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

Gaj, Thomas; Sirk, Shannon J.; Barbas, Carlos F.

doi:10.1002/bit.25096

Cited by 72 publications

(64 citation statements)

References 156 publications

(205 reference statements)

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“…54 Within this family of enzymes, the catalytic domain promotes DNA recombination, while the DNA-binding domain mediates target specificity, as both domains are structurally and functionally distinct. This modularity allows for replacement of the native DNA-binding domain with engineered zinc finger proteins.…”

Section: Recombinasesmentioning

confidence: 99%

Synthetic Zinc Finger Proteins: The Advent of Targeted Gene Regulation and Genome Modification Technologies

2014

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ConspectusThe understanding of gene regulation and the structure and function of the human genome increased dramatically at the end of the 20th century. Yet the technologies for manipulating the genome have been slower to develop. For instance, the field of gene therapy has been focused on correcting genetic diseases and augmenting tissue repair for more than 40 years. However, with the exception of a few very low efficiency approaches, conventional genetic engineering methods have only been able to add auxiliary genes to cells. This has been a substantial obstacle to the clinical success of gene therapies and has also led to severe unintended consequences in several cases. Therefore, technologies that facilitate the precise modification of cellular genomes have diverse and significant implications in many facets of research and are essential for translating the products of the Genomic Revolution into tangible benefits for medicine and biotechnology. To address this need, in the 1990s, we embarked on a mission to develop technologies for engineering protein–DNA interactions with the aim of creating custom tools capable of targeting any DNA sequence. Our goal has been to allow researchers to reach into genomes to specifically regulate, knock out, or replace any gene. To realize these goals, we initially focused on understanding and manipulating zinc finger proteins. In particular, we sought to create a simple and straightforward method that enables unspecialized laboratories to engineer custom DNA-modifying proteins using only defined modular components, a web-based utility, and standard recombinant DNA technology. Two significant challenges we faced were (i) the development of zinc finger domains that target sequences not recognized by naturally occurring zinc finger proteins and (ii) determining how individual zinc finger domains could be tethered together as polydactyl proteins to recognize unique locations within complex genomes. We and others have since used this modular assembly method to engineer artificial proteins and enzymes that activate, repress, or create defined changes to user-specified genes in human cells, plants, and other organisms. We have also engineered novel methods for externally controlling protein activity and delivery, as well as developed new strategies for the directed evolution of protein and enzyme function. This Account summarizes our work in these areas and highlights independent studies that have successfully used the modular assembly approach to create proteins with novel function. We also discuss emerging alternative methods for genomic targeting, including transcription activator-like effectors (TALEs) and CRISPR/Cas systems, and how they complement the synthetic zinc finger protein technology.

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Section: Recombinasesmentioning

confidence: 99%

Synthetic Zinc Finger Proteins: The Advent of Targeted Gene Regulation and Genome Modification Technologies

2014

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“…The estimated 450 human genomic sites identified in silico for recCas9 might be expanded substantially by replacing the Gin recombinase catalytic domain with other natural or manmade small serine recombinases that recognize different core sequences; many of these related enzymes have also been directed to novel sites via fusion to zinc finger proteins (19,63). Moreover, recent work altering Cas9 PAM binding specificity and the recent discovery of numerous Cas9 orthologs raise the possibility of further expanding the number of potential recCas9 sites (64–67).…”

Section: Discussionmentioning

confidence: 99%

“…Tyrosine and serine recombinases such as Cre, Flp and ΦC31 integrase have been widely used to catalyze the recombination of exogenous DNA into model organisms (18,19). However, the use of these enzymes has been limited by their intrinsic, non-programmable DNA sequence specificity.…”

Section: Introductionmentioning

confidence: 99%

“…Most small serine recombinases, for example, recognize a specific pseudo-palindromic core DNA sequence of approximately 20 base pairs (20). Recombination using these enzymes at endogenous DNA sequences only occurs at ‘pseudo-sites’ that resemble the recombinase's natural DNA recognition sequence or at genomic sequences for which the recombinase has been experimentally evolved (19,21–26). …”

Section: Introductionmentioning

confidence: 99%

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A programmable Cas9-serine recombinase fusion protein that operates on DNA sequences in mammalian cells

et al. 2016

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We describe the development of ‘recCas9’, an RNA-programmed small serine recombinase that functions in mammalian cells. We fused a catalytically inactive dCas9 to the catalytic domain of Gin recombinase using an optimized fusion architecture. The resulting recCas9 system recombines DNA sites containing a minimal recombinase core site flanked by guide RNA-specified sequences. We show that these recombinases can operate on DNA sites in mammalian cells identical to genomic loci naturally found in the human genome in a manner that is dependent on the guide RNA sequences. DNA sequencing reveals that recCas9 catalyzes guide RNA-dependent recombination in human cells with an efficiency as high as 32% on plasmid substrates. Finally, we demonstrate that recCas9 expressed in human cells can catalyze in situ deletion between two genomic sites. Because recCas9 directly catalyzes recombination, it generates virtually no detectable indels or other stochastic DNA modification products. This work represents a step toward programmable, scarless genome editing in unmodified cells that is independent of endogenous cellular machinery or cell state. Current and future generations of recCas9 may facilitate targeted agricultural breeding, or the study and treatment of human genetic diseases.

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“…There is a wide diversity of recombinase systems, both bidirectional and unidirectional, that differ in their underlying biochemical mechanisms [43, 44]. Unidirectional recombinase systems are composed of recombinase enzymes (and sometimes their cofactors) that catalyze one-time recombination between two recognition sites.…”

Section: Digital Circuitsmentioning

confidence: 99%

Digital and analog gene circuits for biotechnology

Roquet

2014

Biotechnology Journal

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Biotechnology offers the promise of valuable chemical production via microbial processing of renewable and inexpensive substrates. Thus far, static metabolic engineering strategies have enabled this field to advance industrial applications. However, the industrial scaling of statically engineered microbes inevitably creates inefficiencies due to variable conditions present in large-scale microbial cultures. Synthetic gene circuits that dynamically sense and regulate different molecules can resolve this issue by enabling cells to continuously adapt to variable conditions. These circuits also have the potential to enable next-generation production programs capable of autonomous transitioning between steps in a bioprocess. Here, we review the design and application of two main classes of dynamic gene circuits, digital and analog, for biotechnology. Within the context of these classes, we also discuss the potential benefits of digital-analog interconversion, memory, and multi-signal integration. Though synthetic gene circuits have largely been applied for cellular computation to date, we envision that utilizing them in biotechnology will enhance the efficiency and scope of biochemical production with living cells.

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

Cited by 72 publications

References 156 publications

Synthetic Zinc Finger Proteins: The Advent of Targeted Gene Regulation and Genome Modification Technologies

Synthetic Zinc Finger Proteins: The Advent of Targeted Gene Regulation and Genome Modification Technologies

A programmable Cas9-serine recombinase fusion protein that operates on DNA sequences in mammalian cells

Digital and analog gene circuits for biotechnology

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