Description
Homologous recombination (HR) is considered the "gold standard" in methods for gene-targeting, and has been used to generate many knock-out and knock-in mice essential in understanding gene function and disease. The establishment of mouse embryonic stem (ES) cell lines has allowed for positive selection of ES cells in which HR has occurred, thus circumventing a cardinal disadvantage of HR; low frequency of occurrence. ES cell lines of many organisms are unavailable, therefore, other strategies are necessary to make genomic alterations. Here we show evidence that zinc finger nuclease (ZFN) technology is an effective means to make precise genetic modifications in the model organism, Ciona intestinalis. ZFNs are chimeric proteins consisting of an engineered DNA-binding domain and the nonspecific endonuclease, FokI, that when bound to genomic targets as heterodimers, can induce double stranded breaks (DSB). If error-prone non homologous end joining (NHEJ) incorporates indels at the target site, a frame shift may occur, thus disrupting the targeted gene. As a proof of concept, we assembled ZFNs through two methods, modular design and randomized combinatorial libraries, that target tyrosinase, a catalytic protein essential for the production of melanin in the pigmented cells of Ciona larvae. When expressing the ZFNs in pigmented cells, we observe high frequencies of albinism, the expected phenotype if tyrosinase is disrupted via NHEJ errors. A targeting construct was designed to insert GFP in frame with tyrosinase, at the ZFN target site, if used as a template in HR-mediated DSB repair. When including the targeting construct along with ZFNs, we observe embryos at frequencies up to 15%, that express GFP in their sensory vesicles. These results suggest that ZFN induced DSBs can stimulate recombination frequencies in Ciona at levels that should allow for the future generation of stable knock-out lines
