title: FusX: A Rapid One-Step Transcription Activator-Like Effector Assembly System for Genome Science.
abstract: 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.
authors: 1 Department of Biochemistry and Molecular Biology, Mayo Clinic , Rochester, Minnesota.|2 Department of Medicine, Li Ka Shing Faculty of Medicine, University of Hong Kong , Hong Kong.
authors: Alvin C
authors: AC
authors: Ma
authors: 1 Department of Biochemistry and Molecular Biology, Mayo Clinic , Rochester, Minnesota.
authors: Melissa S
authors: MS
authors: McNulty
authors: 1 Department of Biochemistry and Molecular Biology, Mayo Clinic , Rochester, Minnesota.
authors: Tanya L
authors: TL
authors: Poshusta
authors: 1 Department of Biochemistry and Molecular Biology, Mayo Clinic , Rochester, Minnesota.
authors: Jarryd M
authors: JM
authors: Campbell
authors: 3 Department of Physiology and Biomedical Engineering, Mayo Clinic , Rochester, Minnesota.
authors: Gabriel
authors: G
authors: Martínez-Gálvez
authors: 1 Department of Biochemistry and Molecular Biology, Mayo Clinic , Rochester, Minnesota.
authors: David P
authors: DP
authors: Argue
authors: 1 Department of Biochemistry and Molecular Biology, Mayo Clinic , Rochester, Minnesota.
authors: Han B
authors: HB
authors: Lee
authors: 1 Department of Biochemistry and Molecular Biology, Mayo Clinic , Rochester, Minnesota.
authors: Mark D
authors: MD
authors: Urban
authors: 1 Department of Biochemistry and Molecular Biology, Mayo Clinic , Rochester, Minnesota.
authors: Cassandra E
authors: CE
authors: Bullard
authors: 1 Department of Biochemistry and Molecular Biology, Mayo Clinic , Rochester, Minnesota.
authors: Patrick R
authors: PR
authors: Blackburn
authors: 2 Department of Medicine, Li Ka Shing Faculty of Medicine, University of Hong Kong , Hong Kong.
authors: Toni K
authors: TK
authors: Man
authors: 1 Department of Biochemistry and Molecular Biology, Mayo Clinic , Rochester, Minnesota.
authors: Karl J
authors: KJ
authors: Clark
authors: 1 Department of Biochemistry and Molecular Biology, Mayo Clinic , Rochester, Minnesota.
authors: Stephen C
authors: SC
authors: Ekker
mesh_terms: D000818:Animals; D004268:DNA-Binding Proteins / Q000378:metabolism; D000072669:Gene Editing; D005786:Gene Expression Regulation; D018390:Gene Targeting; D005818:Genetic Engineering / Q000379:methods*; D023281:Genomics / Q000379:methods*; D006801:Humans; D000071336:Transcription Activator-Like Effectors / Q000235:genetics*; D014157:Transcription Factors / Q000378:metabolism*; D015027:Zebrafish / Q000235:genetics / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D000071336:Transcription Activator-Like Effectors; D014157:Transcription Factors