Evolution of Programmable Zinc Finger-recombinases with Activity in Human Cells

Gordley, Russell M.; Smith, Justin D.; Gräslund, Torbjörn; Barbas, Carlos F.

doi:10.1016/j.jmb.2007.01.017

Cited by 61 publications

(92 citation statements)

References 66 publications

(77 reference statements)

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“…Synthetic zinc finger DNA-binding proteins have been generated for many sequences, using several approaches (15-17). Capitalizing on this work, researchers have incorporated synthetic zinc finger proteins into a wide variety of molecular tools (7, 18-29).The DBD of a hyperactive serine recombinase can be replaced with a zinc finger protein of higher affinity and specificity (7,25). This substitution retargets recombination to sequences flanked by zinc finger binding sites (ZFBS) (Fig.…”

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confidence: 99%

“…Our recent work with serine resolvases and invertases led us to hypothesize that we could use a modular approach that capitalizes on cooperative specificity to design synthetic enzymes that would uniquely recognize a single site within the 3 billion-base-pair human genome and allow us to deliver DNA specifically to this site ( Fig. 1A) (7).…”

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confidence: 99%

“…Synthetic zinc finger DNA-binding proteins have been generated for many sequences, using several approaches (15)(16)(17). Capitalizing on this work, researchers have incorporated synthetic zinc finger proteins into a wide variety of molecular tools (7,(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29).…”

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confidence: 99%

“…The DBD of a hyperactive serine recombinase can be replaced with a zinc finger protein of higher affinity and specificity (7,25). This substitution retargets recombination to sequences flanked by zinc finger binding sites (ZFBS) (Fig.…”

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confidence: 99%

“…The serine catalytic domain imposes its own sequence requirements on the interior of the RecZF target site (20-bp core, Fig. 1B) (7). Functional RecZF recombination sites must contain sequences compatible with both the zinc finger DNAbinding protein and recombinase catalytic domain.…”

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Synthesis of programmable integrases

Gordley

Gersbach

Barbas

2009

Proc. Natl. Acad. Sci. U.S.A.

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104

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Accurate modification of the 3 billion-base-pair human genome requires tools with exceptional sequence specificity. Here, we describe a general strategy for the design of enzymes that target a single site within the genome. We generated chimeric zinc finger recombinases with cooperative DNA-binding and catalytic specificities that integrate transgenes with >98% accuracy into the human genome. These modular recombinases can be reprogrammed: New combinations of zinc finger domains and serine recombinase catalytic domains generate novel enzymes with distinct substrate sequence specificities. Because of their accuracy and versatility, the recombinases/integrases reported in this work are suitable for a wide variety of applications in biological research, medicine, and biotechnology where accurate delivery of DNA is desired.recombinases ͉ zinc finger ͉ gene delivery ͉ gene targeting ͉ protein engineering T he postgenomic era of medicine will be defined by our ability to achieve biological control through genetic reprogramming. New tools are needed to accurately rewrite the genomic script and specifically alter genes, gene expression, and epigenetic state at any desired loci. To date, no enzyme-natural or synthetic-has been able to accurately modify only a single targeted site within the human genome (1). Scientists in biology, biotechnology, stem cell research, and gene therapy currently rely on naturally occurring enzymes to perform functions like DNA integration and excision. However, these enzymes recognize multiple sites within the human genome, often resulting in off-target DNA integration and chromosomal translocation (2-6). Our recent work with serine resolvases and invertases led us to hypothesize that we could use a modular approach that capitalizes on cooperative specificity to design synthetic enzymes that would uniquely recognize a single site within the 3 billion-base-pair human genome and allow us to deliver DNA specifically to this site (Fig. 1A) (7).In their native contexts, serine resolvases and invertases selectively recombine target sites within the same DNA molecule. This intramolecular specificity is assured by obligate assembly of large protein complexes, wherein accessory factors bound at neighboring sites impose topological and spatial constraints on the recombination reaction (8). Hyperactive mutants of several serine resolvases and invertases have been discovered that efficiently catalyze unrestricted recombination between minimal dimer-binding sites (Table S1) (9, 10). Furthermore, unlike other site-specific recombinases, serine resolvases and invertases are well suited to synthetic reengineering. These enzymes are modular in both structure and function, each comprised of a distinct catalytic domain flexibly tethered to a small helix-turnhelix DNA-binding domain (DBD). However, these DBDs are poorly suited for accurate genomic recombination (11) because the recognition motifs are short (4-6 bp) and degenerate (12, 13).In contrast, zinc finger DNA-binding proteins recognize target sites of v...

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Synthesis of programmable integrases

Gordley

Gersbach

Barbas

2009

Proc. Natl. Acad. Sci. U.S.A.

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104

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Metalloprotein Design and Engineering

Chakraborty

Hosseinzadeh

2014

Encyclopedia of Inorganic and Bioinorganic Chemistry

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This article covers recent advances in metalloprotein design, focusing on different approaches to their design. Impressive progress has been made in designing metal‐binding sites in peptides, de novo designed proteins, and native protein scaffolds. The design approach can be rational or combinatorial. Under rational design, redesigning an existing metal‐binding site to a new site with dramatically different structure and function complements well the design of new metal‐binding sites by revealing the role of specific residues responsible for a particular structural or functional feature of the metal‐binding site of interest. To create a new metal‐binding site, several approaches have been used, including design based on structural homology, by inspection, using automated computer search algorithms, or combination of the above approaches. In addition, a modular approach involving transplanting a conserved structural unit from one protein into another has also been shown to be effective. Design through combinatorial and evolutionary methods has also been successful, as it requires little prior knowledge of the protein structure. Finally, introducing unnatural amino acids or nonnative metal ions/prosthetic groups to expand the repertoires of metalloproteins has been demonstrated. Successful examples of each of the approaches are given, advantages and disadvantages of the approaches are discussed, and the outlook for future research is also presented.

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Expanding the scope of site‐specific recombinases for genetic and metabolic engineering

Gaj

Sirk

Barbas

2013

Biotech & Bioengineering

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Site-specific recombinases are tremendously valuable tools for basic research and genetic engineering. By promoting high-fidelity DNA modifications, site-specific recombination systems have empowered researchers with unprecedented control over diverse biological functions, enabling countless insights into cellular structure and function. The rigid target specificities of many sites-specific recombinases, however, have limited their adoption in fields that require highly flexible recognition abilities. As a result, intense effort has been directed toward altering the properties of site-specific recombination systems by protein engineering. Here, we review key developments in the rational design and directed molecular evolution of site-specific recombinases, highlighting the numerous applications of these enzymes across diverse fields of study.

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Evolution of Programmable Zinc Finger-recombinases with Activity in Human Cells

Cited by 61 publications

References 66 publications

Synthesis of programmable integrases

Synthesis of programmable integrases

Metalloprotein Design and Engineering

Expanding the scope of site‐specific recombinases for genetic and metabolic engineering

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