Site-specific Recombination System Research Articles

N-methyl-D-aspartate Receptor Subunits 2A and 2B Mediate Connexins and Pannexins in the Trigeminal Ganglion Involved in Orofacial Inflammatory Allodynia during Temporomandibular Joint Inflammation.

Temporomandibular joint osteoarthritis (TMJOA) is a severe form of temporomandibular joint disorders (TMD), and orofacial inflammatory allodynia is one of its common symptoms which lacks effective treatment. N-methyl-D-aspartate receptor (NMDAR), particularly its subtypes GluN2A and GluN2B, along with gap junctions (GJs), are key players in the mediation of inflammatory pain. However, the precise regulatory mechanisms of GluN2A, GluN2B, and GJs in orofacial inflammatory allodynia during TMJ inflammation still remain unclear. Here, we established the TMJ inflammation model by injecting Complete Freund's adjuvant (CFA) into the TMJ and used Cre/loxp site-specific recombination system to conditionally knock out (CKO) GluN2A and GluN2B in the trigeminal ganglion (TG). Von-frey test results indicated that CFA-induced mechanical allodynia in the TMJ region was relieved in GluN2A and GluN2B deficient mice. In vivo, CFA significantly up-regulated the expression of GluN2A and GluN2B, Gjb1, Gjb2, Gjc2 and Panx3 in the TG, and GluN2A and GluN2B CKO played different roles in mediating the expression of Gjb1, Gjb2, Gjc2 and Panx3. In vitro, NMDA up-regulated the expression of Gjb1, Gjb2, Gjc2 and Panx3 in satellite glial cells (SGCs) as well as promoted the intercellular communication between SGCs, and GluN2A and GluN2B knocking down (KD) altered the expression and function differently. NMDAR regulated Gjb1 and Panx3 through ERK1/2 pathway, and mediated Gjb2 and Gjc2 through MAPK, PKA, and PKC intracellular signaling pathways. These findings shed light on the distinct functions of GluN2A and GluN2B in mediating peripheral sensitization induced by TMJ inflammation in the TG, offering potential therapeutic targets for managing orofacial inflammatory allodynia.

Rational construction of a high-quality and high-efficiency biosynthetic system and fermentation optimization for A82846B based on combinatorial strategies in Amycolatopsis orientalis

BackgroundOritavancin is a new generation of semi-synthetic glycopeptide antibiotics against Gram-positive bacteria, which served as the first and only antibiotic with a single-dose therapeutic regimen to treat ABSSSI. A naturally occurring glycopeptide A82846B is the direct precursor of oritavancin. However, its application has been hampered by low yields and homologous impurities. This study established a multi-step combinatorial strategy to rationally construct a high-quality and high-efficiency biosynthesis system for A82846B and systematically optimize its fermentation process to break through the bottleneck of microbial fermentation production.ResultsFirstly, based on the genome sequencing and analysis, we deleted putative competitive pathways and constructed a better A82846B-producing strain with a cleaner metabolic background, increasing A82846B production from 92 to 174 mg/L. Subsequently, the PhiC31 integrase system was introduced based on the CRISPR-Cas12a system. Then, the fermentation level of A82846B was improved to 226 mg/L by over-expressing the pathway-specific regulator StrR via the constructed PhiC31 system. Furthermore, overexpressing glycosyl-synthesis gene evaE enhanced the production to 332 mg/L due to the great conversion of the intermediate to target product. Finally, the scale-up production of A82846B reached 725 mg/L in a 15 L fermenter under fermentation optimization, which is the highest reported yield of A82846B without the generation of homologous impurities.ConclusionUnder approaches including blocking competitive pathways, inserting site-specific recombination system, overexpressing regulator, overexpressing glycosyl-synthesis gene and optimizing fermentation process, a multi-step combinatorial strategy for the high-level production of A82846B was developed, constructing a high-producing strain AO-6. The combinatorial strategies employed here can be widely applied to improve the fermentation level of other microbial secondary metabolites, providing a reference for constructing an efficient microbial cell factory for high-value natural products.

Redefining the bacteriophage mv4 site-specific recombination system and the sequence specificity of its attB and core-attP sites.

