coeliac disease patients: towards bread wheat with hypoimmunogenic gluten by gene editing of α- and γ-gliadin gene families

Abstract Background Polycomb repressive complexes 1 and 2 play important roles in epigenetic gene regulation by posttranslationally modifying specific histone residues. Polycomb repressive complex 2 is responsible for the trimethylation of lysine 27 on histone H3; Polycomb repressive complex 1 catalyzes the monoubiquitination of histone H2A at lysine 119. Both complexes have been thoroughly studied in Arabidopsis, but the evolution of polycomb group gene families in monocots, particularly those with complex allopolyploid origins, is unknown. Results Here, we present the in silico identification of the Polycomb repressive complex 1 and 2 (PRC2, PRC1) subunits in allohexaploid bread wheat, the reconstruction of their evolutionary history and a transcriptional analysis over a series of 33 developmental stages. We identified four main subunits of PRC2 [E(z), Su(z), FIE and MSI] and three main subunits of PRC1 (Pc, Psc and Sce) and determined their chromosomal locations. We found that most of the genes coding for subunit proteins are present as paralogs in bread wheat. Using bread wheat RNA-seq data from different tissues and developmental stages throughout plant ontogenesis revealed variable transcriptional activity for individual paralogs. Phylogenetic analysis showed a high level of protein conservation among temperate cereals. Conclusions The identification and chromosomal location of the Polycomb repressive complex 1 and 2 core components in bread wheat may enable a deeper understanding of developmental processes, including vernalization, in commonly grown winter wheat.

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230 Genome editing applications in plants: high-throughput CRISPR/Cas editing for crop improvement

Journal of Animal Science ◽

10.1093/jas/skz258.115 ◽

2019 ◽

Vol 97 (Supplement_3) ◽

pp. 56-56

Author(s):

Michael Thomson

Keyword(s):

Genome Editing ◽

High Throughput ◽

Positional Cloning ◽

Gene Editing ◽

Crop Improvement ◽

Cost Effective ◽

Gene Families ◽

Ease Of Use ◽

Plant Genome ◽

Wide Range

Abstract The precision and ease of use of CRISPR nucleases, such as Cas9 and Cpf1, for plant genome editing has the potential to accelerate a wide range of applications for crop improvement. For upstream research on gene discovery and validation, rapid gene knock-outs can enable testing of single genes and multi-gene families for functional effects. Large chromosomal deletions can test the function of tandem gene arrays and assist with positional cloning of QTLs by helping to narrow down the target region. Nuclease-deactivated Cas9 fusion proteins with transcriptional activators and repressors can be used to up and down-regulate gene expression. Even more promising, gene insertions and allele replacements can provide the opportunity to rapidly test the effects of different alleles at key loci in the same genetic background, providing a more precise alternative to marker-assisted backcrossing. Recently, Texas A&M AgriLife Research has supported the development of a Crop Genome Editing Lab at Texas A&M working towards optimizing a high-throughput gene editing pipeline and providing an efficient and cost-effective gene editing service for research and breeding groups. The lab is using rice as a model to test and optimize new approaches aimed towards overcoming current bottlenecks. For example, a wealth of genomics data from the rice community enables the development of novel approaches to predict which genes and target modifications may be most beneficial for crop improvement, taking advantage of known major genes, high-resolution GWAS data, multiple high-quality reference genomes, transcriptomics data, and resequencing data from the 3,000 Rice Genomes Project. Current projects have now expanded to work across multiple crops to provide breeding and research groups with a rapid gene editing pipeline to test candidate genes in their programs, with the ultimate goal of developing nutritious, high-yielding, stress-tolerant crops for the future.

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Integration of promoters, inverted repeat sequences and proteomic data into a model for high silencing efficiency of coeliac disease related gliadins in bread wheat

BMC Plant Biology ◽

10.1186/1471-2229-13-136 ◽

2013 ◽

Vol 13 (1) ◽

pp. 136 ◽

Cited By ~ 18

Author(s):

Fernando Pistón ◽

Javier Gil-Humanes ◽

Francisco Barro

Keyword(s):

Coeliac Disease ◽

Bread Wheat ◽

Inverted Repeat ◽

Repeat Sequences ◽

Proteomic Data

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Optimization of T-DNA architecture for Cas9-mediated mutagenesis in Arabidopsis

10.1101/419952 ◽

2018 ◽

Author(s):

Baptiste Castel ◽

Laurence Tomlinson ◽

Federica Locci ◽

Ying Yang ◽

Jonathan D G Jones

Keyword(s):

