Zuidhof, H.R. (2016) Promoter studies using CRISPR/dCas9 and LC-MS/MS based proteomics. Research Project 2 (major thesis), Molecular Biology and Biotechnology (2016-2019).
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Abstract
Many different types of gene regulation studies exist. However, most of these either look at DNA binding of a protein, or study the downstream effects of e.g. overexpression of a certain factor. So far it has been very challenging to uncover the complete set of proteins that bind to an endogenous promoter and govern its activation. This study aimed to do just that: develop a system based on CRISPR/dCas9 in order to specifically pull down promoters. Potentially, subsequent mass spectrometry analysis could identify associated proteins. A restriction deficient Cas9 (dCas9) fused to an avi-tag was transduced into human chronic myelogenous leukemia (K562) cells along with a BirA biotin ligase. Furthermore, two sets of lentiviral vectors were created, expressing ten different gRNAs each. Both sets of gRNAs spanned a region of 200bp from either the PDK1 or SLC2A1 promoter, two genes critically involved in glucose metabolism control. After crosslinking using a Chromatin Immunoprecipitation (ChIP) protocol, avi-tagged dCas9 protein was precipitated using streptavidin beads for subsequent LC-MS/MS analysis to identify proteins that were co-precipitated. Using this protocol, we show that targeted endogenous promoters can efficiently be precipitated providing a means to identify sets of unique proteins that might be specific regulators of the targeted genes.
| Item Type: | Thesis (Research Project 2 (major thesis)) |
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| Degree programme: | Molecular Biology and Biotechnology (2016-2019) |
| Thesis type: | Research Project 2 (major thesis) |
| Language: | English |
| Date Deposited: | 15 Feb 2018 08:25 |
| Last Modified: | 15 Feb 2018 08:25 |
| URI: | https://fse.studenttheses.ub.rug.nl/id/eprint/14632 |
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