48 results about "Site-specific recombination" patented technology

The present invention relates generally to the field of site-specific DNA recombination mediated by lambda integrase, and more specifically, to a method for producing eukaryotic cells and cell lines comprising a genomic landing pad for lambda integrase-mediated recombination, and methods of using the eukaryotic cells themselves and subsequently their use in lambda integrase mediated recombination and as bioreactors for cell therapy.

The present disclosure provides methods of generating landing pad cells for targeted gene integration comprising integrating a landing pad plasmid into the genome of a parent cell at a targeted integration site, for example, using homologous recombination. In one aspect, two site-specific recombination sites (SSRSs) are flanked by a polynucleotide sequence; and two homologous recombination sites are located at the 5'and 3 'ends of the SSRS. Two homologous recombination sites of the landing pad plasmid can be recombined with corresponding homologous recombination sites on the parental plasmid, so that the landing pad plasmid is integrated at an internal position in the parental plasmid. The disclosed methods allow for the generation of high expression cell lines by identifying hotspots for targeted integration in thermal cell lines. The present disclosure also provides cells for generating landing pad cells of the disclosure, as well as kits comprising cells and / or agents for generating landing pad cells of the disclosure. Novel hotspots for targeted integration are also provided.

Among other things, provided herein are systems that replace the natural random integration activity of a retrovirus with site-specific integration machinery. This approach allows for a more precise targeting of a gene of interest into a human genome, e.g., for therapeutic purposes. The system may include integration-deficient retrovirus (e.g., lentivirus) (IDLV), in which the natural integration activity has been reduced (e.g., by mutation to the viral integrase polypeptide). Instead, the system may comprise a site-specific recombinase (e.g., a serine recombinase, e.g., a serine integrase) capable of directing insertion of a template DNA, or portion thereof, into a desired site in the human genome.

Disclosed herein are engineered LSR sequences, compositions, and uses including in genome-editing systems and therapeutic compositions. The LSRs can facilitate nucleic acid recombination at particular attachment sites, using suitable donor sequences. The disclosed LSRs may be used to site-specifically recombine or integrate nucleic acid sequences for a variety of purposes, including treatment of human diseases.

A homologous recombination method of excising a specific DNA sequence from a DNA molecule. The method includes providing the DNA molecule including a pair of target sequences that are a target of a site-specific recombination enzyme and a pair of homologous sequences that are DNA sequences sharing homology with each other. The pair of target sequences are oriented in different directions from each other and arranged on opposite sides of the specific DNA sequence. The pair of homologous sequences are arranged on opposite sides of the specific DNA sequence at positions further outside of the pair of target sequences. The method further includes inducing homologous recombination between the pair of homologous sequences by action of the site-specific recombination enzyme on the DNA molecule so that the specific DNA sequence and the pair of target sequences are excised from the DNA molecule.

Disclosed are altered recombinases wherein at least one amino acid is different from a parent, wildtype recombinase and the altered recombinase has improved recombination efficiency towards wild-type and / or pseudo att site sequences relative to the parent, wildtype recombinase. Disclosed are altered modular recombinases comprising binding (or interacting) regions from a different recombinase. Disclosed are methods of modifying the genomes of cells using the altered recombinases, including methods of site-specifically integrating a polynucleotide sequence of interest in a genome of a eucaryotic cell.

Disclosed herein is a nanorod production system (NPS) that can be used to produce bioscalable functionalized ready nanorods (BSFnano). The produced nanorod is derived from a filamentous bacteriophage Ff (f1, M13 or fd). The NPS disclosed herein allows for efficient biological production of non-infectious, thermally stable isomorphic protein nanorods comprising modifications that allow for site-specific recombination, chemical and enzymatic attachment of peptide and non-peptide functional groups in an orthogonal fashion. Also disclosed are methods of making and using these nanorods, such as in methods of detecting target molecules.

The present invention provides a DNA construct for expressing a genome editing system in plant cells, which comprises an excised region sandwiched by two recombinase recognition sites arranged in the same direction, a gene encoding a site-specific recombinase that specifically recognizes these recombinase recognition sites is arranged outside the excised region, and the excised region comprises a marker gene divided into a 5' region and a 3' region, and a gene encoding the genome editing system arranged between the 3' region and the 5' region.