Through their involvement in the integration and excision of a large number of mobile genetic elements, such as phages and integrative and conjugative elements (ICEs), site-specific recombination systems based on heterobivalent tyrosine recombinases play a major role in genome dynamics and evolution. However, despite hundreds of these systems having been identified in genome databases, very few have been described in detail, with none from phages that infect Bacillota (formerly Firmicutes). In this study, we reanalyzed the recombination module of Lactobacillus delbrueckii subsp. bulgaricus phage mv4, previously considered atypical compared with classical systems. Our results reveal that mv4 integrase is a 369 aa protein with all the structural hallmarks of recombinases from the Tn916 family and that it cooperatively interacts with its recombination sites. Using randomized DNA libraries, NGS sequencing, and other molecular approaches, we show that the 21-bp core-attP and attB sites have structural similarities to classical systems only if considering the nucleotide degeneracy, with two 7-bp inverted regions corresponding to mv4Int core-binding sites surrounding a 7-bp strand-exchange region. We also examined the different compositional constraints in the core-binding regions, which define the sequence space of permissible recombination sites.

Construction and application of a multifunctional CHO cell platform utilizing Cre/lox and Dre/rox site-specific recombination systems.

During the development of traditional Chinese hamster ovary (CHO) cell lines, target genes randomly integrate into the genome upon entering the nucleus, resulting in unpredictable productivity of cell clones. The characterization and screening of high-yielding cell lines is a time-consuming and expensive process. Site-specific integration is recognized as an effective approach for overcoming random integration and improving production stability. We have designed a multifunctional expression cassette, called CDbox, which can be manipulated by the site-specific recombination systems Cre/lox and Dre/rox. The CDbox expression cassette was inserted at the Hipp11(H11) locus hotspot in the CHO-K1 genome using CRISPR/Cas9 technology, and a compliant CHO-CDbox cell platform was screened and obtained. The CHO-CDbox cell platform was transformed into a pool of EGFP-expressing cells using Cre/lox recombinase-mediated cassette exchange (RMCE) in only 2weeks, and this expression remained stable for at least 75 generations without the need for drug stress. Subsequently, we used the Dre/rox system to directly eliminate the EGFP gene. In addition, two practical applications of the CHO-CDbox cell platform were presented. The first was the quick construction of the Pembrolizumab antibody stable expression strain, while the second was a protocol for the integration of surface-displayed and secreted antibodies on CHO cells. The previous research on site-specific integration of CHO cells has always focused on the single functionality of insertion of target genes. This newly developed CHO cell platform is expected to offer expanded applicability for protein production and gene function studies.

An Optogenetics-based Approach to Regulate Colonic Contractions by Modulating the Activity of the Interstitial Cells of Cajal in Mice.

The interstitial cells of Cajal (ICC) are pacemaker cells in the gastrointestinal (GI) tract. We examined whether the activity of ICC could be stimulated to control colonic contractions. An optogenetics-based mouse model in which the light-sensitive protein channelrhodopsin-2 (ChR2) was expressed was used to accomplish cell specific, direct stimulation of ICC. An inducible site-specific Cre-loxP recombination system was used to generate KitCreERT2/+;ROSAChR2(H134R)/tdTomato/+ mice in which ChR2(H134R), a variant of ChR2, was genetically expressed in ICC after tamoxifen administration. Genotyping and immunofluorescence analysis were performed to confirm gene fusion and expression. Isometric force recordings were performed to measure changes in contractions in the colonic muscle strips. ChR2 was specifically expressed in Kit-labeled ICC. The isometric force recordings showed that the contractions of the colonic muscle strips changed under 470 nm blue light. Light stimulation evoked premature low-frequency and high amplitude (LFHA) contractions and enhanced the frequency of the LFHA contractions. The light-evoked contractions were blocked by T16Ainh-A01, an antagonist of anoctamin 1 channels that are expressed selectively in ICC in colonic muscles. Our study demonstrates a potentially feasible approach to stimulate the activity of ICC by optogenetics. The colonic motor patterns of muscle strips, especially LFHA contractions, can be regulated by 470 nm light via ChR2, which is expressed in ICC.

VCre/VloxP and SCre/SloxP as Reliable Site-Specific Recombination Systems for Genome Engineering.

