Analysis of Recombinational Repair of DNA Double-Strand Breaks in Mammalian Cells With I-SceI Nuclease

Eukaryotes repair DNA double-strand breaks (DSBs) by homologous recombination (HR) or by nonhomologous end-joining (NHEJ). DSBs are a natural consequence of DNA metabolism, occurring, for example, during DNA replication and meiosis. DSBs are also induced by chemicals and radiation. I-Sce I endonuclease recognizes an 18-bp sequence with little degeneracy; therefore I-Sce I is highly specific, and its recognition sequence is predicted to occur by chance less than once in even the largest known genomes. As such, I-Sce I can be used to introduce a DSB into a defined (engineered) site in a mammalian chromosome, and this facilitates detailed studies of DSB repair. DSBs induced in repeated regions can be repaired by several different HR processes, including gene conversion with or without associated crossovers, or single-strand annealing. The specific types of HR events that can be scored depend on the configuration of the repeated regions and whether selection for recombinants is imposed. Non-selective assays detect both HR and NHEJ events. This chapter focuses on the systems for delivering I-Sce I nuclease to mammalian cells and the strategies for detecting various outcomes of DSB repair.