This disclosure provides methods and landing pad constructs for generation of parental cell lines suitable for targeted integration. A method is provided by the parental cell line development; this is, the introduction of binding sites of BPV1 E2 protein to landing pad vectors so that expressed BPV1 E2 protein could locate the vector to transcriptionally active region in the genome. Cells with high expression level of reporter genes are selected for the next stage and will be used in the development of cell lines expressing another recombinant protein by recombination mediated cassette exchange (RMCE). Landing pad constructs include recombination target sites for site-specific recombinases, and therefore, it could be replaced with gene-of-interest expression construct containing the same set of recombination target sites. This yields the generation of producer cell lines with less effort compared to traditional cell line development by random integration.

Disclosed herein is a nanorod generation system (NPS) useful for the generation of biologically scalable functionalized nanorods (BSF nanorods). The nanorods generated are derived from the filamentous phage Ff (f1, M13, or fd). The NPS disclosed herein allows for the efficient biological generation of non-infectious, thermostable, homotypic protein nanorods that contain modifications that orthogonally enable site-specific recombination, chemical and enzymatic attachment of peptide and non-peptide functionalities. Also disclosed are methods of generating and using these nanorods, such as in methods for detecting target molecules.

The present invention relates generally to the field of site-specific DNA recombination mediated by lambda integrases, and more specifically to methods of producing eukaryotic cells and cell lines comprising a genomic landing pad for lambda integrase mediated recombination, as well as the eukaryotic cells themselves and subsequent methods of their use for lambda integrase mediated recombination and as bioreactors for cell therapies.

In a homologous recombination method in which a specific DNA sequence is deleted from a DNA molecule, the DNA molecule is used as the DNA molecule. A DNA molecule is prepared in which a pair of target sequences, which are targets for a site-specific recombinase, are disposed so as to sandwich a specific DNA sequence therebetween and so as to have different orientations from each other, and a pair of homologous sequences, which are DNA sequences having homology, are disposed on the outside of the pair of target sequences disposed so as to sandwich the specific DNA sequence. The site-specific recombinase is caused to act on a DNA molecule, and a specific DNA sequence and a pair of target sequences are deleted from the DNA molecule by homologous recombination between a pair of homologous sequences.

Recombinant DNA molecules and constructs are provided that are useful for modulating gene expression in plants. One or more expression cassette(s) of a recombinant DNA molecule or construct may be excised from transgenic plants following transformation by the presence of flanking site-specific recombination sites in the recombinant DNA molecule or construct by expression of a recombinase enzyme encoded by the recombinant DNA molecule or construct. Such a recombinase system may be used to remove such expression cassette(s) from plants transformed with the recombinant DNA construct or vector. The recombinase transgene may be operably linked to a tissue-preferred or tissue-specific promoter for autoexcision in transformed plants without crossing to a different transgenic line expressing the recombinase. Methods for causing autoexcision of one or more expression cassette(s) in a transgenic plant, and plants and cells containing or transformed with a recombinant DNA molecule or construct of the present disclosure, are also provided.

The present invention relates to a method for integrating one or more recombinant sites in a sole target gene of a cell genome comprising carrying out sequentially multiple site-specific recombination by using alternatively two different serine-integrases, wherein the selection marker is removed after each site-specific recombination by different transposases: An in vitro method for inserting at least one recombinant site in a target gene / locus from an isolated cell genome, said method comprising integrating a nucleotide sequence A into the target gene from an isolated cell genome by homologous recombination, wherein said nucleotide sequence A comprises a recombination cassette, flanked at 5′ and 3′ by target gene homology arms, and removing the selection marker by using the second transposase, thus obtaining a cell genome A comprising (i), (ii) and (iii) of nucleotide sequence A.

Recombinant DNA molecules and constructs are provided that are useful for modulating gene expression in plants. One or more expression cassette(s) of a recombinant DNA molecule or construct may be excised from transgenic plants following transformation by the presence of flanking site-specific recombination sites in the recombinant DNA molecule or construct by expression of a recombinase enzyme encoded by the recombinant DNA molecule or construct. Such a recombinase system may be used to remove such expression cassette(s) from plants transformed with the recombinant DNA construct or vector. The recombinase transgene may be operably linked to a tissue-preferred or tissue-specific promoter for autoexcision in transformed plants without crossing to a different transgenic line expressing the recombinase. Methods for causing autoexcision of one or more expression cassette(s) in a transgenic plant, and plants and cells containing or transformed with a recombinant DNA molecule or construct of the present disclosure, are also provided.

