Duy Vo

The CYP3A5 gene encodes for a human cytochrome P450 enzyme that metabolizes approximately 50 percent of drugs. An important substrate for this enzyme is tacrolimus (Tac), the primary immunosuppressant used in solid organ transplantation. One of its variant, CYP3A5*6, is a non-functional allele caused by exon skipping. It is particularly frequent in African American populations and rarely occurs in the Caucasian population. Engineering a CYP3A5*6 cell line from the parental CYP3A5*1 cell line using CRISPR/Cas9 to test its function is essential to the in vitro evaluation of the metabolism of many drugs including Tac. To engineer the CYP3A5*6 cell line gBlocks (a 2.5 kb double-stranded gene fragment) containing exon 7 of the CYP3A5*1 gene with a single nucleotide polymorphism (SNP) A to G related to exon skipping were ligated to an rAAV backbones by adapters created for digestion by AscI and MluI to create plasmids. The plasmids were grown in dam-/dcm- E.coli cells. The plasmids were then characterized using restriction digest with enzymes and Sanger sequencing. The plasmid collected was successfully characterized and had a length of 5408 base pairs with the correct enzyme cut site through gel electrophoresis. Sanger sequencing confirmed the correct gBlock sequence and insertion position into the backbone. The engineered plasmid will now be inserted into a virus, which will be combined with a CRISPR/Cas9 system for homology-directed repair on CYP3A5*1/*1 DD cell lines to create the CYP3A5*6 cell line.