Investigating the Role of the KLHL9 Gene Through CRISPR/Cas9-Mediated Deletion, Poster 49

Cytokines are proteins that play a direct role in regulating host immune responses. Type I interferons are the largest family of cytokines in humans. In this way, type I interferons are an important area of study for... [ view full abstract ]

Cytokines are proteins that play a direct role in regulating host immune responses. Type I interferons are the largest family of cytokines in humans. In this way, type I interferons are an important area of study for understanding how organisms respond to pathogens. These signaling proteins are encoded in a 16 gene locus that is interrupted by a single non-interferon gene, Kelch-like 9 (KLHL9). We are interested in elucidating the role of this gene that is constitutively expressed, has been conserved for over 100 million years, and is about 99% conserved at the amino acid level among mammals. We hypothesize that the KLHL9 gene influences type I interferon gene expression, and consider two models. KLHL9 could either encode for proteins that control type I interferon expression (trans-activating); or it could directly enable the activation of type I interferon expression (cis-activating). To test these models we performed CRISPR/Cas9-mediated deletion of the approximately 1kb KLHL9 promoter and enhancer region. We identified five guide RNAs that successfully targeted the KLHL9 promoter and enhancer in a plasmid template. We subsequently transfected four of the guide RNAs in pairs into mouse embryonic fibroblasts. Assays reveal that the deletion of the KLHL9 promoter and enhancer was successful. Additionally, the mouse embryonic fibroblasts were able to survive despite the deletion. It is now possible to measure changes in type I interferon expression in these cells post-immune challenge to determine whether the absence of the KLHL9 promoter and enhancer influences type I interferon expression.