Generation of a triple‐gene knockout mammalian cell line using engineered zinc‐finger nucleases

Pei‐Qi Liu, Edmond M. Chan, Gregory J. Cost, Lin Zhang, Jianbin Wang, Jeffrey C. Miller, Dmitry Y. Guschin, Andreas Reik, Michael C. Holmes, John E. Mott, Trevor N. Collingwood, Philip D. Gregory

Mammalian cells with multi-gene knockouts could be of considerable utility in research, drug discovery, and cell-based therapeutics. However, existing methods for targeted gene deletion require sequential rounds of homologous recombination and drug selection to isolate rare desired events--a process sufficiently laborious to limit application to individual loci. Here we present a solution to this problem. Firstly, we report the development of zinc-finger nucleases (ZFNs) targeted to cleave three independent genes with known null phenotypes. Mammalian cells exposed to each ZFN pair in turn resulted in the generation of cell lines harboring single, double, and triple gene knockouts, that is, the successful disruption of two, four, and six alleles. All three biallelic knockout events were obtained at frequencies of >1% without the use of selection, displayed the expected knockout phenotype(s), and harbored DNA mutations centered at the ZFN binding sites. These data demonstrate the utility of ZFNs in multi-locus genome engineering.