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

• DOI: 10.1002/bit.22654
• Publication Date: 2010-01-01T04:38:49Z
• Authors: 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. Colling...; Philip D. Gregory
• Keywords: Zinc finger nuclease; Gene knockout; Gene targeting; Genome Engineering; Null allele

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 homologous recombination selection to isolate rare desired events--a process sufficiently laborious limit application individual loci. Here we present a solution this problem. Firstly, report the development zinc-finger nucleases (ZFNs) cleave three independent genes known null phenotypes. exposed each ZFN pair turn resulted generation cell lines harboring single, double, triple knockouts, that is, successful disruption two, four, six alleles. All biallelic knockout events were obtained at frequencies >1% without use selection, displayed expected phenotype(s), harbored DNA mutations centered binding sites. These data demonstrate ZFNs multi-locus genome engineering.