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Nucleic Acids Research Jun 2017Delivery of DNA to cells and its subsequent integration into the host genome is a fundamental task in molecular biology, biotechnology and gene therapy. Here we describe...
Delivery of DNA to cells and its subsequent integration into the host genome is a fundamental task in molecular biology, biotechnology and gene therapy. Here we describe an IP-free one-step method that enables stable genome integration into either prokaryotic or eukaryotic cells. A synthetic mariner transposon is generated by flanking a DNA sequence with short inverted repeats. When purified recombinant Mos1 or Mboumar-9 transposase is co-transfected with transposon-containing plasmid DNA, it penetrates prokaryotic or eukaryotic cells and integrates the target DNA into the genome. In vivo integrations by purified transposase can be achieved by electroporation, chemical transfection or Lipofection of the transposase:DNA mixture, in contrast to other published transposon-based protocols which require electroporation or microinjection. As in other transposome systems, no helper plasmids are required since transposases are not expressed inside the host cells, thus leading to generation of stable cell lines. Since it does not require electroporation or microinjection, this tool has the potential to be applied for automated high-throughput creation of libraries of random integrants for purposes including gene knock-out libraries, screening for optimal integration positions or safe genome locations in different organisms, selection of the highest production of valuable compounds for biotechnology, and sequencing.
Topics: Base Sequence; Cloning, Molecular; DNA Transposable Elements; DNA-Binding Proteins; Electroporation; Escherichia coli; Genes, Synthetic; HEK293 Cells; HeLa Cells; Humans; Inverted Repeat Sequences; Lipids; Mutagenesis, Insertional; Plasmids; Sequence Analysis, DNA; Transfection; Transposases
PubMed: 28204586
DOI: 10.1093/nar/gkx113 -
Current Protocols in Microbiology Nov 2018Human papillomaviruses (HPVs) are frequently integrated in HPV-associated cancers. HPV genomes can be integrated in three patterns: A single integrated HPV genome (type...
Human papillomaviruses (HPVs) are frequently integrated in HPV-associated cancers. HPV genomes can be integrated in three patterns: A single integrated HPV genome (type I), multiple, tandemly integrated HPV genomes (type II), and multiple, tandemly integrated HPV genomes interspersed with host DNA (type III). Analysis of the organization of type II and type III integration sites is complicated by their repetitive nature, as sequences of individual repeats are difficult to distinguish from each other. This article presents a method for directly visualizing HPV integration sites using molecular combing combined with fluorescent in situ hybridization, also known as fiber-FISH. In this technique, genomic DNA is stretched across a glass coverslip and individual integrated HPV sequences are detected and directly visualized by in situ hybridization with a resolution of ∼1 kb. Fiber-FISH allows comprehensive characterization of the genomic organization of HPV integration sites containing type II and type III integration. © 2018 by John Wiley & Sons, Inc.
Topics: DNA, Viral; Humans; In Situ Hybridization, Fluorescence; Molecular Biology; Papillomaviridae; Virus Integration
PubMed: 30129235
DOI: 10.1002/cpmc.61 -
Journal of Virology Apr 2017All retroviruses need to integrate a DNA copy of their genome into the host chromatin. Cellular proteins regulating and targeting lentiviral and gammaretroviral...
All retroviruses need to integrate a DNA copy of their genome into the host chromatin. Cellular proteins regulating and targeting lentiviral and gammaretroviral integration in infected cells have been discovered, but the factors that mediate alpharetroviral avian leukosis virus (ALV) integration are unknown. In this study, we have identified the FACT protein complex, which consists of SSRP1 and Spt16, as a principal cellular binding partner of ALV integrase (IN). Biochemical experiments with purified recombinant proteins show that SSRP1 and Spt16 are able to individually bind ALV IN, but only the FACT complex effectively stimulates ALV integration activity Likewise, in infected cells, the FACT complex promotes ALV integration activity, with proviral integration frequency varying directly with cellular expression levels of the FACT complex. An increase in 2-long-terminal-repeat (2-LTR) circles in the depleted FACT complex cell line indicates that this complex regulates the ALV life cycle at the level of integration. This regulation is shown to be specific to ALV, as disruption of the FACT complex did not inhibit either lentiviral or gammaretroviral integration in infected cells. The majority of human gene therapy approaches utilize HIV-1- or murine leukemia virus (MLV)-based vectors, which preferentially integrate near genes and regulatory regions; thus, insertional mutagenesis is a substantial risk. In contrast, ALV integrates more randomly throughout the genome, which decreases the risks of deleterious integration. Understanding how ALV integration is regulated could facilitate the development of ALV-based vectors for use in human gene therapy. Here we show that the FACT complex directly binds and regulates ALV integration efficiency and in infected cells.
