A diversity of serine phage integrases mediate site-specific recombination in mammalian cells

Keravala, Annahita; Groth, Amy C.; Jarrahian, Sohail; Thyagarajan, Bhaskar; Hoyt, Jason J.; Kirby, Patrick J.; Calos, Michèle P.

doi:10.1007/s00438-006-0129-5

Cited by 94 publications

(99 citation statements)

References 45 publications

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“…By contrast, serine integrases use a doublestrand break mechanism during recombination and can have shorter attachment sites (∼50 bp). In addition, some serine integrases do not require host cofactors, a feature that has led to their successful reuse across a range of organisms (24). We thus chose to explore the engineering of rewritable genetic data storage systems using a bacteriophage serine integrase.…”

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confidence: 99%

Rewritable digital data storage in live cells via engineered control of recombination directionality

Bonnet

Subsoontorn²,

Endy³

2012

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

324

350

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The use of synthetic biological systems in research, healthcare, and manufacturing often requires autonomous history-dependent behavior and therefore some form of engineered biological memory. For example, the study or reprogramming of aging, cancer, or development would benefit from genetically encoded counters capable of recording up to several hundred cell division or differentiation events. Although genetic material itself provides a natural data storage medium, tools that allow researchers to reliably and reversibly write information to DNA in vivo are lacking. Here, we demonstrate a rewriteable recombinase addressable data (RAD) module that reliably stores digital information within a chromosome. RAD modules use serine integrase and excisionase functions adapted from bacteriophage to invert and restore specific DNA sequences. Our core RAD memory element is capable of passive information storage in the absence of heterologous gene expression for over 100 cell divisions and can be switched repeatedly without performance degradation, as is required to support combinatorial data storage. We also demonstrate how programmed stochasticity in RAD system performance arising from bidirectional recombination can be achieved and tuned by varying the synthesis and degradation rates of recombinase proteins. The serine recombinase functions used here do not require cell-specific cofactors and should be useful in extending computing and control methods to the study and engineering of many biological systems.DNA inversion | synthetic biology | genetic engineering | standard biological parts M ost engineered genetic data storage systems use auto-or cross-regulating bistable systems of transcription repressors or activators to define and hold state via continuous gene expression (1-4). Such epigenetic storage systems can be subject to evolutionary counter selection due to resource burdens placed on the host cell or spontaneous switching due to putatively stochastic fluctuations in cellular processes, including gene expression. Moreover, heterologous expression-based systems are difficult to redeploy given differences in gene regulatory mechanisms across organisms.Another approach for storing data inside organisms is to code extrinsic information within genetic material (5). Nucleic acids have undergone natural selection to serve as heritable data storage material in organismal lineages. Moreover, DNA provides attractive features in terms of data storage robustness, scalability, and stability (6). In addition, engineered transmission of DNA molecules could support data exchange between organisms as needed to implement higher-order multicellular behaviors within programmed consortia (6, 7).Practically, researchers have begun to use enzymes that modify DNA, typically site-specific recombinases, to study and control engineered genetic systems. For example, recombinases can catalyze strand exchange between specific DNA sequences and enable precise manipulation of DNA in vitro and in vivo (8). Depending on the relative location or...

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confidence: 99%

Rewritable digital data storage in live cells via engineered control of recombination directionality

Bonnet

Subsoontorn²,

Endy³

2012

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

324

350

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“…Serine integrases are being used in a variety of genetic engineering and synthetic biology applications (15)(16)(17)(18)(19)(20)(21)(22). A major attribute of serine integrases is that they perform unidirectional recombination, as regulated by their dedicated RDF.…”

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confidence: 99%

Control of Recombination Directionality by the Listeria Phage A118 Protein Gp44 and the Coiled-Coil Motif of Its Serine Integrase

et al. 2017

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The serine integrase of phage A118 catalyzes integrative recombination between attP on the phage and a specific attB locus on the chromosome of Listeria monocytogenes, but it is unable to promote excisive recombination between the hybrid attL and attR sites found on the integrated prophage without assistance by a recombination directionality factor (RDF). We have identified and characterized the phage-encoded RDF Gp44, which activates the A118 integrase for excision and inhibits integration. Gp44 binds to the C-terminal DNA binding domain of integrase, and we have localized the primary binding site to be within the mobile coiled-coil (CC) motif but distinct from the distal tip of the CC that is required for recombination. This interaction is sufficient to inhibit integration, but a second interaction involving the N-terminal end of Gp44 is also required to activate excision. We provide evidence that these two contacts modulate the trajectory of the CC motifs as they extend out from the integrase core in a manner dependent upon the identities of the four att sites. Our results support a model whereby Gp44 shapes the Int-bound complexes to control which att sites can synapse and recombine.IMPORTANCE Serine integrases mediate directional recombination between bacteriophage and bacterial chromosomes. These highly regulated site-specific recombination reactions are integral to the life cycle of temperate phage and, in the case of Listeria monocytogenes lysogenized by A118 family phage, are an essential virulence determinant. Serine integrases are also utilized as tools for genetic engineering and synthetic biology because of their exquisite unidirectional control of the DNA exchange reaction. Here, we identify and characterize the recombination directionality factor (RDF) that activates excision and inhibits integration reactions by the phage A118 integrase. We provide evidence that the A118 RDF binds to and modulates the trajectory of the long coiled-coil motif that extends from the large carboxyl-terminal DNA binding domain and is postulated to control the early steps of recombination site synapsis.KEYWORDS site-specific DNA recombination, serine integrase, recombination directionality factor, Listeria monocytogenes T emperate phage genomes integrate into and excise out of bacterial chromosomes using phage-specific site-specific recombination systems. Many phage integrases are members of a subfamily of serine recombinases (SRs) that are known as large SRs due to their very long C-terminal DNA binding regions relative to other SRs (1-4). In the few serine integrase systems that have been studied, phage-encoded recombination

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“…Integrase enzymes from a variety of sources have been used to catalyze integration of transgenes in heterologous systems (14,15). Integrases catalyze irreversible recombination between appropriate attB and attP sites (14,16).…”

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confidence: 99%

Site-specific integrase-mediated transgenesis in mice via pronuclear injection

Tasic

Hippenmeyer

Wang

et al. 2011

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

221

264

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Microinjection of recombinant DNA into zygotic pronuclei has been widely used for producing transgenic mice. However, with this method, the insertion site, integrity, and copy number of the transgene cannot be controlled. Here, we present an integrasebased approach to produce transgenic mice via pronuclear injection, whereby an intact single-copy transgene can be inserted into predetermined chromosomal loci with high efficiency (up to 40%), and faithfully transmitted through generations. We show that neighboring transgenic elements and bacterial DNA within the transgene cause profound silencing and expression variability of the transgenic marker. Removal of these undesirable elements leads to global high-level marker expression from transgenes driven by a ubiquitous promoter. We also obtained faithful marker expression from a tissue-specific promoter. The technique presented here will greatly facilitate murine transgenesis and precise structure/function dissection of mammalian gene function and regulation in vivo.

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A diversity of serine phage integrases mediate site-specific recombination in mammalian cells

Cited by 94 publications

References 45 publications

Rewritable digital data storage in live cells via engineered control of recombination directionality

Rewritable digital data storage in live cells via engineered control of recombination directionality

Control of Recombination Directionality by the Listeria Phage A118 Protein Gp44 and the Coiled-Coil Motif of Its Serine Integrase

Site-specific integrase-mediated transgenesis in mice via pronuclear injection

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