Construction and Evaluation of Zinc Finger Nucleases

Zinc-finger nucleases (ZFNs) are programmable nucleases that have opened the door to the genome editing era. The construction of ZFNs recognizing a target sequence of interest is laborious, and has not been widely used recently. However, key ZFN patents are expiring over the next 2-4 years, enabling a wide range of deployments for clinical and industrial applications. This article introduces a ZFN construction protocol that uses bacterial one-hybrid (B1H) selection and single-stranded annealing (SSA) assay.