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 toward 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 multitube, 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, and (4) is cost-effective. More than 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 flexible and is compatible with standard molecular biology laboratory tools, but can be scaled 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 TALE toolkit. Together, the FusX system accelerates TALE-based genomic science applications from basic science screening work for functional genomics testing and molecular medicine applications.
Bibliographical noteFunding Information:
The authors thank Will Canine and the Open- Trons team for help in troubleshooting hardware and software glitches with the new liquid-handling robot system; Chris Ward, Colby Starker, Dan Carlson, and Dan Voytas for critical suggestions; and all the staff in the Mayo Clinic Zebrafish Facility and HKU Zebrafish Core Facility (FCF, LKS Faculty of Medicine) for excellent zebrafish husbandry. This work is supported by the State of Minnesota (University of Minnesota Mayo Clinic Partnership grant [H001274506] to SCE), National Institutes of Health grants [GM63904, P30DK084567] to SCE, [DA032194] grant to KJC, the Mayo Foundation, NIH Training grant [UL1 TR000135] to PRB and JMC, Marriott Mitochondrial Network, FHB HMRF  to ACM, and HKU Seed Funding Programme for Basic Research [201401159004, 201411159098] to ACM.
© Alvin C. Ma, et al., 2016; Published by Mary Ann Liebert, Inc. 2016.
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