Mutagenese des endogenen TP53-Lokus durch CRISPR/Cas9-vermittelte homologe Rekombination & Base Editing in Lungenkarzinomzellen

Das Bronchialkarzinom ist eine der häufigsten Tumorarten und der führende Grund Krebs-assoziierter Sterbefälle weltweit. In bis zu 70 % aller Bronchialkarzinome liegt das Tumorsuppressorgen TP53 mutiert vor. Ein besseres Verständnis des Genprodukts p53 und dessen Mutanten ist somit ein essenzieller...

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Bibliographic Details
Main Author: Drangenstein, Daniel
Contributors: Stiewe, Thorsten (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2023
Online Access:PDF Full Text
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Lung cancer is one of the most common cancer types and the leading cause of cancer-related mortality worldwide. In approximately 70 % of all lung cancer cases, the tumor suppressor gene TP53 is mutated. Therefore, a better understanding of p53 mutants is an essential step towards the improvement of patient prognosis. That is one reason why p53 is intensively studied. In this project, a cell clone of the lung cancer cell line NCI-H460 (H460) was established in a series of steps. This allowed an efficient mutagenesis of the endogenous TP53 locus of this cell clone via CRISPR/Cas9 and homology directed repair (HDR). Initially, a specific DNA-sequence, called LSL (LoxP-Stop-LoxP) - cassette, was introduced into one TP53 allele. The other two TP53-alleles present in this cell line were knocked out permanently, resulting in monoallelic TP53 expression. Thereafter, efficient editing of the endogenous TP53 locus was possible, so a generation of single TP53 mutants and a saturated mutagenesis of the hotspot codon R175 were implemented in this project. Cellular fitness of those generated mutants was then observed under treatment with the Mdm2-inhibitor Nutlin-3a, which specifically activates the p53 pathway under physiological circumstances. This cell clone is now suitable for introducing most clinically relevant TP53 mutations and analyzing their impact at a physiological expression level and within its native gene structure. This characterization can be used to optimize therapeutic schemes by individually adjusting the treatment according to the p53 mutant present in a patient’s tumor. Furthermore, a mutagenesis of TP53 was performed by using a base editor which consists of a Cas9 nickase connected to an APOBEC3A deaminase. This construct was able to induce cystosine-to-thymine transitions with efficiencies over 90 %. In this project, the hotspot codon R273 of TP53 was successfully edited in H460- and colorectal HCT116 cells. Additionally, a mutagenesis of exon 8 of TP53 was performed in HCT116 cells by exploiting every PAM sequence present and targeting it with an appropriate sgRNA. In general, the base editor showed high efficiency combined with very high specificity.