RESUMEN
The ability to deliver large transgenes to a single genomic sequence with high efficiency would accelerate biomedical interventions. Current methods suffer from low insertion efficiency and most rely on undesired double-strand DNA breaks. Serine integrases catalyze the insertion of large DNA cargos at attachment (att) sites. By targeting att sites to the genome using technologies such as prime editing, integrases can target safe loci while avoiding double-strand breaks. We developed a method of phage-assisted continuous evolution we call IntePACE, that we used to rapidly perform hundreds of rounds of mutagenesis to systematically improve activity of PhiC31 and Bxb1 serine integrases. Novel hyperactive mutants were generated by combining synergistic mutations resulting in integration of a multi-gene cargo at rates as high as 80% of target chromosomes. Hyperactive integrases inserted a 15.7 kb therapeutic DNA cargo containing von Willebrand Factor. This technology could accelerate gene delivery therapeutics and our directed evolution strategy can easily be adapted to improve novel integrases from nature.
Asunto(s)
Evolución Molecular Dirigida , Integrasas , Transgenes , Evolución Molecular Dirigida/métodos , Integrasas/metabolismo , Integrasas/genética , Humanos , Mutagénesis Insercional , Mutación , Bacteriófagos/genética , Bacteriófagos/enzimología , Sitios de Ligazón Microbiológica/genéticaRESUMEN
Standard zebrafish transgenesis involves random transgene integration with resource-intensive screening. While phiC31 integrase-based attP/attB recombination has streamlined transgenesis in mice and Drosophila, validated attP-based landing sites for universal applications are lacking in zebrafish. Here, we developed phiC31 Integrase Genomic Loci Engineered for Transgenesis (pIGLET) as transgenesis approach, with two attP landing sites pIGLET14a and pIGLET24b from well-validated Tol2 transgenes. Both sites facilitate diverse transgenesis applications including reporters and Cre/loxP transgenes. The pIGLET14a and pIGLET24b landing sites consistently yield 25 to 50% germline transmission, substantially reducing the resources needed for transgenic line generation. Transgenesis into these sites enables reproducible expression patterns in F0 zebrafish embryos for enhancer discovery and testing of gene regulatory variants. Together, our new landing sites streamline targeted, reproducible zebrafish transgenesis as a robust platform for various applications while minimizing the workload for generating transgenic lines.
Asunto(s)
Animales Modificados Genéticamente , Técnicas de Transferencia de Gen , Transgenes , Pez Cebra , Animales , Pez Cebra/genética , Integrasas/genética , Integrasas/metabolismo , Sitios de Ligazón Microbiológica/genéticaRESUMEN
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.
Asunto(s)
Sitios de Ligazón Microbiológica , Bacteriófagos , Integrasas , Recombinación Genética , Bacteriófagos/genética , Integrasas/metabolismo , Integrasas/genética , Sitios de Ligazón Microbiológica/genética , Lactobacillus delbrueckii/virología , Lactobacillus delbrueckii/genética , Recombinasas/metabolismo , Recombinasas/genética , Sitios de UniónRESUMEN
Integrated hepatitis B virus (HBV) DNA, found in more than 85% of HBV-associated hepatocellular carcinomas (HBV-HCCs), can play a significant role in HBV-related liver disease progression. HBV-host junction sequences (HBV-JSs), created through integration events, have been used to determine HBV-HCC clonality. Here, we investigate the feasibility of analyzing HBV integration in a noninvasive urine liquid biopsy. Using an HBV-targeted next-generation sequencing (NGS) assay, we first identified HBV-JSs in eight HBV-HCC tissues and designed short-amplicon junction-specific polymerase chain reaction assays to detect HBV-JSs in matched urine. We detected and validated tissue-derived junctions in five of eight matched urine samples. Next, we screened 32 urine samples collected from 25 patients infected with HBV (5 with hepatitis, 10 with cirrhosis, 4 with HCC, and 6 post-HCC). Encouragingly, all 32 urine samples contained HBV-JSs detectable by HBV-targeted NGS. Of the 712 total HBV-JSs detected in urine, 351 were in gene-coding regions, 11 of which, including TERT (telomerase reverse transcriptase), had previously been reported as recurrent integration sites in HCC tissue and were found only in the urine patients with cirrhosis or HCC. The integration breakpoints of HBV DNA detected in urine were found predominantly (~70%) at a previously identified integration hotspot, HBV DR1-2 (down-regulator of transcription 1-2). Conclusion: HBV viral-host junction DNA can be detected in urine of patients infected with HBV. This study demonstrates the potential for a noninvasive urine liquid biopsy of integrated HBV DNA to monitor patients infected with HBV for HBV-associated liver diseases and the efficacy of antiviral therapy.
