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TAL Nucleases (Talns): Hybrid Proteins Composed of TAL Effectors and Foki DNA- Cleavage Domain Ting Li Iowa State University

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TAL Nucleases (Talns): Hybrid Proteins Composed of TAL Effectors and Foki DNA- Cleavage Domain Ting Li Iowa State University Genetics, Development and Cell Biology Genetics, Development and Cell Biology Publications 8-2010 TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA- cleavage domain Ting Li Iowa State University Sheng Huang Iowa State University Wen Zhi Jiang Iowa State University David Wright Iowa State University Martin H. Spalding Iowa State University, [email protected] Follow this and additional works at: http://lib.dr.iastate.edu/gdcb_las_pubs See next page for additional authors Part of the Genetics and Genomics Commons The ompc lete bibliographic information for this item can be found at http://lib.dr.iastate.edu/ gdcb_las_pubs/174. For information on how to cite this item, please visit http://lib.dr.iastate.edu/ howtocite.html. This Article is brought to you for free and open access by the Genetics, Development and Cell Biology at Iowa State University Digital Repository. It has been accepted for inclusion in Genetics, Development and Cell Biology Publications by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain Abstract DNA double-strand breaks enhance homologous recombination in cells and have been exploited for targeted genome editing through use of engineered endonucleases. Here we report the creation and initial characterization of a group of rare-cutting, site-specific DNA uclen ases produced by fusion of the restriction enzyme FokI endonuclease domain (FN) with the high-specificity DNA-binding domains of AvrXa7 and PthXo1. AvrXa7 and PthXo1 are members of the transcription activator-like (TAL) effector family whose central repeat units dictate target DNA recognition and can be modularly constructed to create novel DNA specificity. The yh brid FN-AvrXa7, AvrXa7-FN and PthXo1-FN proteins retain both recognition specificity for their target DNA (a 26 bp sequence for AvrXa7 and 24 bp for PthXo1) and the double-stranded DNA cleaving activity of FokI and, thus, are called TAL nucleases (TALNs). With all three TALNs, DNA is cleaved adjacent to the TAL-binding site under optimal conditions in vitro . When expressed in yeast, the TALNs promote DNA homologous recombination of a LacZ gene containing paired AvrXa7 or asymmetric AvrXa7/ PthXo1 target sequences. Our results demonstrate the feasibility of creating a tool box of novel TALNs with potential for targeted genome modification in organisms lacking facile mechanisms for targeted gene knockout and homologous recombination. Disciplines Genetics and Genomics Comments This article is published as Li, Ting, Sheng Huang, Wen Zhi Jiang, David Wright, Martin H. Spalding, Donald P. Weeks, and Bing Yang. "TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain." Nucleic acids research 39, no. 1 (2010): 359-372. 10.1093/nar/gkq704. Posted with permission. Creative Commons License This work is licensed under a Creative Commons Attribution-Noncommercial 4.0 License Authors Ting Li, Sheng Huang, Wen Zhi Jiang, David Wright, Martin H. Spalding, Donald P. Weeks, and Bing Yang This article is available at Iowa State University Digital Repository: http://lib.dr.iastate.edu/gdcb_las_pubs/174 Published online 10 August 2010 Nucleic Acids Research, 2011, Vol. 39, No. 1 359–372 doi:10.1093/nar/gkq704 TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain Ting Li1, Sheng Huang1, Wen Zhi Jiang2, David Wright1, Martin H. Spalding1, Donald P. Weeks2 and Bing Yang1,* 1Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA 50011, USA and 2Department of Biochemistry, University of Nebraska, Lincoln, NE 68588, USA Received June 5, 2010; Accepted July 23, 2010 ABSTRACT custom-engineered proteins that recognize long stretches of DNA sequences and their application for targeting DNA double-strand breaks enhance homologous specific genes for knockout or gene replacement (1). The recombination in cells and have been exploited for key component of these engineered nucleases is the DNA targeted genome editing through use of engineered recognition domain that is capable of precisely directing endonucleases. Here we report the creation and the nuclease to the target site for the purpose of initial characterization of a group of rare-cutting, introducing a DNA double-strand break (DSB). These site-specific DNA nucleases produced by fusion of breaks are principally repaired by one of two pathways, the restriction enzyme FokI endonuclease domain non-homologous end-joining (NHEJ) or homologous (FN) with the high-specificity DNA-binding domains recombination (HR). Repair by NHEJ often results in of AvrXa7 and PthXo1. AvrXa7 and PthXo1 are mutagenic deletions/insertions in the targeted gene. members of the transcription activator-like (TAL) Moreover, DSBs can stimulate HR between the endogen- ous target gene locus and an exogenously introduced hom- effector family whose central repeat units dictate ologous DNA fragment with desired genetic information, target DNA recognition and can be modularly a process called gene replacement or genome editing (2–4). constructed to create novel DNA specificity. The To date the most promising method involving genome hybrid FN-AvrXa7, AvrXa7-FN and PthXo1-FN editing is the use of custom-designed zinc finger nucleases proteins retain both recognition specificity for their (ZFNs) (5). ZFN technology involves the use of hybrid target DNA (a 26 bp sequence for AvrXa7 and 24 bp proteins derived from the DNA-binding domains of zinc for PthXo1) and the double-stranded DNA cleaving finger (ZF) proteins fused with the non-specific DNA- activity of FokI and, thus, are called TAL nucleases cleavage domain of the endonuclease FokI (6,7). (TALNs). With all three TALNs, DNA is cleaved FokI, a type IIS nuclease, was first isolated from the adjacent to the TAL-binding site under optimal bacterium Flavobacterium okeanokoites (8). The nuclease conditions in vitro. When expressed in yeast, the consists of two separate domains, an N-terminal DNA- binding domain and a C-terminal DNA-cleavage TALNs promote DNA homologous recombination domain. The DNA-binding domain recognizes the of a LacZ gene containing paired AvrXa7 or asym- non-palindromic sequence 50-GGATG-30 while the cata- metric AvrXa7/PthXo1 target sequences. Our results lytic domain cleaves double-stranded DNA non- demonstrate the feasibility of creating a tool box of specifically at a fixed distance of 9 and 13 nucleotides novel TALNs with potential for targeted genome downstream of the recognition site (9,10). FokI exists as modification in organisms lacking facile mechan- an inactive monomer in solution and becomes an active isms for targeted gene knockout and homologous dimer upon binding to its target DNA and in the presence recombination. of specific divalent metals (11). It has been proposed that to form a functional complex, two molecules of FokI each bind to their recognition sequence in a double INTRODUCTION stranded DNA molecule and then dimerize in such a way Perhaps the most significant application of endonucleases that the two nuclease domains unite to form a functional in the post genome era is their coupling with endonuclease that cleaves both DNA strands at *To whom correspondence should be addressed. Tel: +515 294 2968; Fax: +515 294 5256; Email: [email protected] The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. ß The Author(s) 2010. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Downloaded from https://academic.oup.com/nar/article-abstract/39/1/359/2409075/TAL-nucleases-TALNs-hybrid-proteins-composed-of by 04860000 user on 17 October 2017 360 Nucleic Acids Research, 2011, Vol. 39, No. 1 sites downstream of the FokI recognition sequence custom-design of novel TAL effectors with the required (12,13,14). specificity to target a single locus in a genome. ZFNs can be assembled as modules that are custom- AvrXa7 and PthXo1 are TAL Type III effectors from designed to recognize selected DNA sequences, typically Xanthomonas oryzae pv. oryzae (Xoo), the causal 18–24 bp with a 4–7 bp spacer between two half sites pathogen of bacterial blight of rice. They each contain a (15–17). Following binding at the preselected site, a DSB unique combination of RVDs in 26 and 24 repeats, re- is produced by the action of the ZFN’s FokI cleavage spectively (Figure 1B and C). For some Xoo strains, domain (4). ZFN technology has been successfully AvrXa7 is a key virulence factor in susceptible rice. On applied to genetic modification in a variety of organisms, the other hand, it is also an avirulence determinant in the including yeast, plants, mammals and even human cell otherwise resistant plants containing the cognate resist- lines [reviewed in (18,19)]. Despite the promise of ZFN ance gene Xa7 (30,31). As the essential virulence factor, technology in basic and applied research, widespread AvrXa7 activates the rice gene Os11N3 to induce a state of adoption of this technology has been hampered by a disease susceptibility (G.Anthony et al., manuscript in bottleneck in custom-engineering zinc fingers that preparation). The gene induction by AvrXa7 is mediated possess the requisite high specificity and affinity for prese- through its recognition of a specific DNA element within lected DNA target site. Such engineering is labor inten- the promoter region of Os11N3, an element we refer to sive, time consuming and associated with high rates of here as the effector binding element (EBE) (sequence failure (20,21). Since the effectiveness of these endonucle- shown in Figure 1B). Similarly, PthXo1 is also an essential ases depends almost solely on their DNA-binding specifi- virulence factor for some strains of X.
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