Inhibition of Human Immunodeficiency Virus Type 1 Replication with Artificial Transcription Factors Targeting the Highly Conserved Primer-Binding Site

Eberhardy, Scott R.; Gonçalves, João; Coelho, Sofia; Segal, David J.; Berkhout, Ben; Barbas, Carlos F.

doi:10.1128/jvi.80.6.2873-2883.2006

Cited by 49 publications

(49 citation statements)

References 61 publications

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“…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).…”

mentioning

confidence: 99%

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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confidence: 99%

Synthesis of programmable integrases

Gordley

Gersbach

Barbas

2009

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

Self Cite

104

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“…ZF-TFs have been previously used in strategies to control replication and/or infection by viruses, including the DNA virus herpes simplex virus and the retrovirus HIV (Eberhardy et al 2006;Kim et al 2005;Papworth et al 2003;Reynolds et al 2003;Segal et al 2004), in mammalian cells. Sera (2005) described a ZF-TF that restricts the replication of the single-stranded-DNA-containing Geminivirus beet curly top virus by binding to the origin of replication.…”

Section: Discussionmentioning

confidence: 99%

Negative regulation of the RTBV promoter by designed zinc finger proteins

et al. 2010

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The symptoms of rice tungro disease are caused by infection by a DNA-containing virus, rice tungro bacilliform virus (RTBV). To reduce expression of the RTBV promoter, and to ultimately reduce virus replication, we tested three synthetic zinc finger protein transcription factors (ZF-TFs), each comprised of six finger domains, designed to bind to sequences between -58 and +50 of the promoter. Two of these ZF-TFs reduced expression from the promoter in transient assays and in transgenic Arabidopsis thaliana plants. One of the ZF-TFs had significant effects on plant regeneration, apparently as a consequence of binding to multiple sites in the A. thaliana genome. Expression from the RTBV promoter was reduced by approximately 45% in transient assays and was reduced by up to 80% in transgenic plants. Co-expression of two different ZF-TFs did not further reduce expression of the promoter. These experiments suggest that ZF-TFs may be used to reduce replication of RTBV and thereby offer a potential method for control of an important crop disease.

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“…Early work on the use of engineered zinc-finger transcription factors revealed that synthetic transcriptional modulators are effective tools for a broad range of applications, enabling such tasks as inhibiting viral replication (Papworth et al 2003;Reynolds et al 2003;Segal et al 2004;Eberhardy et al 2006), modulating the expression of disease-associated loci (Graslund et al 2005;Wilber et al 2010), inducing angiogenesis for accelerated wound healing (Rebar et al 2002), and genomic screening of cellular targets for cancer progression and drug resistance Blancafort et al 2005Blancafort et al , 2008. Facilitated by many of the insights gained from zinc-finger transcription factor technology, both TALEs and CRISPR-Cas9 have now further expanded the possibilities of engineered transcriptional activators and repressors.…”

Section: Applications Of Targeted Transcriptional Regulationmentioning

confidence: 99%

Genome-Editing Technologies: Principles and Applications

Gaj

Sirk

Shui

et al. 2016

Cold Spring Harb Perspect Biol

297

142

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Targeted nucleases have provided researchers with the ability to manipulate virtually any genomic sequence, enabling the facile creation of isogenic cell lines and animal models for the study of human disease, and promoting exciting new possibilities for human gene therapy. Here we review three foundational technologies-clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9), transcription activator-like effector nucleases (TALENs), and zinc-finger nucleases (ZFNs). We discuss the engineering advances that facilitated their development and highlight several achievements in genome engineering that were made possible by these tools. We also consider artificial transcription factors, illustrating how this technology can complement targeted nucleases for synthetic biology and gene therapy.

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Inhibition of Human Immunodeficiency Virus Type 1 Replication with Artificial Transcription Factors Targeting the Highly Conserved Primer-Binding Site

Cited by 49 publications

References 61 publications

Synthesis of programmable integrases

Synthesis of programmable integrases

Negative regulation of the RTBV promoter by designed zinc finger proteins

Genome-Editing Technologies: Principles and Applications

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