Asunto(s)
Carcinoma Hepatocelular/orina , ADN Viral/orina , Virus de la Hepatitis B/genética , Neoplasias Hepáticas/orina , Integración Viral/genética , Adulto , Anciano , Sitios de Ligazón Microbiológica/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/virología , ADN Viral/genética , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/virología , Masculino , Persona de Mediana Edad , Reacción en Cadena de la PolimerasaRESUMEN
We apply an oligo-library and machine learning-approach to characterize the sequence and structural determinants of binding of the phage coat proteins (CPs) of bacteriophages MS2 (MCP), PP7 (PCP), and Qß (QCP) to RNA. Using the oligo library, we generate thousands of candidate binding sites for each CP, and screen for binding using a high-throughput dose-response Sort-seq assay (iSort-seq). We then apply a neural network to expand this space of binding sites, which allowed us to identify the critical structural and sequence features for binding of each CP. To verify our model and experimental findings, we design several non-repetitive binding site cassettes and validate their functionality in mammalian cells. We find that the binding of each CP to RNA is characterized by a unique space of sequence and structural determinants, thus providing a more complete description of CP-RNA interaction as compared with previous low-throughput findings. Finally, based on the binding spaces we demonstrate a computational tool for the successful design and rapid synthesis of functional non-repetitive binding-site cassettes.
Asunto(s)
Allolevivirus/genética , Proteínas de la Cápside/metabolismo , Escherichia coli/virología , Levivirus/genética , ARN/metabolismo , Sitios de Ligazón Microbiológica/genética , Sitios de Unión/genética , Línea Celular Tumoral , Escherichia coli/genética , Biblioteca de Genes , Humanos , Aprendizaje Automático , Plásmidos/genéticaRESUMEN
We describe a simple and efficient technique that allows scarless engineering of Drosophila genomic sequences near any landing site containing an inverted attP cassette, such as a MiMIC insertion. This two-step method combines phiC31 integrase-mediated site-specific integration and homing nuclease-mediated resolution of local duplications, efficiently converting the original landing site allele to modified alleles that only have the desired change(s). Dominant markers incorporated into this method allow correct individual flies to be efficiently identified at each step. In principle, single attP sites and FRT sites are also valid landing sites. Given the large and increasing number of landing site lines available in the fly community, this method provides an easy and fast way to efficiently edit the majority of the Drosophila genome in a scarless manner. This technique should also be applicable to other species.
Asunto(s)
Marcación de Gen/métodos , Mutagénesis Insercional/métodos , Mutagénesis Sitio-Dirigida/métodos , Animales , Sitios de Ligazón Microbiológica/genética , Desoxirribonucleasa I/genética , Desoxirribonucleasa I/metabolismo , Drosophila melanogaster , Genoma de los Insectos , Integrasas/genética , Integrasas/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
The phage-derived phiC31 integrase is a useful tool for mediating sequence-specific genomic integration in mammalian cells, recombining donor plasmids bearing the attB recognition site with introduced genomic attP sites or endogeneous pseudo-attP sites having partial identity to attP. In most prior studies, phiC31 integrase has been introduced as plasmid DNA or mRNA. The current report examines whether phiC31 integrase functions efficiently in mammalian cells when co-nucleofected as a purified protein, along with attB-containing donor plasmids or PCR fragments. We describe preparation of phiC31 integrase protein and evidence that it can mediate genomic integration in human 293 cells, including PCR evidence for integration at an endogenous pseudo-attP site. This work demonstrates for the first time the ability of 605- and 613-amino-acid versions of phiC31 integrase protein to mediate efficient, site-specific integration into the genome of human cells when co-nucleofected with full-sizedattB-containing donor plasmids or linear 2.5-kb PCR fragments. This protein-mediated approach may be especially useful for integration of exogenous sequences into valuable therapeutic target cells, such as hematopoietic stem cells or T cells, that are sensitive to introduced DNA.
