High-throughput cellular screening of engineered nuclease activity using the single-strand annealing assay and...

Engineered nucleases have been used to generate many model organisms and show great promise for therapeutic genome editing. Current methods to evaluate the activity of these nucleases can be laborious and often are hampered by readouts with small signals and a significant amount of background noise. We present a simple method that utilizes the established single-strand annealing (SSA) assay coupled with a luciferase assay to generate a high-throughput analysis of nuclease activity. Luciferase reporters provide a higher signal and lower background levels than fluorescent reporters. We engineered a commercially available luciferase plasmid (pGL4.51, Promega) to generate a set of nuclease target plasmids that produce a high signal and activity that correlates well with in vitro data. The SSA luciferase assay can discriminate between nucleases that give similar signals with other nuclease activity assays. The target plasmid and nucleases are transfected into cells and are generally cultured for 2 days. Luciferase activity is quantified in the same cell culture plate--streamlining the process from transfection to assay. We have used this robust process to investigate the activity of zinc finger nucleases (ZFNs) and transcription activated-like effector nucleases (TALENs).