title: A single-chain TALEN architecture for genome engineering.
abstract: Transcription-activator like effector nucleases (TALENs) are tailor-made DNA endonucleases and serve as a powerful tool for genome engineering. Site-specific DNA cleavage can be made by the dimerization of FokI nuclease domains at custom-targeted genomic loci, where a pair of TALENs must be positioned in close proximity with an appropriate orientation. However, the simultaneous delivery and coordinated expression of two bulky TALEN monomers (>100 kDa) in cells may be problematic to implement for certain applications. Here, we report the development of a single-chain TALEN (scTALEN) architecture, in which two FokI nuclease domains are fused on a single polypeptide. The scTALEN was created by connecting two FokI nuclease domains with a 95 amino acid polypeptide linker, which was isolated from a linker library by high-throughput screening. We demonstrated that scTALENs were catalytically active as monomers in yeast and human cells. The use of this novel scTALEN architecture should reduce protein payload, simplify design and decrease production cost.
authors: Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
authors: Ning
authors: N
authors: Sun
authors: Huimin
authors: H
authors: Zhao
mesh_terms: D001483:Base Sequence; D002384:Catalysis; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation; D015723:Gene Library; D023061:Gene Order; D005822:Genetic Vectors / Q000235:genetics; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D008969:Molecular Sequence Data; D015202:Protein Engineering; D055503:Protein Multimerization
chemical_list: D004247:DNA; C035042:endodeoxyribonuclease FokI; D003850:Deoxyribonuclease I; D015252:Deoxyribonucleases, Type II Site-Specific