Asunto(s)
Sitios de Ligazón Microbiológica/genética , Integrasas/genética , Siphoviridae/genética , Integración Viral/genética , Línea Celular , Genoma Humano/genética , Genómica/métodos , Humanos , Plásmidos/genética , Siphoviridae/enzimologíaRESUMEN
Epstein-Barr virus (EBV) genomes persist in latently infected cells as extrachromosomal episomes that attach to host chromosomes through the tethering functions of EBNA1, a viral encoded sequence-specific DNA binding protein. Here we employ circular chromosome conformation capture (4C) analysis to identify genome-wide associations between EBV episomes and host chromosomes. We find that EBV episomes in Burkitt's lymphoma cells preferentially associate with cellular genomic sites containing EBNA1 binding sites enriched with B-cell factors EBF1 and RBP-jK, the repressive histone mark H3K9me3, and AT-rich flanking sequence. These attachment sites correspond to transcriptionally silenced genes with GO enrichment for neuronal function and protein kinase A pathways. Depletion of EBNA1 leads to a transcriptional de-repression of silenced genes and reduction in H3K9me3. EBV attachment sites in lymphoblastoid cells with different latency type show different correlations, suggesting that host chromosome attachment sites are functionally linked to latency type gene expression programs.
Asunto(s)
Sitios de Ligazón Microbiológica/genética , Sitios de Ligazón Microbiológica/fisiología , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/fisiología , Interacciones Microbiota-Huesped/genética , Linfoma de Burkitt/genética , Linfoma de Burkitt/virología , Línea Celular Tumoral , Cromosomas Humanos/genética , Cromosomas Humanos/virología , Epigénesis Genética , Antígenos Nucleares del Virus de Epstein-Barr/fisiología , Herpesvirus Humano 4/patogenicidad , Interacciones Microbiota-Huesped/fisiología , Humanos , Modelos Biológicos , Plásmidos/genética , Latencia del Virus/genética , Latencia del Virus/fisiologíaRESUMEN
Multiple gene knockouts are often employed in studies of microbial physiology and genetics. However, the selective markers that confer antibiotic resistance are generally limited, so it is necessary to remove these resistance genes before the next round of using, which is time consuming and labor intensive. Here, we created a universal circular gene knockout system for both the gram-negative bacterial Burkholderiales strain DSM 7029 and the gram-positive bacterial Mycobacterium smegmatis mc2 155, by combining the homologous recombination with multiple serine integrase-meditated site-specific recombination systems. In this system, a resistance gene and an integrase gene were constructed within the two attachment sites corresponding to a second, different integrase to form a cassette for gene disruption, which could be easily removed by the second integrase during the subsequent round of gene knockout. The sacB gene was also employed for negative selection. As the integrase-mediated deletion of the resistance/integrase gene cassette was highly efficient and concurrent with the following knockout round, the cyclic use of three cassettes could achieve multiple gene knockout in a sequential manner. Following the modularity concept in synthetic biology, common components of the knockout plasmids were retained as BioBricks, accelerating the knockout plasmids construction process. The circular gene knockout system can also be used for multiple gene insertions and applied to other microorganisms.
Asunto(s)
Burkholderiales/genética , Farmacorresistencia Microbiana/genética , Técnicas de Inactivación de Genes/métodos , Genes Bacterianos/genética , Mycobacterium smegmatis/genética , Sitios de Ligazón Microbiológica/genética , Burkholderiales/metabolismo , Recombinación Homóloga/genética , Integrasas/genética , Integrasas/metabolismo , Mycobacterium smegmatis/metabolismo , Plásmidos/genética , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND: Few natural product pathways from rare Actinomycetes have been studied due to the difficulty in applying molecular approaches in these genetically intractable organisms. In this study, we sought to identify more integrating vectors, using phage int/attP loci, that would efficiently integrate site-specifically in the rare Actinomycete, Amycolatopsis marina DSM45569. RESULTS: Analysis of the genome of A. marina DSM45569 indicated the presence of attB-like sequences for TG1 and R4 integrases. The TG1 and R4 attBs were active in in vitro recombination assays with their cognate purified integrases and attP loci. Integrating vectors containing either the TG1 or R4 int/attP loci yielded exconjugants in conjugation assays from Escherichia coli to A. marina DSM45569. Site-specific recombination of the plasmids into the host TG1 or R4 attB sites was confirmed by sequencing. CONCLUSIONS: The homologous TG1 and R4 attB sites within the genus Amycolatopsis have been identified. The results indicate that vectors based on TG1 and R4 integrases could be widely applicable in this genus.
