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Enhancing CRISPR-Cas efficiency in Rabl-configured cereal crop genomes (CrisBar)
Project
Project code: 031B0551
Contract period: 01.07.2018
- 30.06.2020
Budget: 456,778 Euro
Purpose of research: Experimental development
Keywords: barley, wheat, Rabl configuration, high throughput pipeline
Gene-editing applications based on clustered regularly interspaced short palindromic repeats (CRISPR) in combination with CRISPR-associated nucleases (Cas) are of enormous importance in basic and applied science. CRISPR-Cas-mediated genome editing opens up possibilities to undertake directed editing, even in organisms that have been resilient to classical approaches. This allows mechanistic studies of genes in e.g. barley (Hordeum vulgare) and wheat (Triticum aestivum). CrisBar aims to optimize the selection of both target genes and corresponding CRISPR-Cas guide RNAs. We will establish high throughput pipelines for target gene selection and gRNA design and screening for convenient and high-efficiency multiplex genome editing in monocotyledonous plants, in particular barley and wheat. Both cereal crops were two of the first domesticated food crops and wheat has been the basic staple food of the major civilizations of Europe, West Asia and North Africa. Today, wheat is grown on more land area than any other commercial crop and continues to be the most important food grain source for humans. We will exploit the recent and game-changing availability of complete reference genomes for barley and hexaploid wheat to systematically analyse chromosomal as well as genomic prerequisites for CRISPR-Cas targets and gRNAs. Both barley and wheat genomes display distinct chromosomal territories that are associated with the so-called Rabl configuration in cereal genomes, a particular chromosomal conformation that organises the chromosomes in fold-back structures with telomeres at one pole of the nucleus and the centromeres of all chromosomes aligned at the opposing pole. This particular configuration aligns with functional characteristics of the different territories of the chromosomes and is expected to influence CRISPR-Cas targeting efficiency. We will combine optimized gRNA design with experimental tools to further enhance the throughput of CRISPR-Cas reporter systems, allowing us to deduce gRNA design rules from an unprecedented minimum of ~1100 target-gRNA combinations, focusing on both specificity and multi-plexing between homeologous gene copies among the subgenomes of wheat. Finally, this project will capitalize on DPPN (German Plant Phenotyping Network; BMBF) phenotyping facilities and establish a high throughput screen for plant immunity-related traits of target-gRNA combinations in elicitor-treated protoplasts. Together, this project will result in experimental and bioinformatic toolkits to optimize (1) gene and gRNA selection based on the chromosomal context of the target genes, (2) mutagenesis efficiency, and (3) the detection of defense-associated phenotypes in cereal crops.
Section overview
Subjects
- Plant Breeding
- Biotechnology
- Process engineering
Framework programme
Funding programme
Excutive institution
Helmholtz Zentrum München, GmbH German Research Center for Environmental Health (GSF)
