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CRISPR/Cas mediated chromosome engineering in Arabidopsis

Primary Supervisor: Dr James Higgins, Department of Genetics and Genome Biology

Secondary supervisor: Ed Louis

PhD project title: CRISPR/Cas mediated chromosome engineering in Arabidopsis

University of Registration: University of Leicester

Project outline:

CRISPR/Cas has emerged as a flexible tool for genome editing in complex eukaryotes. It has been widely used for targeted mutagenesis and DNA repair as well more sophisticated approaches utilising the specificity of guide RNAs to target Cas9-tagged proteins. The effectiveness of this approach now provides opportunities for engineering chromosomes in plants to answer fundamental biological questions as well as potentially translating benefits into crops (Ronspies et al. 2021). Many crop species have complex polyploid genomes with large structural variants such as inversions, insertions and deletions as well as large introgressed foreign DNA from wild relatives. These often provide advantageous traits but may also be associated with negative traits. Genome editing will revolutionise crop improvement by refining introgressed regions of DNA to only the genes required as well as realigning structural variants between varieties. Here we aim to develop these approaches in the tractable model plant Arabidopsis, as well as answer fundamental biological questions on how chromosome structure affects recombination.

Arabidopsis is a useful model system to undertake CRISPR/Cas mediated chromosome engineering in eukaryotes as it is easy to transform and contains five small chromosomes. More specifically, chromosomes 2 and 4 are the smallest and contain Nucleolar Organising Regions (NORs) on their short arms. The NORs are characterized by large repetitive sequences encoding ribosomal 45S subunits. Chromosomes 3 and 5 contain the ribosomal 5S subunit and chromosome 1 has no rDNA repeats. The NORs are inhibitory to meiotic recombination and the small size influences crossover number and position. Chromosome structure is also likely to locally and globally affect gene expression, DNA methylation and chromatin organization. Therefore, the aim of this project will be to exchange the long arm of Arabidopsis chromosome 1 with the short arms of chromosome 2 and 4 and then analyse meiotic recombination and gene expression. Recombination will be assayed using the fluorescent tagged line reporter system (as shown in previous MIBTP graduates paper – France et al. 2021) and gene expression will be performed on a small number of selected genes by qPCR. Chromosome pairing, recombination and formation of the synaptonemal complex will be investigated utilising cytological techniques such as immunolocalisation of antibodies to specific meiotic proteins in conjunction with super-resolution fluorescence microscopy.

Objectives:

  • Design guide RNAs to target break sites on long arm of chromosome 1 and short arms of chromosomes 2 and 4 and then transform into Arabidopsis
  • Screen transformants for engineered chromosomes
  • Analyse engineered lines for chromosome rearrangements utilising recombination markers, qPCR, chromatin markers and immunolocalisation.

References:

  1. France MG, Enderle J, Röhrig S, Puchta H, Franklin FCH, Higgins JD (2021) ZYP1 is required for obligate cross-over formation and cross-over interference in Arabidopsis. PNAS; 118(14):e2021671118. doi: 10.1073/pnas.2021671118.
  2. Rönspies M, Dorn A, Schindele P, Puchta H. (2021). CRISPR-Cas-mediated chromosome engineering for crop improvement and synthetic biology. Nat Plants. (5):566-573. doi: 10.1038/s41477-021-00910-4

BBSRC Strategic Research Priority: Sustainable Agriculture and Food: Plant and Crop Science & Understanding the Rules of Life: Plant Science

Techniques that will be undertaken during the project:

CRISPR/Cas mediated double strand break formation, cloning, DNA sequencing plant and yeast transformation, super-resolution fluorescent microscopy, PCR, qPCR, immunolocalisation.

Contact: Dr James HigginsLink opens in a new window, University of Leicester