Asunto(s)
Actinobacteria/genética , Vectores Genéticos/genética , Genoma Bacteriano/genética , Recombinación Genética , Actinobacteria/virología , Amycolatopsis , Sitios de Ligazón Microbiológica/genética , Secuencia de Bases , Integrasas/genética , Integrasas/metabolismo , Homología de Secuencia de Ácido Nucleico , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
The real value of gas-fermenting clostridia, capable of using CO and CO2, resides in their potential of being developed into cell factories to produce various bulk chemicals and fuels. This process requires rapid chromosomal integration of heterologous chemical biosynthetic pathways, which is impeded by the absence of genetic tools competent for efficient genome engineering in these anaerobes. Here, we developed a phage serine integrase-mediated site-specific genome engineering technique in Clostridium ljungdahlii, one of the major acetogenic gas-fermenting microbes. Two heterologous phage attachment/integration (Att/Int) systems (from Clostridium difficile and Streptomyces) were introduced into C. ljungdahlii and proven to be highly active, achieving efficient chromosomal integration of a whole donor vector via single-crossover recombination. Based on this, we further realized markerless chromosomal integration of target DNA fragments through a "dual integrase cassette exchange" (DICE) strategy with the assistance of the CRISPR-Cas9 editing system. As a proof of concept, a butyric acid production pathway from Clostridium acetobutylicum was integrated into the C. ljungdahlii genome without the introduction of extra markers, enabling stable expression of the pathway genes. The resulting engineered strain produced 1.01â¯g/L of butyric acid within 3 days by fermenting synthesis gas (CO2/CO). More importantly, the engineered strain showed good genetic stability and maintained butyric acid production ability after continuous subculturing. The system developed in this study overcomes the deficiencies of currently available genetic tools in the chromosomal integration of large DNA fragments (rapid, markerless and stable) in C. ljungdahlii, and may be extended to other Clostridium species.
Asunto(s)
Bacteriófagos/enzimología , Bacteriófagos/genética , Clostridium/genética , Clostridium/metabolismo , Integrasas/genética , Integrasas/metabolismo , Ingeniería Metabólica/métodos , Sitios de Ligazón Microbiológica/genética , Ácido Butírico/metabolismo , Sistemas CRISPR-Cas , ADN Bacteriano/genética , Fermentación , Genoma Bacteriano/genética , Redes y Vías Metabólicas/genética , Plásmidos/genética , Serina/metabolismoRESUMEN
A predominant tool for adaptation in Gram-negative bacteria is the functional genetic platform called integron. Integrons capture and rearrange promoterless gene cassettes in a unique recombination process involving the recognition of folded single-stranded DNA hairpins-so-called attC sites-with a strong preference for the attC bottom strand. While structural elements have been identified to promote this preference, their mechanistic action remains incomplete. Here, we used high-resolution single-molecule optical tweezers (OT) to characterize secondary structures formed by the attC bottom (${{att}}{{{C}}_{{\rm{bs}}}}$) and top (${{att}}{{{C}}_{{\rm{ts}}}}$) strands of the paradigmatic attCaadA7 site. We found for both sequences two structures-a straight, canonical hairpin and a kinked hairpin. Remarkably, the recombination-preferred ${{att}}{{{C}}_{{\rm{bs}}}}$ predominantly formed the straight hairpin, while the ${{att}}{{{C}}_{{\rm{ts}}}}$ preferentially adopted the kinked structure, which exposes only one complete recombinase binding box. By a mutational analysis, we identified three bases in the unpaired central spacer, which could invert the preferred conformations and increase the recombination frequency of the ${{att}}{{{C}}_{{\rm{ts}}}}$in vivo. A bioinformatics screen revealed structural bias toward a straight, canonical hairpin conformation in the bottom strand of many antibiotic resistance cassettes attC sites. Thus, we anticipate that structural fine tuning could be a mechanism in many biologically active DNA hairpins.
