Use of CRISPR/base editors for the generation of TP53 p.R337H knocking cells using, mutation associated with Li-Fraumeni Syndrome

Abstract

TP53 (p53) is a tumor suppressor whose functions and mutations are widely studied. TP53 germinative mutations can lead to Li-Fraumeni syndrome (LFS), which is characterized by the development of tumors in multiple organs at an early age. The R337H substitution (TP53R337H) in the oligomerization domain impairs the formation of tetramers, necessary for the correct function of TP53. The few published papers, describe that TP53R337H function was not different from wild type (WT) when expressed in protein-deficient cell lines, while other mutations in the same region (R337C, L344P) were deleterious for the function. We observed that those articles about R337H mutation employed transient transfection in TP53-null cells to express TP53R337H, generating cells whit aberrant expression of the mutant when compared with LFS patient cells, which could have masked the effects of the R337H substitution on TP53 function. In this project we aim to analyze the molecular changes induced by the R337H mutation using a new variation of the genomic editing CRISPR/Cas9 technique, which allows base editing without the need for homologous recombination to generate knocking cells for the R337H mutation. It will be evaluate TP53R337H expression and stability using the knockin cells, as well as interactions with other proteins (e.g. MDM2) and aspects related to TP53-dependent responses (DNA damage activation, cell cycle arrest induction and apoptosis). As a result, we hope to generate knowledge and techniques to improve the acquaintance and formulation of new treatments for Brazilian families with this mutation.