The invention relates generally to the field of genome editing and provides DNA recombinases, which efficiently and specifically recombine genomic target sequences via the fusion of recombinase monomers. More specifically, the invention provides a method to generate a fusion protein for efficient and specific genome editing, comprising a complex of recombinases comprising at least a first recombinase enzyme, a second recombinase enzyme and at least one linker, wherein said first recombinase enzyme and said second recombinase enzyme specifically recognize a first half-site and a second half-site of an upstream target site and / or a downstream target site of a recombinase; wherein said first recombinase enzyme and said second recombinase enzyme are interconnected via a linker; and wherein said linker comprises or consists of an oligopeptide. The invention further relates to fusion proteins generated with this method. The invention also discloses designer-recombinases, which catalyze the inversion of a DNA sequence present in the int1h regions on the human X chromosome. The invention further relates to nucleic acid molecules encoding said DNA recombinases and fusion proteins, as well as to the use of said fusion proteins, DNA recombinases and nucleic acid molecules in a pharmaceutical composition.

Disclosed herein are nanorod productions systems (NPS) useful for the production of biological scalable functionalization-ready nanorods (BSFnano). The nanorods produced are derived from filamentous phage Ff (f1, M13 or fd). The NPS disclosed herein permits efficient biological production of non-infectious, heat-stable isomorphic proteinaceous nanorods comprising modifications allowing site-specific recombinant, chemical and enzymatic attachment of peptide and non-peptide functionalities in an orthogonal manner. Also disclosed are methods of making and using these nanorods, such as in methods of detecting target molecules.

To provide compositions and methods capable of delivering and expressing multiple genes from a biosynthetic pathway in a recipient cell using a synthetic chromosome.SOLUTION: A method comprises: transfecting a recipient cell line with synthetic chromosome production components including nucleic acid sequences that allow site-specific integration of target nucleic acid sequences; producing a synthetic platform chromosome with multiple site-specific recombination sites; transfecting the recipient cell line with a delivery vector including multiple genes having the ability to realize a biosynthetic pathway; activating site-specific recombination between the synthetic platform chromosome and the delivery vector, so that the multiple genes having the ability to realize the biosynthetic pathway are loaded onto the synthetic platform chromosome to produce a synthetic chromosome expressing the biosynthetic pathway; and isolating a recipient cell including the synthetic chromosome expressing the biosynthetic pathway.SELECTED DRAWING: None

The invention relates to the field of molecular biology, and discloses a method for rapidly screening recombinase site variants. An expression vector pUCBN comprising a Bxb1 recombinase expression cassette, a 35S promoter and an attP site located at the downstream of the 35S promoter is constructed, the plasmid and an attB site fragment subjected to PCR amplification are co-transformed into a protoplast, the attB site and the attP site are specifically recognized by utilizing serine recombinase Bxb1 to generate site-specific recombination, recombination between the vector and the fragment is achieved, and the expression vector pUCBN is obtained. Therefore, the recombinase recognition site variant is rapidly screened. Compared with a conventional method for screening recombinase sites by respectively constructing an attB site and an attP site on two vectors, the method has the advantages that the step of frequently constructing the vectors is omitted, a large amount of time and cost are saved, the method is also suitable for high-throughput screening research, recognition sites of Bxb1 recombinase can be rapidly screened in cotton, and the method is suitable for popularization and application. And a basis is provided for widening the application of the serine recombinase Bxb1 in cotton.

Recombinant DNA molecules and constructs are provided that are useful for modulating gene expression in plants. One or more expression cassette(s) of a recombinant DNA molecule or construct may be excised from transgenic plants following transformation by the presence of flanking site-specific recombination sites in the recombinant DNA molecule or construct by expression of a site-specific recombinase enzyme encoded by the recombinant DNA molecule or construct. Such a recombinase system may be used to remove expression cassette(s) from plants transformed with the recombinant DNA construct or vector. The recombinase transgene may be operably linked to a promoter suitable for autoexcision in transformed plants without crossing to a different transgenic line expressing the recombinase. Methods for causing autoexcision of one or more expression cassette(s) in a transgenic plant, and plants and cells containing or transformed with a recombinant DNA molecule or construct of the present disclosure, are also provided.