Asunto(s)
ADN/genética , Farmacorresistencia Bacteriana/genética , Integrones/genética , Recombinación Genética , Sitios de Ligazón Microbiológica/genética , ADN/química , ADN de Cadena Simple/genética , Escherichia coli/genética , Integrasas/genética , Conformación de Ácido Nucleico , Pinzas ÓpticasRESUMEN
In the current report, we describe the identification of three genetically distinct groups of prophages integrated into three different chromosomal sites of human gut-associated Bifidobacterium breve and Bifidobacterium longum strains. These bifidobacterial prophages are distantly related to temperate actinobacteriophages of several hosts. Some prophages, integrated within the dnaJ2 gene, are competent for induction, excision, replication, assembly and lysis, suggesting that they are fully functional and can generate infectious particles, even though permissive hosts have not yet been identified. Interestingly, several of these phages harbor a putative phase variation shufflon (the Rin system) that generates variation of the tail-associated receptor binding protein (RBP). Unlike the analogous coliphage-associated shufflon Min, or simpler Cin and Gin inversion systems, Rin is predicted to use a tyrosine recombinase to promote inversion, the first reported phage-encoded tyrosine-family DNA invertase. The identification of bifidobacterial prophages with RBP diversification systems that are competent for assembly and lysis, yet fail to propagate lytically under laboratory conditions, suggests dynamic evolution of bifidobacteria and their phages in the human gut.
Asunto(s)
Bifidobacterium/virología , Microbioma Gastrointestinal , Profagos/fisiología , Sitios de Ligazón Microbiológica/genética , Secuencia de Bases , Bifidobacterium/efectos de los fármacos , Evolución Biológica , Microbioma Gastrointestinal/efectos de los fármacos , Genoma Viral , Especificidad del Huésped/efectos de los fármacos , Especificidad del Huésped/genética , Humanos , Mitomicina/farmacología , Profagos/efectos de los fármacos , Profagos/genética , Profagos/ultraestructura , Virión/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
The marine actinomycete genus Salinispora is a remarkably prolific source of structurally diverse and biologically active secondary metabolites. Herein, we select the model organism Salinispora tropica CNB-440 for development as a heterologous host for the expression of biosynthetic gene clusters (BGCs) to complement well-established Streptomyces host strains. In order to create an integratable host with a clean background of secondary metabolism, we replaced three genes (salA-C) essential for salinosporamide biosynthesis with a cassette containing the Streptomyces coelicolor ΦC31 phage attachment site attB to generate the mutant S. tropica CNB-4401 via double-crossover recombination. This mutagenesis not only knocks-in the attachment site attB in the genome of S. tropica CNB-440 but also abolishes production of the salinosporamides, thereby simplifying the strain's chemical background. We validated this new heterologous host with the successful integration and expression of the thiolactomycin BGC that we recently identified in several S. pacifica strains. When compared to the extensively engineered superhost S. coelicolor M1152, the production of thiolactomycins from S. tropica CNB-4401 was approximately 3-fold higher. To the best of our knowledge, this is the first example of using a marine actinomycete as a heterologous host for natural product BGC expression. The established heterologous host may provide a useful platform to accelerate the discovery of novel natural products and engineer biosynthetic pathways.
Asunto(s)
Productos Biológicos/metabolismo , Micromonosporaceae/genética , Micromonosporaceae/metabolismo , Familia de Multigenes/genética , Actinobacteria/genética , Sitios de Ligazón Microbiológica/genética , Vías Biosintéticas/genética , Metabolismo Secundario/genética , Streptomyces/genética , Streptomyces/metabolismo , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Tiofenos/metabolismoRESUMEN
This study was designed to screen for SCCmec types and to characterize the attachment site (attB) and universal insertion site (orfX) of SCCmec in a collection of 27 isolates (nâ¯=â¯11) methicillin resistant S. aureus and (nâ¯=â¯16) methicillin susceptible S. aureus isolates in Malaysia. Screening of SCCmec types and characterization of the attachment site was carried out using PCR amplification and Sanger's sequencing method. The result showed that a large proportion of the MRSA isolates carried SCCmec type III 7/11 (63%). Three isolates 3/11 (27%) and 1/11 (9.0%) carried SCCmec type II and IVd respectively. Amplification of the universal insertion site of the SCCmec (orfX) and attachment site (attB) showed that all 16 S. aureus isolates were positive for the orfX gene, while only 7 were positive for the attB gene. Phylogenetic diversity showed that the isolates clustered around strains with features similar to a community acquired MRSA. In conclusion, a high carriage rate of SCCmec type III was observed. The result also showed that all the S. aureus isolates have the orfX structure; however, not all isolates possesses the attB site on the 3' end of the orfX region.