Functional Analysis ◽

Gene Editing ◽

First Generation ◽

Gene Families ◽

Loss Of Function ◽

Site Specific ◽

Systematic Comparison ◽

Multiple Promoters ◽

Highly Active ◽

Homozygous Mutations

ABSTRACTBacterial CRISPR systems have been widely adopted to create operator-specified site-specific nucleases. Such nuclease action commonly results in loss-of-function alleles, facilitating functional analysis of genes and gene families We conducted a systematic comparison of components and T-DNA architectures for CRISPR-mediated gene editing in Arabidopsis, testing multiple promoters, terminators, sgRNA backbones and Cas9 alleles. We identified a T-DNA architecture that usually results in stable (i.e. homozygous) mutations in the first generation after transformation. Notably, the transcription of sgRNA and Cas9 in head-to-head divergent orientation usually resulted in highly active lines. Our Arabidopsis data may prove useful for optimization of CRISPR methods in other plants.

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A dual sgRNA approach for functional genomics in Arabidopsis thaliana[OPEN]

10.1101/172676 ◽

2017 ◽

Author(s):

Laurens Pauwels ◽

Rebecca De Clercq ◽

Jonas Goossens ◽

Sabrina Iñigo ◽

Clara Williams ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

Gene Editing ◽

Reverse Genetics ◽

Gene Families ◽

Null Alleles ◽

Loss Of Function ◽

Knock Out ◽

Nonsense Mediated Decay ◽

Dna Insertion ◽

Entire Gene

AbstractReverse genetics uses loss-of-function alleles to interrogate gene function. The advent of CRISPR/Cas9-based gene editing now allows to generate knock-out alleles for any gene and entire gene families. Even in the model plant Arabidopsis thaliana, gene editing is welcomed as T-DNA insertion lines do not always generate null alleles. Here, we show efficient generation of heritable mutations in Arabidopsis using CRISPR/Cas9 with a workload similar to generating overexpression lines. We obtain Cas9 null-segregants with bi-allelic mutations in the T2 generation. Out of seven new mutant alleles we report here, one allele for GRXS17, the ortholog of human GRX3/PICOT, did not phenocopy previously characterized nulls. Notwithstanding, the mutation caused a frameshift and triggered nonsense-mediated decay. We demonstrate that our workflow is also compatible with a dual sgRNA approach in which a gene is targeted by two sgRNAs simultaneously. This paired nuclease method can result in a more reliable loss-of-function alleles that lack a large essential part of the gene. The ease in the CRISPR/Cas9 workflow should help in the eventual generation of true null alleles of every gene in the Arabidopsis genome, which will advance both basic and applied plant research.One-sentence summaryWe present a dual sgRNA approach to delete Arabidopsis gene 34 fragments in order to obtain reliable functional knock-outs.

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Identification of Polycomb Repressive Complex 1 and 2 Core Components in Hexaploid Bread Wheat

10.1101/703546 ◽

2019 ◽

Author(s):

Beáta Strejčková ◽

Radim Čegan ◽

Ales Pecinka ◽

Zbyněk Milec ◽

Jan Šafář

Keyword(s):

Bread Wheat ◽

Developmental Stages ◽

Gene Families ◽

Polycomb Group ◽

Transcriptional Analysis ◽

Histone H2a ◽

Polycomb Repressive Complex ◽

Polycomb Repressive Complex 2 ◽

Core Components ◽

Polycomb Repressive Complex 1

ABSTRACTPolycomb repressive complex 1 and 2 play important roles in epigenetic gene regulation by posttranslationally modifying specific histone residues. Polycomb repressive complex 2 is responsible for the trimethylation of lysine 27 on histone H3, while Polycomb repressive complex 1 catalyzes the monoubiquitination of histone H2A at lysine 119. Although these biochemical functions are evolutionarily conserved, studies in animals and plants, mainly Arabidopsis thaliana, showed that specific subunits have evolved into small gene families, with individual members acting at different developmental stages or responding to specific environmental stimuli. However, the evolution of polycomb group gene families in monocots, particularly those with complex allopolyploid origins, is unknown. Here, we present the in silico identification of the Polycomb repressive complex 1 and 2 subunits in allohexaploid bread wheat, the reconstruction of their evolutionary history and a transcriptional analysis over a series of 33 developmental stages. The identification and chromosomal location of the Polycomb repressive complex 1 and 2 core components in bread wheat may enable a deeper understanding of developmental processes, including vernalization in commonly grown winter wheat.

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