The Cre/loxP system is a versatile and powerful tool that has been used to develop many kinds of genetically modified mice, such as conditional knockout mice and mutant protein-expressing mice through the excision of a STOP cassette. However, while numerous in vivo and in vitro applications of the Cre/loxP system have been reported, it remains difficult to target at one time more than one set of recognition sites in an identical single cell in mice using the Cre/loxP system. To overcome this barrier, we developed two novel site-specific recombination systems called VCre/VloxP and SCre/SloxP. These systems allow multiple independent site-specific recombination, for example, multiple targeted deletions in the same cell at different times. In this chapter, I describe the features of VCre/VloxP and SCre/SloxP, practical protocols and tips on how to use them in genomic engineering applications, potential problems in their use, and how problems can be identified and solved.

Structural basis for topological regulation of Tn3 resolvase.

Site-specific DNA recombinases play a variety of biological roles, often related to the dissemination of antibiotic resistance, and are also useful synthetic biology tools. The simplest site-specific recombination systems will recombine any two cognate sites regardless of context. Other systems have evolved elaborate mechanisms, often sensing DNA topology, to ensure that only one of multiple possible recombination products is produced. The closely related resolvases from the Tn3 and γδtransposons have historically served as paradigms for the regulation of recombinase activity by DNA topology. However, despite many proposals, models of the multi-subunit protein-DNA complex (termed the synaptosome) that enforces this regulation have been unsatisfying due to a lack of experimental constraints and incomplete concordance with experimental data. Here, we present new structural and biochemical data that lead to a new, detailed model of the Tn3 synaptosome, and discuss how it harnesses DNA topology to regulate the enzymatic activity of the recombinase.

Identification and Characterisation of pST1023 A Mosaic, Multidrug-Resistant and Mobilisable IncR Plasmid.

We report the identification and characterisation of a mosaic, multidrug-resistant and mobilisable IncR plasmid (pST1023) detected in Salmonella ST1023, a monophasic variant 4,[5],12:i: strain of widespread pandemic lineage, reported as a Southern European clone. pST1023 contains exogenous DNA regions, principally gained from pSLT-derivatives and IncI1 plasmids. Acquisition from IncI1 included oriT and nikAB and these conferred the ability to be mobilisable in the presence of a helper plasmid, as we demonstrated with the conjugative plasmids pST1007-1D (IncFII) or pVC1035 (IncC). A sul3-associated class 1 integron, conferring resistance to aminoglycosides, chloramphenicol and trimethoprim-sulphonamides, was also embedded in the acquired IncI1 DNA segment. pST1023 also harboured an additional site-specific recombination system (rfsF/rsdB) and IS elements of the IS1, IS5 (IS903 group) and IS6 families. Four of the six IS26 elements present constituted two pseudo-compound-transposons, named PCT-sil and PCT-Tn10 (identified here for the first time). The study further highlighted the mosaic genetic architecture and the clinical importance of IncR plasmids. Moreover, it provides the first experimental data on the ability of IncR plasmids to be mobilised and their potential role in the horizontal spread of antimicrobial-resistant genes.

Genome engineering of the Corynebacterium glutamicum chromosome by the Extended Dual-In/Out strategy

A novel genome editing method for repeated introduction of foreign DNA, including insertion of rather large DNA fragments, into predesigned points in the Corynebacterium glutamicum chromosome was developed. The method is based on the implementation of the Dual-In/Out strategy, which was previously provided in Escherichia coli according to recombineering-based methods (Minaeva et al., 2008) and allowed step-by-step construction of marker-less plasmid free recombinant strains. The strategy, suggested in the current study, is based on (i) E. coli Rac prophage RecE564/RecT-dependent recombineering; (ii) corynephage ϕ16 (Int/Xis)- and E. coli phage P1 Cre-mediated site-specific recombination systems; and (iii) the development of a C. glutamicum electrotransformation protocol with donor chromosomal DNA for combining of obtained modifications. It was found, that for each tested C. glutamicums strain, the efficiency of the different modifications for electrotransformation fluctuated significantly (up to two orders of magnitude), likely due to the recombinogenic accessibility of the corresponding locus of the bacterial chromosome. To avoid this difficulty, we proposed the phage Mu-driven transposition as a powerful approach for pre-selection of chromosomal regions convenient for single insertions and their further combination in a one strain. Additionally, it was found that the expression of RecE564/RecT coding genes in the recipient strain facilitated the inheritance of the penetrated DNA. It is proposed that the developed strategy in general and its separate elements should be helpful for broadening the genetic toolbox needed for genome editing of targeted C. glutamicum strains.