Asunto(s)
Sitios de Ligazón Microbiológica/efectos de los fármacos , Sitios de Ligazón Microbiológica/genética , Proteínas Bacterianas/genética , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/genética , Meticilina/farmacología , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/genética , Antibacterianos/farmacología , Secuencia de Bases , ADN Bacteriano/genética , Variación Genética/genética , Humanos , Malasia , Staphylococcus aureus Resistente a Meticilina/clasificación , Staphylococcus aureus Resistente a Meticilina/aislamiento & purificación , Pruebas de Sensibilidad Microbiana , Tipificación Molecular/métodos , Proteínas de Unión a las Penicilinas/genética , Filogenia , Reacción en Cadena de la Polimerasa/métodos , Análisis de Secuencia de ADN , Infecciones Estafilocócicas/diagnóstico , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/clasificación , Staphylococcus aureus/aislamiento & purificación , Factores de Transcripción/genéticaRESUMEN
Genetic analysis is facilitated by the efficient production of transgenic strains expressing a DNA of interest as a single copy at a designated chromosomal location. However, technical progress toward this goal in medaka fish (Oryzias latipes), a vertebrate model organism, has been slow. It is well known that phiC31 integrase enables efficient site-directed transgenesis by catalyzing the recombination of an attP DNA motif in a host genome with an attB motif in a targeting vector. This system was pioneered in medaka using the Sleeping Beauty transposon system, and the attP site was established at three chromosomal locations. However, this number appeared insufficient with regard to genetic linkage between the attP-landing site and a genetically modified locus of interest. Here, to establish a collection of transgenic strains of medaka, we introduced an attP motif into the medaka genome using the Ac/Ds maize transposon system and established 12 independent transgenic strains harboring a single copy of the attP motif in at least 11 of the 24 medaka chromosomes. We designed an attB-targeting vector that was integrated efficiently and precisely into the attP-landing site, and with which the DNA of interest was efficiently transmitted to germline cells. Extraneous sequences in the integrants derived from the bacterial backbone of the attB-targeting vector as well as a transgenic fluorescence marker present in the attP-landing site were removable through flippase-mediated recombination. Further, an advanced targeting vector with a heart-specific recombination marker served as a useful tool for easily screening phiC31 integrase-mediated recombinant G0 embryos, leading to the efficient establishment of transgenic strains. Thus, our resources advance genetic research in medaka.
Asunto(s)
Animales Modificados Genéticamente/genética , Técnicas de Transferencia de Gen , Oryzias/genética , Animales , Sitios de Ligazón Microbiológica/genética , Elementos Transponibles de ADN , Vectores Genéticos/genética , Integrasas/genética , Integrasas/metabolismo , Siphoviridae/enzimología , Siphoviridae/genética , Proteínas Virales/genética , Proteínas Virales/metabolismo , Zea mays/genéticaRESUMEN
Effective genome engineering should lead to a desired locus change with minimal adverse impact to the genome itself. However, flanking loci with site-directed recombinase recognition sites, such as those of the phage ΦC31 integrase, allows for creation of platforms for cassette exchange and manipulation of genomic regions in an iterative manner, once specific loci have been targeted. Here we show that a genomic locus engineered with inverted minimal phage ΦC31 attP/attB sites can undergo efficient recombinase-mediated cassette exchange (RMCE) in the fruit fly Drosophila melanogaster.
Asunto(s)
Sitios de Ligazón Microbiológica/genética , Bacteriófagos/metabolismo , Drosophila melanogaster/genética , Ingeniería Genética/métodos , Genoma de los Insectos , Animales , Proteínas Fluorescentes Verdes/metabolismo , Recombinasas/metabolismoRESUMEN
Developmental deconvolution of complex organs and tissues at the level of individual cells remains challenging. Non-invasive genetic fate mapping has been widely used, but the low number of distinct fluorescent marker proteins limits its resolution. Much higher numbers of cell markers have been generated using viral integration sites, viral barcodes, and strategies based on transposons and CRISPR-Cas9 genome editing; however, temporal and tissue-specific induction of barcodes in situ has not been achieved. Here we report the development of an artificial DNA recombination locus (termed Polylox) that enables broadly applicable endogenous barcoding based on the Cre-loxP recombination system. Polylox recombination in situ reaches a practical diversity of several hundred thousand barcodes, allowing tagging of single cells. We have used this experimental system, combined with fate mapping, to assess haematopoietic stem cell (HSC) fates in vivo. Classical models of haematopoietic lineage specification assume a tree with few major branches. More recently, driven in part by the development of more efficient single-cell assays and improved transplantation efficiencies, different models have been proposed, in which unilineage priming may occur in mice and humans at the level of HSCs. We have introduced barcodes into HSC progenitors in embryonic mice, and found that the adult HSC compartment is a mosaic of embryo-derived HSC clones, some of which are unexpectedly large. Most HSC clones gave rise to multilineage or oligolineage fates, arguing against unilineage priming, and suggesting coherent usage of the potential of cells in a clone. The spreading of barcodes, both after induction in embryos and in adult mice, revealed a basic split between common myeloid-erythroid development and common lymphocyte development, supporting the long-held but contested view of a tree-like haematopoietic structure.
