Abstract 214: Functional Analysis of the Low Density Lipoprotein Receptor Using Saturation Mutagenesis Strategies

Low density lipoprotein receptor (LDLR) genetic mutations are well-documented to cause familial hypercholesterolemia (FH) in patients. These mutations affect a broad range of LDLR functions, including LDL binding and receptor trafficking, and five classes exist to describe their functional consequences. Although over 1,000 LDLR mutants have been identified, an additional 15,000+ LDLR mutants are possible by only a single amino acid substitution in the ~160kDa protein. Since cataloging mutants would undoubtedly be of therapeutic benefit, we are systematically classifying the effect of every single amino acid substitution on LDLR function using saturation mutagenesis. LDLR was knocked out of HEK293 cells using standard CRISPR/Cas9 practices and decreased protein levels were assessed by flow cytometry and immunoblot staining. In order to introduce each individual mutation into the genomic DNA sequence for high-throughput, functional analyses, the PiggyBac transposon system was used to insert a serine-integrase attachment site into a single allele in the LDLR locus, whereby efficient knock in of LDLR mutants from a pooled cDNA library can be achieved. Mutant LDLR libraries containing ~800 single amino acid substitutions were generated using commercial oligo pools and Gibson cloning. Of particular therapeutic interest is the identification of mutations that alter LDL’s ability to bind to the receptor. In our first set of experiments, fluorescently labeled LDL was incubated with cells containing pooled single amino acid substitutions in LDLR, and cells were sorted using flow cytometry. Sorted groups ranging from fluorescent negative to positive were sent for next generation sequencing in order to identify severe vs benign mutants on receptor function. As some of these mutations may decrease LDL binding activity due to inhibited receptor trafficking, we also stained pooled cells with an antibody directed against LDLR, sorted by flow cytometry and analyzed using next generation sequencing with similar strategies. Ultimately, cataloging LDLR genetic mutations should be of great therapeutic benefit, as it will allow better diagnosis and treatment of patients suffering from FH.