Chimeric recombinases with designed DNA sequence recognition

Akopian, Aram; He, Jin; Boocock, Martin R.; Stark, W. Marshall

doi:10.1073/pnas.1533177100

Cited by 93 publications

(109 citation statements)

References 24 publications

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“…Exciting data are appearing from labs exploring the use of rare-cutting endonucleases such as I-SceI and zinc-finger/FokI chimeras that can cleave genomic DNA at fixed (and Targeted gene repair: progress and prospects H Parekh-Olmedo et al few) locations. [15][16][17][18][19] It is possible that by minimizing the number of DSBs, the gene repair frequency will be elevated without the concurrent pleiotropic effects. The events of the second phase where the 'correction' of the targeted mutant gene actually occurs are poorly understood.…”

Section: Prospectsmentioning

confidence: 99%

Gene therapy progress and prospects: targeted gene repair

et al. 2005

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The capacity to correct a mutant gene within the context of the chromosome holds great promise as a therapy for inherited disorders but fulfilling this promise has proven to be challenging. However, steady progress is being made and the development of gene repair as a viable and robust approach is underway. Here, we present some of the recent advances that are helping to shape our thinking about the feasibility and the limitations of this technique. For the most part, these advances center on understanding the regulation of the reaction and validating its application in animal models. Gene Therapy (2005) 12, 639-646.

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Section: Prospectsmentioning

confidence: 99%

Gene therapy progress and prospects: targeted gene repair

et al. 2005

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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.…”

mentioning

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%

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Synthesis of programmable integrases

Gordley

Gersbach

Barbas

2009

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

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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“…First and foremost are the development of new tools capable of introducing genomic modifications in the absence of DNA breaks. Targeted recombinases (Akopian et al 2003;Mercer et al 2012), which can be programmed to recognize specific DNA sequences (Gaj et al 2013;Sirk et al 2014;Wallen et al 2015) and even integrate therapeutic factors into the human genome (Gaj et al 2014b), are one such option. More recent work has indicated that single-base editing without DNA breaks can be achieved using an engineered Cas9 nickase complex (Komor et al 2016), although it remains unknown how effective this technology is in therapeutically relevant settings.…”

Section: Discussionmentioning

confidence: 99%

Genome-Editing Technologies: Principles and Applications

Gaj

Sirk

Shui

et al. 2016

Cold Spring Harb Perspect Biol

270

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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Chimeric recombinases with designed DNA sequence recognition

Cited by 93 publications

References 24 publications

Gene therapy progress and prospects: targeted gene repair

Gene therapy progress and prospects: targeted gene repair

Synthesis of programmable integrases

Genome-Editing Technologies: Principles and Applications

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