Construction of chimeric viruses based on pepper mild mottle virus using a modified Cre/loxP system

Cre/loxP, a site-specific recombination system, has been widely used for various purposes, including chromosomal translocations, generation of marker-free transgenic plants, tissue-specific activation of a reporter gene and efficient heterologous gene expression in plants. However, stable or transient expression of Cre recombinase in plants can cause chlorosis or necrosis. Here, we describe a modified Cre/loxP recombination system using a DNA fragment flanked with loxP sites in the same orientation in which necrosis induced by Cre recombinase in Nicotiana benthamiana leaves was alleviated. The modified system was successfully used to create functional GFP-tagged pepper mild mottle virus (PMMoV) and a chimeric virus with coat protein (CP) substitution assembled from separate pro-vector modules. Our results provide a new strategy and flexible technique to construct chimeric virus and infectious clones for plant viruses with large genomes.

Predicting algorithm of attC site based on combination optimization strategy

Site-specific recombination systems are widely used as bioengineering tools. However, the traditional site-specific recombination system requires a consensus sequence for the specific site. Such sequence-level constraints limit effective recombination between sites. Therefore, in order to develop an efficient site-specific recombination system, we investigated the attC site of the bacterial integration subsystem and built a predictive model to infer the important features that contribute to recombination. Here, we design an attC site prediction algorithm based on a combination optimisation strategy. Based on the structural features of attC sites, the prediction algorithm realises the high-precision prediction of the recombination frequencies between sites and the screening of the top 20 important features that play a role in recombination, which are effective for improving the design method of attC sites. The algorithm has better portability and higher prediction accuracy compared with the existing advanced algorithms, among which the Pearson correlation coefficient is 0.87, explained variance score is 0.73, root mean square error is 0.006 and mean absolute error is 0.041. This can not only provide ideas for the research of efficient recombination systems but also provide a theoretical basis for developing genetic engineering further.

Activation of the N-methyl-D-aspartate receptor contributes to orofacial neuropathic and inflammatory allodynia by facilitating calcium-calmodulin-dependent protein kinase II phosphorylation in mice

Neuropathic and inflammatory pain are major clinical challenges due to their ambiguous mechanisms and limited treatment approaches. N-methyl-D-aspartate receptor (NMDAR) and calcium-calmodulin-dependent protein kinase II (CaMKII) are responsible for nerve system sensation and are required for the induction and maintenance of pain. However, the roles of NMDAR and CaMKII in regulating orofacial pain are still less well known. Here, we established a neuropathic pain model by transecting a mouse inferior alveolar nerve (IAN) and an inflammatory pain model by injecting complete Freund’s adjuvant (CFA) into its whisker pad. The Cre/loxp site-specific recombination system was used to conditionally knock out (KO) NR2B in the trigeminal ganglion (TG). Von Frey filament behavioral tests showed that IANX and CFA-induced mechanical allodynia were altered in NR2B-deficient mice. CFA upregulated CaMKIIα and CaMKIIβ in the mouse TG and spinal trigeminal caudate nucleus (SpVc). CaMKIIα first decreased and then increased in the TG after IANX, and CaMKIIβ decreased in the TG and SpVc. CFA and IANX both greatly enhanced the expression of phospho (p)-NR2B, p-CaMKII, cyclic adenosine monophosphate (cAMP), p-ERK, and p-cAMP response element binding protein (CREB) in the TG and SpVc. These neurochemical signal pathway alterations were reversed by the conditional KO of NR2B and inhibition of CaMKII. Similarly, IANX- and CFA-related behavioral alterations were reversed by intra-ganglionic (i.g.) -application of inhibitors of CaMKII, cAMP, and ERK. These findings revealed novel molecular signaling pathways (NR2B-CaMKII-cAMP-ERK-CREB) in the TG- and SpVc-derived latent subsequent peripheral and spinal central sensitization under nerve injury and inflammation, which might be beneficial for the treatment of orofacial allodynia.

Marker-Free Genome Engineering in Amycolatopsis Using the pSAM2 Site-Specific Recombination System.

