Ailesel Akdeniz ateşine özgü uyarılmış pluripotent kök hücrelerdeki MEFV geni için CRISPR/Cas temelli genom yazılım sisteminin oluşturulması

The ability to derive induced pluripotent stem cells (iPSCs) from any individual has opened up the possibility to generate patient- and disease-specific cell culture models. Such models facilitate research into disease mechanisms and provide a platform for in vitro drug screens. A drawback of disease-specific iPSC models is the lack of genetically matched controls. Recent implementation of bacterial adaptive immune system known as clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems in mammalian cells as a new genome editing tool has provided unparalleled simplicity and precision to introduce site-specific changes into host DNA. A central aim of this thesis was to generate tools to implement a Crispr/Cas9 based system in an iPSC model of genetic inflammatory disease with a goal to replace disease-causing allele with its wild type counterpart. Familial Mediterranean Fever (FMF) is a autosomal recessive autoinflammatory disease caused by the mutations in the mefv gene. FMF has a high carrier rate in Turkey and the most severe form of the disease result from homozygous M694V mutations. iPSCs were derived from one such FMF patient and the presence of the disease-associated mutations was confirmed by DNA sequencing. To target the disease allele, single chimeric guide RNAs targeting mefv sequence at the vicinity of the M694V mutation were designed and cloned into expression vectors. The functionality of Crispr/Cas9 components in mammalian cells were confirmed using a Green fluorescent protein (GFP) reporter assay in 293T cells. To monitor homologous recombination (HR), a reporter construct in which GFP sequence was interrupted by the targeted mefv sequences was generated. Different HR donor templates were tested and linearized plasmids were observed to be the most efficient. Next, the ability of the selected guide RNAs to cleave the endogenous mefv locus was confirmed by a mismatch-specific DNA endonuclease assay. However, we failed to detect a corrected allele upon introduction of the Crispr components and a repair template bearing the wild type mefv exon 10 sequences into patient-specific iPSCs. To facilitate detection of corrected allele, a new donor template bearing silent mutations that create new restriction sites was designed. This donor template will enable the use of restriction fragment length polymorphism assay (RFLP) for screening rare iPSC clones that are successfully corrected.