Ma, A.C., McNulty, M.S., Tanya, P.L., Jarryd, C.M., Martínez-Gálvez, G., Argue, D.P., Lee, H.B., Urban, M.D., Bullard, C.E., Blackburn, P.R., Man, T.K., Clark, K.J., Ekker, S.C. (2016) FusX: A rapid one-step TALE assembly system for genome science. Human gene therapy. 27(6):451-63.
Transcription activator-like effectors (TALEs) are extremely effective single-molecule DNA-targeting molecular cursors used for locus-specific genome science applications, including high precision molecular medicine and other genome engineering applications. TALEs are used in genome engineering for locus-specific DNA editing and imaging, as artificial transcriptional activators and repressors, and for targeted epigenetic modification. TALEs as nucleases (TALENs) are effective editing tools and offer high binding specificity and fewer sequence constraints towards the targeted genome than other custom nuclease systems. One bottleneck of broader TALE use is reagent accessibility. For example, one commonly deployed method uses a multi-tube, 5-day assembly protocol. Here we describe FusX, a streamlined Golden Gate TALE assembly system that (1) is backward compatible with popular TALE backbones, (2) is functionalized as a single-tube 3-day TALE assembly process (3) requires only commonly used basic molecular biology reagents (4) and is cost-effective. Over 100 TALEN pairs have been successfully assembled using FusX, and 27 pairs were quantitatively tested in zebrafish with each showing high somatic and germline activity. Furthermore, this assembly system is very flexible and is compatible with standard molecular biology lab tools but can be scaled using with automated laboratory support. To demonstrate, we use a highly accessible and commercially available liquid handling robot to rapidly and accurately assemble TALEs using the FusX systemTALE toolkit. Together, the FusX system accelerates TALE-based genomic science applications from basic science screening work for functional genomics testing and molecular medicine applications.