ExperimentName: PAH Variant Rescued by FokI-Cas9

RescueCellLine: Cytotoxic T cell (CL:0000910) AKA COS-7 Cell Line

GeneAlteration: A wild-type PAH cDNA was introduced into a vector via XbaI and BamHI cleavage after amplification from the plasmid. The mutation was introduced via site-directed mutagenesis and confirmed via Sanger sequencing. COS-7 cells were seeded and cultured before being transfected with either the mutant vector or the wild-type control. The resulting clone was characterized through RT-PCR and expanded.

RescueCellPhenotype: HP:0005982 (Reduced phenylalanine hydroxylase activity)

RescueMethod: The FokI-dCas9-IRES-ZsGreen1 plasmid was created by designing gRNAs to target the 1222C>T variant and synthesizing a final sgRNA expression fragment which was cloned into the base vector. Electroporation was then performed on the COS-7 cells with the expression vectors incorporated. Flow cytometry analysis confirmed that 18.4% of cells co-expressed both the FokI-dCas9 and the sgRNA.The cells were treated with the ssODN that represents the replacement sequence and confirmed via Sanger sequencing. Any off-target sites were anlyzed via Sanger sequencing and displayed no evidence of off-target cleavage.

RescueEvidence: Western blot analysis performed on cell expressing the wild-type PAH and those treated with differing levels of ODN and RS-1 showed that those treated with the replacement sequence had significantly higher PAH expression than the mutant cells that were not transfected with the vectors. Further, liquid chromatography-electrospray ionization tandem mass spectrometry was used to evaluate PAH activity after repair showing an activity of 22.08 +/- 1.67 % with the RS-1 expressed as opposed to 16.86+/- 1.31 % without the replacement sequence.

RescueCellVariant: NM_000277.3(PAH):c.1222C>T (p.Arg408Trp)

ClinVarID: 577 https://www.ncbi.nlm.nih.gov/clinvar/variation/577/