Generating Isogenic Models of Li-Fraumeni Syndrome and Restoring p53 using CRISPR

Li-Fraumeni syndrome (LFS) is a highly penetrant familial cancer predisposition disorder characterized by a spectrum of multiple early-onset primary cancers in first- and second-degree relatives, usually caused by a germline mutation in the TP53 tumor suppressor gene. The discovery of CRISPR has led to precision gene editing strategies which provides new opportunities to model and treat genetic disorders. By applying CRISPR gene editing strategies to correct germline TP53 mutations in patient derived LFS fibroblast models and in a mouse LFS osteosarcoma model, this thesis demonstrates the feasibility and explores the limitations of mutant TP53 allele specific targeting. First, to understand the role of heterozygous germline TP53 mutations (H193P and S240G) in the context of normal cell function and the development of cancer, we used a CRISPR gene editing approach to specifically target mutant TP53 alleles in LFS patient-derived fibroblast lines and restore two functional wild-type TP53 alleles. Our approach was highly specific (no detectable editing in wild-type alleles); surprisingly, repaired TP53 fibroblast lines exhibited a reduced response to DNA damage (low p53 and p21 expression) when compared to unedited fibroblast lines harbouring either mutant or wild-type TP53. Repaired fibroblast lines were significantly more susceptible to senescence, suggesting that the restoration of a homozygous wild-type TP53 genotype in heterozygous mutant TP53 LFS fibroblast lines disrupts the balance of p53 signaling and prevents the escape of LFS fibroblasts from replicative senescence. In a second study, we designed a CRISPR-mediated approach to target mutant Trp53 and restore p53 function as a therapeutic in tumour cell lines derived from a p53R172H/+ LFS mouse model. After generating double-strand breaks with sgRNAs targeting the R172H Trp53 mutation in an osteosarcoma cell line, single-cell clonal populations were generated and examined. 76% of clonal lines had multiple short indels, indicating a high preference for NHEJ repair. sgRNA efficiency for generating targeted double stranded breaks approached 100% in generated clones, but none had observed p53 restoration. Overall, these results highlight the feasibility of precision gene editing in mutant p53 systems and shed light on the complexities of germline p53 mutant signaling in both normal and cancer cells.Ph.D