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Functional characterization of the TP53 mutome using CRISPR/Cas9 saturating mutagenesis
TP53 is an essential tumor suppressor gene which is inactivated in every second tumor. Most frequently TP53 is disabled by missense mutations which result in the expression of a mutant p53 protein. Mutant p53 protein is unable to prevent uncontrolled proliferation and can additionally increase cance...
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| Format: | Dissertation |
| Sprache: | Englisch |
| Veröffentlicht: |
Philipps-Universität Marburg
2021
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| Online Zugang: | PDF-Volltext |
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| Zusammenfassung: | TP53 is an essential tumor suppressor gene which is inactivated in every second tumor. Most frequently TP53 is disabled by missense mutations which result in the expression of a mutant p53 protein. Mutant p53 protein is unable to prevent uncontrolled proliferation and can additionally increase cancer growth by dominant negative and gain of function effects. Mutations in TP53 are frequently associated with aggressive tumor growth, chemotherapy resistance and shortened survival. Therefore, the TP53 gene status has important clinical implication. Moreover, information about TP53 mutations will be essential for application of emerging p53-targeted therapeutics: Mdm2 inhibitors and p53 reactivators.
Eight most frequent hotspot mutations account for nearly 30% of all missense TP53 variants found in tumors and are extensively studied. Hotspot TP53 mutations lead to production of a transcriptionally inactive loss of function protein. The rest 70% of TP53-mutated tumors contain one of >2000 distinct mutant p53 variants, most of which are uncharacterized. Such a broad spectrum of mutants makes prediction of their impact on disease outcome a very challenging task. Therefore, for advancing personalized cancer treatment it would be of utmost importance to study how the hundreds of individual p53 mutations influence a therapy response. Functional characterization of hundreds of mutations in a gene of interest is a tall order task which requires time-consuming in vitro and in vivo experiments. Thus, an experimental approach for a massive parallel phenotypic screening of mutations in TP53 gene would be of a great value.
In the present work we took advantage of the CRISPR-Cas9 gene editing technology to develop the CSMS – CRISPR-based saturated mutagenesis screening of TP53 gene, an improved system for massive parallel functional screening of p53 mutants.
We have established rapid and flexible protocol of targeting p53 mutations into endogenous TP53 locus using CRISPR-Cas9-induced homology-directed repair. We have validated CSMS by performing saturation mutagenesis of the short protein motif and demonstrated outstanding performance. We have scaled our protocol up to establish a high-throughput method that allows precise functional characterization of thousands of p53 variants. We have tested effects of p53 mutations on response to Mdm2 inhibitors and irradiation and revealed excellent correlation of screening results with known structural, functional and clinical data. Furthermore, we have demonstrated that CMSM is able to highlight even subtle functional difference between mutants and identify partially loss of function mutants. Manipulating the endogenous TP53 locus allowed us to study effects of mutation in non-coding regions, which was previously unachievable. A detailed comparison of our data with the previously published studies provided compelling evidence, that the procedure established in our study is significantly more accurate in categorization of pathogenic TP53 mutations.
In summary, we have attested CSMS as a powerful tool to catalogue TP53 mutations. This tool can be used in the future to increase the utility of mutations in TP53 as clinical biomarkers. |
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| Umfang: | 230 Seiten |
| DOI: | 10.17192/z2021.0294 |