CRISPR/Cas9 ex vivo gene editing of rat primary hepatocytes as a potential therapeutic model for genetic disease

In this project, we used CRISPR/Cas9 to knock out several genes that are highly expressed in the liver: Fah is an enzyme involved in the catabolism of tyrosine, and is defective in the human genetic disease Hereditary Tyrosinemia Type 1. CYP2E1 is an important liver metabolic enzyme involved in Cytochrome P450 drug metabolism. We targeted these genes because of their potential as a model for gene therapy and their value in further studies of metabolism. Hypothesis CRISPR/Cas9 based gene editing can be used for ex vivo gene therapy to edit multiple genes in rat primary hepatocytes. Specific Aims This project aimed to successfully isolate and culture rat primary hepatocytes, generate CRISPR constructs targeting specific genes, and test the knock‐out of those genes by transfecting CRISPR plasmids into immortalized Rat C6 cells and primary hepatocytes. Methods Surgeries were performed on wild type rats to isolate and culture primary hepatocytes in Williams E media. Multiple CRISPR/Cas9 constructs utilizing unique guide RNAs (gRNA) were generated using the UCSC Genome Browser to target the Rat Fah gene and CYP2E1 . The CRISPR/Cas9 plasmids were designed using a px330 plasmid backbone, and replicated in E. coli colonies. These CRISPR plasmids were then transfected into the Rat C6 immortalized cell line using the Lonza Nucleofector system (Cologne, Germany), and tested with the Cel‐1 assay to confirm activity. Primary hepatocytes were also transfected with GFP plasmid and CRISPR plasmid to confirm transfection efficiency in these cells. Results Multiple targeted CRISPR/Cas9 plasmids were constructed and tested using polymerase chain reaction. The PCR results confirmed banding patterns consistent with CRISPR/Cas9 plasmids targeting the Fah gene, as well as CYP2E1 . After transfection of these CRISPR plasmids into Rat C6 cells, Cel‐1 assay results showed non‐homologous end joining (NHEJ) activity for the CYP2E1 CRISPR, but not Fah CRISPR, suggesting that only the CYP2E1 CRISPR was active. Transfection of GFP plasmid and CRISPR plasmid into primary hepatocytes showed positive GFP fluorescence under microscopy, indicating that the Nucleofector system was successful for transfecting plasmid DNA into primary hepatocytes. Conclusions and Next Steps We have surgically isolated and cultured rat primary hepatocytes to be used for CRSIPR/Cas9 gene editing. We have constructed and confirmed CRISPR plasmids to knock out the Rat CYP2E1 gene, and will continue to optimize our targeting of the Fah gene. Next steps will include testing for NHEJ in primary hepatocytes to confirm CRISPR/Cas9 activity and Sanger sequencing of the modified DNA to identify the precise sequence edits made by CRISPR/Cas9. These tools allow for targeted molecular editing of the rat genome, which may be valuable in modeling future clinical approaches to genetic disease, drug metabolism, and cancer therapy. Support or Funding Information National Institute of Diabetes and Digestive and Kidney Diseases R25 Grant 5R25DK098104‐03