Diversity in the serine recombinases

We explored the potential of using a bacteriophage integrase system to achieve site-specific genome integration of murine phenylalanine hydroxylase cDNA in the livers of phenylketonuric (PKU) mice. The phiBT1 phage integrase is an enzyme that catalyses the efficient recombination between unique sequences in the phage and bacterial genomes, leading to the site-specific integration of the former into the latter in a unidirectional manner. Here we showed that this phage integrase functions efficiently in mouse cells, and several naturally occurring pseudo-attP sites located in the intergenic regions of the mouse genome have been identified and molecularly characterized. We further demonstrated that the addition of nuclear localization signal sequences to the C terminus of the phage integrase enhanced the efficiency for transgene integration into the mouse genome. Using this phage integration system, we delivered mouse phenylalanine hydroxylase cDNA to the livers of PKU mice by hydrodynamic injection of plasmid DNA and showed that the severity of the hyperphenylalaninemic phenotype in the treated mice decreased significantly. After three applications, serum phenylalanine levels in all treated PKU mice were reduced to the normal range and remained stable thereafter. Their fur color also changed from gray to black, indicating the reconstitution of melanin biosynthesis as a result of available tyrosine derived from reconstituted phenylalanine hydroxylation in the liver. Thus, the phiBT1 bacteriophage integrase represents an effective site-specific genome integration system in mammalian cells and can be of great value in DNA-mediated gene therapy for a multitude of genetic disorders.

mentioning

confidence: 99%

RETRACTED: Complete and persistent phenotypic correction of phenylketonuria in mice by site-specific genome integration of murine phenylalanine hydroxylase cDNA

Chen

Woo

2005

“…Additional biochemically independent RAD modules could likely be identified from the increasing set of known natural recombinases, (37)(38)(39) or perhaps by engineering synthetic integrase excisionase pairs with altered DNA recognition specificity (40)(41)(42). Such work, along with puzzles of integrating dozens of competing biochemical functions, suggest that engineering increased capacity in vivo data storage systems will help define and challenge the limits of synthetic biology.…”

Section: Discussionmentioning

confidence: 99%

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

Bonnet

Subsoontorn²,

Endy³

2012

326

350

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...

“…ZFRs are artificial DNA-modifying enzymes generated by fusing site-specific DNA-binding zincfinger proteins (ZFPs) to highly selective serine resolvase or invertase catalytic domains (21). A ZFR target site consists of two zinc-finger protein binding-sites (ZFBS) flanking a 20-bp core sequence recognized by the serine resolvase or invertase catalytic domain.…”

mentioning

confidence: 99%

Structure-guided reprogramming of serine recombinase DNA sequence specificity

Gaj

Mercer

Gersbach

et al. 2010

Routine manipulation of cellular genomes is contingent upon the development of proteins and enzymes with programmable DNA sequence specificity. Here we describe the structure-guided reprogramming of the DNA sequence specificity of the invertase Gin from bacteriophage Mu and Tn3 resolvase from Escherichia coli. Structure-guided and comparative sequence analyses were used to predict a network of amino acid residues that mediate resolvase and invertase DNA sequence specificity. Using saturation mutagenesis and iterative rounds of positive antibiotic selection, we identified extensively redesigned and highly convergent resolvase and invertase populations in the context of engineered zinc-finger recombinase (ZFR) fusion proteins. Reprogrammed variants selectively catalyzed recombination of nonnative DNA sequences >10,000-fold more effectively than their parental enzymes. Alanine-scanning mutagenesis revealed the molecular basis of resolvase and invertase DNA sequence specificity. When used as rationally designed ZFR heterodimers, the reprogrammed enzyme variants site-specifically modified unnatural and asymmetric DNA sequences. Early studies on the directed evolution of serine recombinase DNA sequence specificity produced enzymes with relaxed substrate specificity as a result of randomly incorporated mutations. In the current study, we focused our mutagenesis exclusively on DNA determinants, leading to redesigned enzymes that remained highly specific and directed transgene integration into the human genome with >80% accuracy. These results demonstrate that unique resolvase and invertase derivatives can be developed to site-specifically modify the human genome in the context of zinc-finger recombinase fusion proteins.gene targeting | protein engineering | site-specific recombination | zinc-finger recombinase S ite-specific recombinases are essential for a variety of diverse biological processes, including the integration and excision of viral genomes, the transposition of mobile genetic elements, and the regulation of gene expression (1). Recently, site-specific recombinases have emerged as powerful tools for advanced genome engineering (2, 3). The exquisite sequence specificities of recombination systems such as Cre/lox, FLP/FRT, and φC31/ att allow researchers to accurately modify genetic information for a variety of applications (4-6). However, DNA sequence constraints imposed by site-specific recombinases make routine modification of cellular genomes contingent on the presence of artificially introduced recognition sequences. As a result, a number of attempts have been made to circumvent or reprogram the strict DNA sequence specificities observed in these systems (7-9). Despite these efforts, engineered site-specific recombinase variants often exhibit considerably relaxed DNA sequence specificities (8, 10-14), a detrimental byproduct that often results in adverse off-target chromosomal modification (15-17). Thus, there is significant interest in the development of generalized protein engineering strategies capable...