title: CRISPR RNA-guided FokI nucleases repair a PAH variant in a phenylketonuria model.
abstract: The CRISPR/Cas9 system is a recently developed genome editing technique. In this study, we used a modified CRISPR system, which employs the fusion of inactive Cas9 (dCas9) and the FokI endonuclease (FokI-dCas9) to correct the most common variant (allele frequency 21.4%) in the phenylalanine hydroxylase (PAH) gene - c.1222C>T (p.Arg408Trp) - as an approach toward curing phenylketonuria (PKU). PKU is the most common inherited diseases in amino acid metabolism. It leads to severe neurological and neuropsychological symptoms if untreated or late diagnosed. Correction of the disease-causing variants could rescue residual PAH activity and restore normal function. Co-expression of a single guide RNA plasmid, a FokI-dCas9-zsGreen1 plasmid, and the presence of a single-stranded oligodeoxynucleotide in PAH_c.1222C>T COS-7 cells - an in vitro model for PKU - corrected the PAH variant and restored PAH activity. Also in this system, the HDR enhancer RS-1 improved correction efficiency. This proof-of-concept indicates the potential of the FokI-dCas9 system for precision medicine, in particular for targeting PKU and other monogenic metabolic diseases.
authors: Division of Functional Genome Analysis, German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors: Yi
authors: Y
authors: Pan
authors: Dietmar-Hopp-Metabolic Center, Department of General Pediatrics, University Hospital, Heidelberg, Germany.
authors: Nan
authors: N
authors: Shen
authors: Dietmar-Hopp-Metabolic Center, Department of General Pediatrics, University Hospital, Heidelberg, Germany.
authors: Sabine
authors: S
authors: Jung-Klawitter
authors: Department of General Pediatrics I, University Hospital, Heidelberg, Germany.
authors: Christian
authors: C
authors: Betzen
authors: Department of General Pediatrics I, University Hospital, Heidelberg, Germany.
authors: Georg F
authors: GF
authors: Hoffmann
authors: Division of Functional Genome Analysis, German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors: Jörg D
authors: JD
authors: Hoheisel
authors: Dietmar-Hopp-Metabolic Center, Department of General Pediatrics, University Hospital, Heidelberg, Germany.|University Children's Hospital, Zürich, Switzerland.
authors: Nenad
authors: N
authors: Blau
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics* / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D009154:Mutation; D010651:Phenylalanine Hydroxylase / Q000235:genetics* / Q000378:metabolism; D010661:Phenylketonurias / Q000209:etiology* / Q000235:genetics; D010957:Plasmids
chemical_list: D010651:Phenylalanine Hydroxylase; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific