1015. Assessment of Zinc Finger-FOKI Chimeric Endonuclease Activity Using a Single Strand Annealing Assay in Mammalian Cells

Abstract

Correction of the genetic mutations that underlie genetic diseases remains an essential goal of gene therapy. Repair by homologous recombination (HR) is a promising therapeutic strategy. In mammalian cells, HR is a rare event that can be stimulated by creating a double strand break (DSB) near the site to be modified. A DSB at specific genomic location can be accomplished using customized zinc finger proteins fused to a FokI endonuclease catalytic domain (ZFP-FOKI). To target non-palindromic sequences, two ZFPs engineered for distinct sequences are needed. Only the asymmetric complex involving the two ZFP-FOKI proteins is able to catalyze DNA cleavage at the specific non-palindromic sequence. To analyze and optimize ZFP-FOKI fusion protein activity in mammalian cells, we developed a cellular assay based on co-transfection of a plasmid- based single strand annealing (SSA) recombination substrate. The DSB generated by ZFP-FOKI at the specific site, allows restoration of luciferase reporter gene activity by homologous recombination. Here we report the development of an assay system to analyze ZFP-FOKI protein activity and toxicity in mammalian cells. Using an inverted gradient concentration of vectors expressing ZFP-FOKI, we are able to characterize conditions for optimal activity of the asymmetric complex and toxicity of the expressed ZFP-FOKI proteins. Correction of the genetic mutations that underlie genetic diseases remains an essential goal of gene therapy. Repair by homologous recombination (HR) is a promising therapeutic strategy. In mammalian cells, HR is a rare event that can be stimulated by creating a double strand break (DSB) near the site to be modified. A DSB at specific genomic location can be accomplished using customized zinc finger proteins fused to a FokI endonuclease catalytic domain (ZFP-FOKI). To target non-palindromic sequences, two ZFPs engineered for distinct sequences are needed. Only the asymmetric complex involving the two ZFP-FOKI proteins is able to catalyze DNA cleavage at the specific non-palindromic sequence. To analyze and optimize ZFP-FOKI fusion protein activity in mammalian cells, we developed a cellular assay based on co-transfection of a plasmid- based single strand annealing (SSA) recombination substrate. The DSB generated by ZFP-FOKI at the specific site, allows restoration of luciferase reporter gene activity by homologous recombination. Here we report the development of an assay system to analyze ZFP-FOKI protein activity and toxicity in mammalian cells. Using an inverted gradient concentration of vectors expressing ZFP-FOKI, we are able to characterize conditions for optimal activity of the asymmetric complex and toxicity of the expressed ZFP-FOKI proteins.