Topics: Amino Acid Sequence; Animals; Avian Leukosis Virus; Cell Cycle Proteins; Chick Embryo; Conserved Sequence; DNA, Viral; DNA-Binding Proteins; HEK293 Cells; High Mobility Group Proteins; Humans; Integrases; Protein Binding; Protein Interaction Domains and Motifs; Transcription Factors; Transcriptional Elongation Factors; Virus Integration
PubMed: 28122976
DOI: 10.1128/JVI.00082-17 -
Genome Biology 2007Viral and transposon vectors have been employed in gene therapy as well as functional genomics studies. However, the goals of gene therapy and functional genomics are... (Review)
Review
Viral and transposon vectors have been employed in gene therapy as well as functional genomics studies. However, the goals of gene therapy and functional genomics are entirely different; gene therapists hope to avoid altering endogenous gene expression (especially the activation of oncogenes), whereas geneticists do want to alter expression of chromosomal genes. The odds of either outcome depend on a vector's preference to integrate into genes or control regions, and these preferences vary between vectors. Here we discuss the relative strengths of DNA vectors over viral vectors, and review methods to overcome barriers to delivery inherent to DNA vectors. We also review the tendencies of several classes of retroviral and transposon vectors to target DNA sequences, genes, and genetic elements with respect to the balance between insertion preferences and oncogenic selection. Theoretically, knowing the variables that affect integration for various vectors will allow researchers to choose the vector with the most utility for their specific purposes. The three principle benefits from elucidating factors that affect preferences in integration are as follows: in gene therapy, it allows assessment of the overall risks for activating an oncogene or inactivating a tumor suppressor gene that could lead to severe adverse effects years after treatment; in genomic studies, it allows one to discern random from selected integration events; and in gene therapy as well as functional genomics, it facilitates design of vectors that are better targeted to specific sequences, which would be a significant advance in the art of transgenesis.
Topics: Animals; Base Sequence; DNA; Genetic Therapy; Genetic Vectors; Genome; Genomics; Humans; Mutagenesis, Insertional
PubMed: 18047689
DOI: 10.1186/gb-2007-8-s1-s12 -
STAR Protocols Mar 2022Here, we describe a reliable approach for targeted DNA integrations in the genome of , one of the main causal agents of mucormycosis. We provide a strategy for stable,...
Here, we describe a reliable approach for targeted DNA integrations in the genome of , one of the main causal agents of mucormycosis. We provide a strategy for stable, targeted integration of DNA templates by homologous recombination (HR) based on the CRISPR-Cas9 technology. This strategy opens a wide range of possibilities for the genetic modification of and will be useful for the study of mucormycosis. For complete details on the use and execution of this protocol, please refer to Lax et al. (2021).
Topics: CRISPR-Cas Systems; DNA; Homologous Recombination; Mucormycosis; Rhizopus
PubMed: 35308131
DOI: 10.1016/j.xpro.2022.101237 -
International Journal of Cancer Apr 2017Recent studies showed that human papillomavirus (HPV) integration contributes to the genomic instability seen in HPV-associated head and neck squamous cell carcinoma...
Identification of human papillomavirus (HPV) 16 DNA integration and the ensuing patterns of methylation in HPV-associated head and neck squamous cell carcinoma cell lines.