Disclosed are altered recombinases wherein at least one amino acid is different from a parent, wildtype recombinase and the altered recombinase has improved recombination efficiency towards wild-type and / or pseudo att site sequences relative to the parent, wildtype recombinase. Disclosed are altered modular recombinases comprising binding (or interacting) regions from a different recombinase. Disclosed are methods of modifying the genomes of cells using the altered recombinases, including methods of site-specifically integrating a polynucleotide sequence of interest in a genome of a eucaryotic cell.

The application discloses a corynebacterium striatum phage and application thereof. The corynebacterium striatum phage is a corynebacterium striatum phage CSP1, the preservation number of which is CGMCC NO: 45598, and the preservation time is July 4, 2023. The corynebacterium striatum phage has strong tolerance, biological safety and carries site-specific recombination functional elements (integrase and recombination sites). Meanwhile, the corynebacterium striatum phage can lyse 20 strains of multi-drug resistant corynebacterium striatum, and lays a foundation for subsequent medical researches using the corynebacterium striatum phage as a test and treatment carrier, a targeted pathogenic bacterium or symbiotic bacterium, and an immune regulator inducer.

The invention discloses a recombinase-based highlight induced photosynthetic electron transfer loop and application thereof, the loop uses a highlight induced ELIP1 promoter to drive FLP recombinase expression, and regulates and controls the expression of a photosynthetic electron transfer key component gene PETE or PETC through an FLP / FRT site specific recombination system. The problem of light inhibition caused by overload of a plant photosynthetic electron transfer chain in a highlight environment is solved, light intensity dependent gene expression regulation and control are realized, the photosynthetic electron transfer characteristic under highlight stress is effectively improved, and the photoadaptability and photosynthetic efficiency of plants are enhanced. The system provides a new tool for crop stress resistance molecular breeding and synthetic biology research.

The present disclosure relates to a novel screening method for screening a plurality of pGREs in parallel and nucleotide constructs for use in the method. The construction body comprises a nucleotide block, the sequence of the nucleotide block is PRIMREG1-pGREE-PRIMREG2-GENEE or GENE-PRIMREG1-pGREE-PRIMREG2, and the PRIMREG1 is a primer recognition sequence 1 or an FLEx box containing the primer recognition sequence 1. PGRE is a presumed gene regulatory element, PRIMREG2 is a primer recognition sequence 2 or a FLEx cassette comprising a primer recognition sequence 2, GENE comprises nucleotide blocks REPSEQ, IRES and SSR, where REPSEQ is a reporter gene and is present or absent, IRES is an internal ribosome entry site or a 2A self-cleaving peptide, and SSR is a site-specific recombinase gene. At least one of the PRIMREG1 and the PRIMREG2 is a FLEx cassette comprising a primer recognition sequence.

Recombinant DNA molecules and constructs are provided that are useful for modulating gene expression in plants. One or more expression cassette(s) of a recombinant DNA molecule or construct may be excised from transgenic plants following transformation by the presence of flanking site-specific recombination sites in the recombinant DNA molecule or construct by expression of a site-specific recombinase enzyme encoded by the recombinant DNA molecule or construct. Such a recombinase system may be used to remove expression cassette(s) from plants transformed with the recombinant DNA construct or vector. The recombinase transgene may be operably linked to a promoter suitable for autoexcision in transformed plants without crossing to a different transgenic line expressing the recombinase. Methods for causing autoexcision of one or more expression cassette(s) in a transgenic plant, and plants and cells containing or transformed with a recombinant DNA molecule or construct of the present disclosure, are also provided.