Asunto(s)
Sitios de Ligazón Microbiológica/genética , Linaje de la Célula/genética , Rastreo Celular/métodos , Código de Barras del ADN Taxonómico/métodos , Células Madre Hematopoyéticas/citología , Recombinación Genética/genética , Análisis de la Célula Individual/métodos , Animales , Células Clonales/citología , Células Clonales/metabolismo , Embrión de Mamíferos/citología , Células Eritroides/citología , Células Eritroides/metabolismo , Femenino , Células Madre Hematopoyéticas/metabolismo , Integrasas/metabolismo , Linfocitos/citología , Linfocitos/metabolismo , Masculino , Ratones , Mosaicismo , Células Mieloides/citología , Células Mieloides/metabolismoRESUMEN
Bacterial prodigiosins are red-colored secondary metabolites with multiple activities, such as anticancer, antimalarial and immunosuppressive, which hold great potential for medical applications. In this study, dramatically enhanced prodigiosins (RED) production in Streptomyces coelicolor was achieved by combinatorial metabolic engineering, including inactivation of the repressor gene ohkA, deletion of the actinorhodin (ACT) and calcium-dependent antibiotic (CDA) biosynthetic gene clusters (BGCs) and multi-copy chromosomal integration of the RED BGC. The results showed that ohkA deletion led to a 1-fold increase of RED production over the wild-type strain M145. Then, the ACT and CDA BGCs were deleted successively based on the ΔohkA mutant (SBJ101). To achieve multi-copy RED BGC integration, artificial ΦC31 attB site(s) were inserted simultaneously at the position where the ACT and CDA BGCs were deleted. The resulting strains SBJ102 (with a single deletion of the ACT BGC and insertion of one artificial attB site) and SBJ103 (with the deletion of both BGCs and insertion of two artificial attB sites) produced 1.9- and 6-fold higher RED titers than M145, respectively. Finally, the entire RED BGC was introduced into mutants from SBJ101 to SBJ103, generating three mutants (from SBJ104 to SBJ106) with chromosomal integration of one to three copies of the RED BGC. The highest RED yield was from SBJ106, which produced a maximum level of 96.8 mg g-1 cell dry weight, showing a 12-fold increase relative to M145. Collectively, the metabolic engineering strategies employed in this study are very efficient for the construction of high prodigiosin-producing strains.
Asunto(s)
Microbiología Industrial/métodos , Ingeniería Metabólica , Familia de Multigenes/genética , Prodigiosina/biosíntesis , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Sitios de Ligazón Microbiológica/genética , Vías Biosintéticas/genética , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica/genética , Mejoramiento Genético , Genoma Bacteriano/genética , Redes y Vías Metabólicas/genéticaRESUMEN
BACKGROUND: Direct manipulation of the genome is a widespread technique for genetic studies and synthetic biology applications. The tyrosine and serine site-specific recombination systems of bacteriophages HK022 and ΦC31 are widely used for stable directional exchange and relocation of DNA sequences, making them valuable tools in these contexts. We have developed site-specific recombination tools that allow the direct selection of recombination events by embedding the attB site from each system within the ß-lactamase resistance coding sequence (bla). RESULTS: The HK and ΦC31 tools were developed by placing the attB sites from each system into the signal peptide cleavage site coding sequence of bla. All possible open reading frames (ORFs) were inserted and tested for recombination efficiency and bla activity. Efficient recombination was observed for all tested ORFs (3 for HK, 6 for ΦC31) as shown through a cointegrate formation assay. The bla gene with the embedded attB site was functional for eight of the nine constructs tested. CONCLUSIONS: The HK/ΦC31 att-bla system offers a simple way to directly select recombination events, thus enhancing the use of site-specific recombination systems for carrying out precise, large-scale DNA manipulation, and adding useful tools to the genetics toolbox. We further show the power and flexibility of bla to be used as a reporter for recombination.