Recent studies showed that human papillomavirus (HPV) integration contributes to the genomic instability seen in HPV-associated head and neck squamous cell carcinoma (HPV-HNSCC). However, the epigenetic alterations induced after HPV integration remains unclear. To identify the molecular details of HPV16 DNA integration and the ensuing patterns of methylation in HNSCC, we performed next-generation sequencing using a target-enrichment method for the effective identification of HPV16 integration breakpoints as well as the characterization of genomic sequences adjacent to HPV16 integration breakpoints with three HPV16-related HNSCC cell lines. The DNA methylation levels of the integrated HPV16 genome and that of the adjacent human genome were also analyzed by bisulfite pyrosequencing. We found various integration loci, including novel integration sites. Integration loci were located predominantly in the intergenic region, with a significant enrichment of the microhomologous sequences between the human and HPV16 genomes at the integration breakpoints. Furthermore, various levels of methylation within both the human genome and the integrated HPV genome at the integration breakpoints in each integrant were observed. Allele-specific methylation analysis suggested that the HPV16 integrants remained hypomethylated when the flanking host genome was hypomethylated. After integration into highly methylated human genome regions, however, the HPV16 DNA became methylated. In conclusion, we found novel integration sites and methylation patterns in HPV-HNSCC using our unique method. These findings may provide insights into understanding of viral integration mechanism and virus-associated carcinogenesis of HPV-HNSCC.
Topics: Alleles; Carcinoma, Squamous Cell; Cell Line, Tumor; DNA Methylation; DNA, Viral; Disease Progression; Genome, Viral; Head and Neck Neoplasms; Human papillomavirus 16; Humans; Long Interspersed Nucleotide Elements; Male; Middle Aged; Oncogene Proteins, Viral; Papillomavirus Infections; Squamous Cell Carcinoma of Head and Neck; Virus Integration
PubMed: 28006857
DOI: 10.1002/ijc.30589 -
Virology Nov 2017The pathogenic autonomous human parvovirus B19 (B19V) productively infects erythroid progenitor cells (EPCs). Functional similarities between B19V nonstructural protein...
The pathogenic autonomous human parvovirus B19 (B19V) productively infects erythroid progenitor cells (EPCs). Functional similarities between B19V nonstructural protein (NS1), a DNA binding endonuclease, and the Rep proteins of Adeno-Associated Virus (AAV) led us to hypothesize that NS1 may facilitate targeted nicking of the human genome and B19 vDNA integration. We adapted an integration capture sequencing protocol (IC-Seq) to screen B19V infected human CD36+ EPCs for viral integrants, and discovered 40,000 unique B19V integration events distributed throughout the human genome. Computational analysis of integration patterns revealed strong correlations with gene intronic regions, H3K9me3 sites, and the identification of 41 base pair consensus sequence with an octanucleotide core motif. The octanucleotide core has homology to a single region of B19V, adjacent to the P6 promoter TATA box. We present the first direct evidence that B19V infection of erythroid progenitor cells disrupts the human genome and facilitates viral DNA integration.
Topics: CD36 Antigens; Cells, Cultured; DNA; DNA-Binding Proteins; Endonucleases; Erythroid Precursor Cells; Humans; Parvovirus B19, Human; Viral Nonstructural Proteins; Virus Integration
PubMed: 28806616
DOI: 10.1016/j.virol.2017.08.011 -
The Plant Journal : For Cell and... Aug 2022High efficiency site-directed chromosomal integration of exogenous DNA in plants remains a challenge despite recent advances in genome editing technologies. One approach...