The invention belongs to the technical field of bioengineering, and particularly relates to a recombinant plasmid for promoting synthesis of tylosin, an engineering bacterium and a preparation method thereof, the genetically engineered bacterium (Streptomyces fradiae G3-F2) is preserved in China Center for Type Culture Collection on February 24, 2025, and the preservation number is CCTCC NO: M 2025278. According to the streptomyces fradiae G3-F2 disclosed by the invention, through site-specific recombination, a recombinant plasmid is constructed by utilizing an optimized ZouA gene DNA amplification system, so that multi-copy increase of a tylF gene is realized, and further, streptomyces fradiae G3-F2 is prepared; the enzymatic reaction rate of the methyltransferase is greatly improved; the efficient conversion of the component C is realized; and low-temperature culture fermentation is combined, so that the tylosin synthesis rate and efficiency are greatly improved. The titer of the strain in a 50L tank can reach 18500U / mL under the low-temperature culture condition of 28 DEG C, so that the production cost can be greatly reduced.

Provided are compositions and methods for making a diploid yeast having heterozygosity for at least one chromosome. The method includes mating haploid yeast having at least a first modified chromosome comprising a synthetic chromosome suitable for recombination-site-mediated evolution (as a SCRaMbLE-ready modification). The SCRaMbLE-ready modification includes introduced site-specific recombinase recognition sites that can be recognized by a recombinase. Yeast that have at least one SCRaMbLE-ready modification of a chromosome are mated with a haploid yeast devoid of the SCRaMbLE-ready modification to obtain diploid SCRaMbLE-ready yeast. Subsequent to mating the haploid SCRaMbLE-ready yeast to yeast devoid of the SCRaMbLE-ready modification the method includes using the recombinase to recombine the first modified chromosome to obtain heterozygous diploid yeast comprising at least one recombined (SCRaMbLEd) chromosome and a homologous non-SCRaMbLEd chromosome. The method further includes identifying heterozygous diploid yeast that comprise the at least one SCRaMbLEd chromosome that have a changed phenotype that is different from the phenotype of the diploid SCRaMbLE-ready yeast. Yeast made by the methods, compositions of matter made by the yeast, spores made by the yeast, and kits containing the yeast and / or their spores are included.

The application discloses a method for continuous editing of multi-site chromosomes in eukaryotic cells. The application provides a method for integrating the genome of eukaryotic cells, comprising: using a knock-in site-specific recombination site (such as noxM) in the genome of eukaryotic cells, realizing integration of a large fragment circular DNA molecule at a specific site of the genome under the action of a knock-in site-specific recombinase (such as Nigri), and simultaneously using an adjacent knock-out site-specific recombination site (such as loxP or a mutant thereof), rearranging the related sites under the action of a knock-out site-specific recombinase (such as Cre), so as to realize continuity of integration. The novel editing platform of high-efficiency site-specific recombination provided by the application can realize directional, continuous and multi-site integration of a large fragment DNA in eukaryotic cells, and the application has important application prospects in the field of biological technologies including plant breeding, animal cell line development, fungus engineering and the like.

This invention belongs to the field of bioengineering technology, specifically relating to a recombinant plasmid for tylosin synthesis, an engineered bacterial strain, and its preparation method. The genetically engineered bacterial strain (Streptomyces freundii G3-F2) was deposited at the China Center for Type Culture Collection (CCTCC) on February 24, 2025, with accession number CCTCC NO: M 2025278. This invention achieves site-specific recombination, utilizes an optimized ZouA gene DNA amplification system to construct the recombinant plasmid, and realizes… tulle Multiple copies of the F gene were increased, resulting in the preparation of *Streptomyces freundii* G3-F2. This significantly improved the rate of methyltransferase enzymatic reaction and achieved efficient conversion of component C. Combined with low-temperature fermentation, the rate and efficiency of tylosin synthesis were greatly enhanced. The strain achieved a titer of 18500 U / mL in a 50L tank under 28℃ low-temperature culture conditions, which can significantly reduce production costs.

The application provides a tumor cell dormancy and awakening marker tracing system (DormTracer), specifically, the system combines a gene switch and a site-specific recombination technology, expression of a reporter gene is driven by a recombination enzyme fused with a dormancy marker protein, the reporter gene is affected by a cell dormancy history, but does not affect the cycle of cell dormancy and proliferation, therefore, the tumor cell dormancy history can be traced, and the current instantaneous state of the cell is not limited to monitoring. The system can distinguish tumor cells in three states of proliferation, dormancy and dormancy awakening.