NLRP3 is innate immune receptor, involved in forming inflammasomes. Inflammasomes are multimeric protein complexes that are formed in a cell to orchestrate host defense mechanisms against pathogen or danger-associated molecular patterns. Inflammasome NLRP3 is also clinically implicated inflammasome, as it can be involved in initiation or progression of autoinflammatory or autoimmune diseases. The scope of the thesis was to alter the Nlrp3 gene in mouse-derived macrophage cells with CRISPR/Cas9 technology and characterize the activity of the Nlrp3 mutant under endogenous promoter. CRISPR/Cas9 technology is a novel system that enables site specific genome modifications. We prepared several site-specific constructs to cut the DNA sequence upstream of LRR-coding region. We tested the activity of constructs with tests T7, plasmid pCAG-EGxxFP and in vitro digestion. Then we electroporated iBMDM cells and sorted them with cell sorter and made single cell clones. We screened the clones with western blot to detect NLRP3 protein. We tested clones of interest for the presence of mutations with T7 test and for inflammasome activation and IL-1β maturation by ELISA. In this work we were unable to obtain a NLRP3 deletion mutant, but we showed how to design, prepare and employ the CRISPR/Cas9 technology for the study of molecular mechanisms of NLRP3 inflammasome activation. NLRP3 inflammasome activation is involved in various inflammatory conditions thus the knowledge of the mechanisms of NLRP3 inflammasome assembly is very important for design of novel therapeutic approaches.
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