High efficiency site-directed chromosomal integration of exogenous DNA in plants remains a challenge despite recent advances in genome editing technologies. One approach to mitigate this problem is to increase the effective concentration of the donor DNA at the target site of interest. HUH endonucleases (ENs) coordinate rolling circle replication. In vitro, they can form stable covalent bonds with DNA that carries their recognition motifs. When fused to a CRISPR-associated endonuclease, HUH ENs may improve integration rates by increasing the local donor concentration through tethering of the donor to the CRISPR nuclease. We tested this hypothesis by using chimeric proteins between LbCas12a as a CRISPR-associated endonuclease and the HUH EN from Faba Bean Necrotic Yellow Virus in soybean (Glycine max). Two fusion protein configurations were tested to integrate a 70-nt oligonucleotide donor into a commercially important target site using protoplasts and in planta transformation. Site-directed integration rates of the donor DNA, when tethered to the fusion protein, reached about 26% in plants and were up to four-fold higher than in untethered controls. Integrations via canonical homology-directed repair or non-homologous end joining were promoted by tethering in a similar fashion. This study is the first demonstration of HUH EN-associated tethering to improve site-directed DNA integration in plants.
Topics: CRISPR-Cas Systems; DNA; Endonucleases; Gene Editing; Genome, Plant; Glycine max
PubMed: 35635764
DOI: 10.1111/tpj.15849 -
PLoS Genetics Sep 2021Some DNA viruses infect host animals usually by integrating their DNAs into the host genome. However, the mechanisms for integration remain largely unknown. Here, we...
Some DNA viruses infect host animals usually by integrating their DNAs into the host genome. However, the mechanisms for integration remain largely unknown. Here, we find that Cotesia vestalis bracovirus (CvBV), a polydnavirus of the parasitic wasp C. vestalis (Haliday), integrates its DNA circles into host Plutella xylostella (L.) genome by two distinct strategies, conservatively and randomly, through high-throughput sequencing analysis. We confirmed that the conservatively integrating circles contain an essential "8+5" nucleotides motif which is required for integration. Then we find CvBV circles are integrated into the caterpillar's genome in three temporal patterns, the early, mid and late stage-integration. We further identify that three CvBV-encoded integrases are responsible for some, but not all of the virus circle integrations, indeed they mainly participate in the processes of early stage-integration. Strikingly, we find two P. xylostella retroviral integrases (PxIN1 and PxIN2) are highly induced upon wasp parasitism, and PxIN1 is crucial for integration of some other early-integrated CvBV circles, such as CvBV_04, CvBV_12 and CvBV_24, while PxIN2 is important for integration of a late-integrated CvBV circle, CvBV_21. Our data uncover a novel mechanism in which CvBV integrates into the infected host genome, not only by utilizing its own integrases, but also by recruiting host enzymes. These findings will strongly deepen our understanding of how bracoviruses regulate and integrate into their hosts.
Topics: Animals; DNA, Viral; Host-Parasite Interactions; Integrases; Moths; Polydnaviridae; Wasps
PubMed: 34492000
DOI: 10.1371/journal.pgen.1009751 -
Molecular Microbiology Nov 2002Elements that excise and integrate, such as prophages, and transfer by conjugation, such as plasmids, have been found in various bacteria. These elements appear to have... (Review)
Review
Elements that excise and integrate, such as prophages, and transfer by conjugation, such as plasmids, have been found in various bacteria. These elements appear to have a diversified set of characteristics including cell-to-cell contact using pili or cell aggregation, transfer of single-stranded or double-stranded DNA, low or high specificity of integration and serine or tyrosine recombinases. This has led to a highly heterogeneous nomenclature, including conjugative transposons, integrative 'plasmids', genomic islands and numerous unclassified elements. However, all these elements excise by site-specific recombination, transfer the resulting circular form by conjugation and integrate by recombination between a specific site of this circular form and a site in the genome of their host. Whereas replication of the circular form probably occurs during conjugation, this replication is not involved in the maintenance of the element. In this review, we show that these elements share very similar characteristics and, therefore, we propose to classify them as integrative and conjugative elements (ICEs). These elements evolve by acquisition or exchanges of modules with various transferable elements including at least ICEs and plasmids. The ICEs are probably widespread among the bacteria.
Topics: Bacteria; Conjugation, Genetic; DNA Transposable Elements; Integrases; Recombination, Genetic
PubMed: 12410819
DOI: 10.1046/j.1365-2958.2002.03191.x