Actinobacteria of the genus Amycolatopsis are important for antibiotic production and other valuable biotechnological applications such as bioconversion or bioremediation. Despite their importance, tools and methods for their genetic manipulation are less developed than in other actinobacteria such as Streptomyces. We report here the use of the pSAM2 site-specific recombination system to delete antibiotic resistance cassettes used in gene replacement experiments or to create large genomic deletions. For this purpose, we constructed a shuttle vector, replicating in Escherichia coli and Amycolatopsis, expressing the integrase and the excisionase from the Streptomyces integrative and conjugative element pSAM2. These proteins are sufficient for site-specific recombination between the attachment sites attL and attR. We also constructed two plasmids, replicative in E. coli but not in Amycolatopsis, for the integration of the attL and attR sites on each side of a large region targeted for deletion. We exemplified the use of these tools in Amycolatopsis mediterranei by obtaining with high efficiency a marker-free deletion of one single gene in the rifamycin biosynthetic gene cluster or of the entire 90-kb cluster. These robust and simple tools enrich the toolbox for genome engineering in Amycolatopsis.

Application of the FLP/LoxP-FRT recombination system to switch the eGFP expression in a model prokaryote.

In prokaryotes, few studies have applied the flippase (FLP)/P1-flippase recombination target (LoxP-FRT) recombination system to switch gene expression. This study developed a new method for switching gene expression by constructing an FLP/LoxP-FRT site-specific recombination system in Escherichia coli. To this end, we placed the Nos terminator flanked by a pair of LoxP-FRT in front of enhanced green fluorescent protein (eGFP). The Nos terminator was used to block the expression of the eGFP. When a plasmid expressing FLP was available, deletion of the Nos terminator would allow expression of eGFP. The regulatory effect was demonstrated by eGFP expression. The efficiency of the gene switch was calculated as high as 89.67%. The results showed that the FLP/LoxP-FRT recombinase system could be used as a gene switch to regulate gene expression in prokaryotes. This new method for switching gene expression could simplify the gene function analysis in E. coli and other prokaryotes, as well as eukaryotes.

New<i> Bacillus subtilis</i> vector, pSSβ, as genetic tool for site-specific integration and excision of cloned DNA, and prophage elimination

Site-specific recombination (SSR) systems are employed in many genetic mobile elements, including temperate phages, for their integration and excision. Recently, they have also been used as tools for applications in fields ranging from basic to synthetic biology. SPβ is a temperate phage of the Siphoviridae family found in the laboratory standard Bacillus subtilis strain 168. SPβ encodes a serine-type recombinase, SprA, and recombination directionality factor (RDF), SprB. SprA catalyzes recombination between the attachment site of the phage, attP, and that of the host, attB, to integrate phage genome into the attB site of the host genome and generate attL and attR at both ends of the prophage genome. SprB works in conjunction with SprA and switches from attB/attP to attL/R recombination, which leads to excision of the prophage. In the present study, we took advantage of this highly efficient recombination system to develop a site-specific integration and excision plasmid vector, named pSSβ. It was constructed using pUC plasmid and the SSR system components, attP, sprA and sprB of SPβ. pSSβ was integrated into the attB site with a significantly high efficiency, and the resulting pSSβ integrated strain also easily eliminated pSSβ itself from the host genome by the induction of SprB expression with xylose. This report presents two applications using pSSβ that are particularly suitable for gene complementation experiments and for a curing system of SPβ prophage, that may serve as a model system for the removal of prophages in other bacteria.

Artificial nondirectional site-specific recombination systems

SummarySite-specific recombination systems (SRSs) are widely used in studies on synthetic biology and related disciplines. Nondirectional SRSs can randomly trigger excision, integration, reversal, and translocation, which are effective tools to achieve large-scale genome recombination. In this study, we designed 6 new nondirectional SRSs named Vika/voxsym1-4 and Dre/roxsym1-2. All 6 artificial nondirectional SRSs were able to generate random deletion and inversion in Saccharomyces cerevisiae. Moreover, all six SRSs were orthogonal to Cre/loxPsym. The pairwise orthogonal nondirected SRSs can simultaneously initiate large-scale and independent gene recombination in two different regions of the genome, which could not be accomplished using previous orthogonal systems. These SRSs were found to be robust while working in the cells at different growth stages, as well as in the different spatial structure of the chromosome. These artificial pairwise orthogonal nondirected SRSs offer newfound potential for site-specific recombination in synthetic biology.

A Novel Cre/lox-Based Genetic Tool for Repeated, Targeted and Markerless Gene Integration in Yarrowia lipolytica.

