A CRISPR/dCas9-based strategy to identify proteins associated to specific DNA loci at København Universitet

Big question:

We can use targeted methods to examine where in the genome a protein binds DNA. These methods require a specific antibody or the addition of a protein tag in Chromatin Immuno Precipitation (ChIP) experiments followed by quantitative comparison of co-precipated DNA, for example in the entire genome by next-generation sequencing (ChIP-seq). While ChIP-seq is very pwerful, it is currently very challenging to identify which proteins bind a certain DNA region in vivo in an unbiased way. This question is often extremely relevant, for example in disease with genetic or epigenetic origin, and plant fitness that correlates with geneticor epigenetic changes. We have started to use ctalytically dead Cas9 (dCas9) fused to protein tags (e.g. Flag) in combination with sequence-specific guide RNA (gRNA) to target dCas9 to genomic regions without cleaving the DNA (as active Cas9 would). This approach gives use the ability to target dCas9 to genomic regions of interest in plants and yeast.

Your role:

You would join an existing projet that aims to use CRISPR/dCas9 and designer TALEN (engineered proteins recognizing specific DNA sequences) to identify proteins co-purifying with specific DNA regions by quanjtitative mass spectrometry. You would help with the method optimization, in particular regadring the design of CRISPR/dCas9 and gRNA components. We currently establish this method to profile differential epigenetic signatures at nuclesomes flanking eukaryotic Transcription Start Sites (TSSs). However, we also target genomic regions that are characterized by "repressive transcription" in yeast (1) and plants (2). It could also be conceivable to target a region with DNA polymorphisms associated to yield or plant fitness in plants, and/or ethanol biology in yeast as part of the project.

Literature:

(1) candidate DNA regions in yeast: https://elifesciences.org/articles/31989

(2) candidate DNA regions in plants: https://journals.plos.org/plosgenetics/article/comments?id=10.1371/journal.pgen.1007969

Methods used:CRISPR/dCas9, proteomics, metabolic labeling, TALEs, molecular biology, biochemistry
Keywords:CRISPR, epigenetics, RNA, chromatin, brewing
Supervisor(s):Sebastian Marquardt
Email:sebastian.marquardt@plen.ku.dk

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