Design and characterization of the activity of Nlrp3 truncation mutant using CRISPR/Cas9 technology

NLRP3 je protein, ki lahko tvori inflamasom. Inflamasomi so znotrajcelični multimerni proteinski kompleksi in vodijo obrambo proti molekulskim vzorcem povezanimi s patogeni in poškodbami. NLRP3 tudi klinično zanimiv, saj sodeluje pri nastanku in napredovanju avtoinflamatornih in avtoimununskih bolezni. Namen naloge je bil spremeniti gen Nlrp3 v celicah mišjih makrofagov s tehnologijo CRISPR/Cas9 in preveriti delovanje mutante pod endogenim promotorjem. Tehnologija CRISPR/Cas9 je nedavno razvita tehnologija, ki omogoča tarčno spreminjanje genoma. Pripravili smo mestno specifične konstrukte, ki bi lahko tarčno rezali zaporedje DNA in bi posledično vnesli stop kodon ali spremenili bralni okvir gena Nlrp3 s čimer bi odstranili domeno LRR iz zrelega proteina NLRP3. Delovanje konstruktov smo preverili s testom T7, test s plazmidom pCAG-EGxxFP in cepitvijo in vitro. Nato smo s konstruktom elektroporirali celice mišjih makrofagov, jih ločili s celičnim ločevalcem in pripravili posamezne klone. S prenosom po westernu smo preverili večje število klonov. Prisotnost mutacij smo nadaljnje preverili s testom T7, za aktivacijo inflamasoma in zorenje IL-1β pa smo uporabili imunoencimski test ELISA. V nalogi nam ni uspelo pridobiti delecijske mutante NLRP3, s katero bi lahko preverjali lastnosti proteina NLRP3, smo pa prikazali potek dela za dizajniranje, pripravo in uporabo tehnologije CRISPR/Cas9 za preverjanje molekularnega mehanizma aktivacije inflamasoma NLRP3. Aktivacija inflamasoma NLRP3 je povezana z vnetnimi boleznimi, tako, da je znanje o mehanizmih potrebnih za izgradnjo inflamasoma NLRP3 zelo pomembno za dizajniranje novih načinov zdravljenja.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