The unconventional yeast Yarrowia lipolytica is extensively applied in bioproduction fields owing to its excellent metabolite and protein production ability. Nonetheless, utilization of this promising host is still restricted by the limited availability of precise and effective gene integration tools. In this study, a novel and efficient genetic tool was developed for targeted, repeated, and markerless gene integration based on Cre/lox site-specific recombination system. The developed tool required only a single selection marker and could completely excise the unnecessary sequences. A total of three plasmids were created and seven rounds of marker-free gene integration were examined in Y. lipolytica. All the integration efficiencies remained above 90%, and analysis of the protein production and growth characteristics of the engineered strains confirmed that genome modification via the novel genetic tool was feasible. Further work also confirmed that the genetic tool was effective for the integration of other genes, loci, and strains. Thus, this study significantly promotes the application of the Cre/lox system and presents a powerful tool for genome engineering in Y. lipolytica.

A DNA Inversion System in Eukaryotes Established via Laboratory Evolution.

DNA inversion is a type of site-specific recombination system that plays an important role in the generation of genetic diversity and phenotypic adaptation by programmed rearrangements in bacteria. However, no such inversion system exhibiting a strong directionality bias has been identified or developed in eukaryotes yet. Here, using directed evolution of Rci recombinase, a tyrosine recombinase from a bacterial DNA inversion system, we identified a mutant Rci8 with a ratio of inversion/deletion up to ∼4320 in yeast. Based on Rci8 recombinase and sfxa101 sites, we have established a DNA inversion system in yeast and mammalian cells, enabling specificity for DNA inversions between inverted sites over deletions between directly repeated sites. Our results validated that the reversible DNA inversion system can act as an on/off transcriptional switch. Moreover, we demonstrate that the inversion system can also work on linear chromosomes. The eukaryotic DNA inversion system would provide a new tool for fields of genetic circuits, cellular barcoding, and synthetic genomes.

Exploiting Polyploidy for Markerless and Plasmid-Free Genome Engineering in Cyanobacteria.

Here we describe a universal approach for plasmid-free genome engineering in cyanobacteria that exploits the polyploidy of their chromosomes as a natural counterselection system. Rather than being delivered via replicating plasmids, genes encoding for DNA modifying enzymes are instead integrated into essential genes on the chromosome by allelic exchange, as facilitated by antibiotic selection, a process that occurs readily and with only minor fitness defects. By virtue of the essentiality of these integration sites, full segregation is never achieved, with the strain instead remaining as a merodiploid so long as antibiotic selection is maintained. As a result, once the desired genome modification is complete, removal of antibiotic selection results in the gene encoding for the DNA modifying enzyme to then be promptly eliminated from the population. Proof of concept of this new and generalizable strategy is provided using two different site-specific recombination systems, CRE-lox and DRE-rox, in the fast-growing cyanobacterium Synechococcus sp. PCC 7002, as well as CRE-lox in the model cyanobacterium Synechocystis sp. PCC 6803. Reusability of the method, meanwhile, is demonstrated by constructing a high-CO2 requiring and markerless Δndh3 Δndh4 ΔbicA ΔsbtA mutant of Synechococcus sp. PCC 7002. Overall, this method enables the simple and efficient construction of stable and unmarked mutants in cyanobacteria without the need to develop additional shuttle vectors nor counterselection systems.

In vitro characterization of the site-specific recombination system based on genus Habenivirus ϕRSM small serine integrase.

The genus Habenivirus which includes Ralstonia virus ϕRSM encodes a site-specific integrase of a small serine recombinase belonging to the resolvase/invertase family. Here we describe the integrative/excisive recombination reactions mediated by ϕRSM integrase using in vitro assays. The products of attP/attB recombination, i.e. attL and attR, were exactly identical to those found in the prophage ϕRSM in R. solanacearum strains. The minimum size of attB required for integration was determined to be 37bp, containing a 13bp core and flanking sequences of 4bp on the left and 20bp on the right. ϕRSM integrative recombination proceeds efficiently in vitro in the absence of additional proteins or high-energy cofactors. Excision of a functional phage genome from a prophage fragment was demonstrated in vitro, demonstrating two-way activity of ϕRSM1 integrase. This is the first example of a small serine recombinase from the resolvase/invertase group that functions in integrative and excisive recombination for filamentous phages. This serine integrase could be used as a tool for several genome engineering applications.