Retargeting Sleeping Beauty Transposon Insertions by Engineered Zinc Finger DNA-binding Domains

摘要: The Sleeping Beauty (SB) transposon is a nonviral, integrating vector system with proven efficacy in preclinical animal models, and thus holds promise for future clinical applications. However, SB has close-to-random insertion profile that could lead to genotoxic effects, thereby presenting potential safety issue. We evaluated zinc finger (ZF) DNA-binding domains (DBDs) their abilities introduce bias into SB's profile. E2C, binds unique site the erbB-2 gene, mediated locus-specific insertions at low frequencies. A novel ZF targeting LINE1 repeats, ZF-B, showed specific binding an 18-bp represented by ~12,000 copies human genome. mapped using linear-amplification (LAM)-PCR Illumina sequencing. Targeted ZF-B peaked approximately fourfold enrichment of transposition around sites yielding ~45% overall frequency LINE1. decrease dataset respect genes was found, suggesting repeats act as sponge “soak up” fraction redirect them away from genes. Improvements technology careful choice targeted genomic regions may improve