246 results about "Transcription Activation" patented technology

The present invention provides RNA-guided endonucleases, which are engineered for expression in eukaryotic cells or embryos, and methods of using the RNA-guided endonuclease for targeted genome modification in eukaryotic cells or embryos. Also provided are fusion proteins, wherein each fusion protein comprises a CRISPR / Cas-like protein or fragment thereof and an effector domain. The effector domain can be a cleavage domain, an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repressor domain. Also provided are methods for using the fusion proteins to modify a chromosomal sequence or regulate expression of a chromosomal sequence.

The invention relates to a gene targeting system, which comprises two parts such as a site-specific cleavage nuclease expression vector and a targeting vector, wherein the targeting vector contains 2-10 donor DNA fragments, 5' ends and 3' ends of every donor DNA fragment are respectively inserted into recognition sequences of the site-specific cleavage nuclease, the donor DNA comprises an upstream homologous arm, a downstream homologous arm and an exogenous DNA sequence positioned between the upstream homologous arm and the downstream homologous arm, and the site-specific cleavage nuclease expression vector is any one selected from an expression vector carrying zinc finger nuclease, a transcription activator-like effector nuclease expression vector, and a RNA-mediated nuclease RNA:Cas9 expression vector.

The present invention relates to Transcription Activator-Like Effector (TALE) derived proteins that allow to efficiently target and / or process double stranded nucleic acid sequences. The proteins of the invention are typically chimeric protein monomers composed of a core scaffold comprising Repeat Variable Dipeptide regions (RVDs) having binding specificity to a DNA target sequence, to which is fused a catalytic domain to its N-terminal. This later catalytic domain, which can be a monomer of a nuclease, is placed at this position to possibly interact with another catalytic domain fused to another TAL monomer, such that, when said monomers are binding to their respective target DNA sequences, both catalytic domains form a catalytic entity likely to process DNA in the proximity of these target sequences. This new TAL architecture makes it possible to target only one DNA strand, which is not the case, for instance, with classical TALEN architectures. The present invention also relates to vectors encoding such proteins and compositions or kits in which Transcription Activator-Like Effector (TALE) proteins of the present invention are used.

The invention discloses a pair of transcription activator-like effector nucleases, and encoding genes and application thereof. The pair of transcription activator-like effector nucleases (TALEN) is obtained by fusing a pair of deoxyribonucleic acid (DNA) recognition proteins and two heterologous subunits of a Flavobacterium okeanokoites 1(Fok 1) DNA incision enzyme and can recognize two adjacent loci on goat or sheep beta lactoglobulin gene (BLG) exon2 specifically. When the transcription activator-like effector nucleases are transferred to a host cell simultaneously, the exon2 loci of the BLG gene of the host cell can be targeted by the transcription activator-like effector nucleases, and the targeted loci are subjected to genetic mutation, so that the targeted modification of the goat or sheet BLG gene is realized, and the transcription activator-like effector nucleases have the advantages of high specificity, high targeting efficiency, high accuracy and the like.

The present invention provides a pair of short peptides, proteins and polynucleotides, a host cell and application thereof, and provides propose an artificial synthetic method for transcriptional activator-like effector nuclease (TALEN) gene of human cell endogenous IL2RG gene efficient targeting, and a method for orientation targeting by using a recombinant plasmid containing the gene. The TALEN comprises a pair of transcription activator-like effector (TALEs) proteins and DNA incision enzyme catalytic subunit respectively fused with the proteins, and can respectively recognize two adjacent sites on human IL2RG gene exon 2. On the basis of designing a TALEs amino acid sequence, a nucleotide sequence encoding the TALEN is synthesized and a vector containing the nucleotide sequence is constructed. By using the TALEN plasmid to transfect cells, the efficiency of cell targeting can be greatly improved.

Some aspects of this disclosure provide compositions, methods, systems, and kits for controlling the activity and / or improving the specificity of RNA-programmable proteins, such as Cas9. For example, provided are guide RNAs (gRNAs) that are engineered to exist in an “on” or “off” state, which control the binding and, in certain instances, cleavage activity of RNA-programmable proteins (e.g., RNA-programmable endonucleases). By incorporating ligand-responsive self-cleaving catalytic RNAs (aptazymes) into guide RNAs, a set of aptazyme-embedded guide RNAs was developed that enable small molecule-controlled nuclease-mediated genome editing and small molecule-controlled base editing, as well as small molecule-dependent transcriptional activation in mammalian cells.

The invention relates to a high-efficiency artificial activating transcription factor dCas9-TV, and a coding gene and applications thereof. According to a construction method, the carboxyl terminal ofnuclease inactivated Cas9 protein (dCas9) is connected with a plurality of copies of VP64 and TAL transcription-activating domains so as to obtain a series of novel artificial activating transcription factors, and obtain dCas9-TV with the best transcriptional activation activity via screening. When only one guide RNA (gRNA) is adopted for targeting a specific gene promoter, dCas9-TV is capable ofrealizing high efficiency activating of transcription of endogenous genes of Arabidopis thaliana and paddy rice; when a plurality of gRNA are adopted for targeting a plurality of target genes, dCas0-TV is capable of realizing transcription activation of a plurality of genes. In addition, it is confirmed that dCas9-TV possesses the same high efficiency targeting transcription activation activity in human cells. An in vitro assembled dCas9-TV / gRNA ribonucleoprotein compound can be adopted for transcription activation of Arabidopis thaliana and paddy rice endogenous genes. The high-efficiency artificial activating transcription factor dCas9-TV can be adopted in the fields such as genome genetic screening, metabolite biosynthesis pathway reconstruction, and crop improvement.

Increasing tolerance to butanol in yeast has been accomplished by increasing activity of the filamentous growth response. Yeast with increased expression of MSS11p, a transcriptional activator of the filamentous growth response pathway had increased tolerance to isobutanol. These yeast may be used for improved butanol production.

The invention relates to materials and methods useful for controlling the unwanted spread of transgenic traits. The methods involve a male-sterile female containing a transgene for a desired trait and a transgene causing seed infertility. The methods also involve a male-fertile plant carrying a transcription activator that activates expression of both transgenes carried by the male-sterile female. Pollination of the male-sterile female by a male-fertile plant activates expression of both transgenes in the female. The resulting seeds express the gene product of the desired trait and are infertile.

The invention discloses a method for improving the winter resistance of Japanese lawngrass. This method includes converting transcription activation factor CBF1 / DREB1b of quasi-mustard induction gene into Japanese lawngrass embryonal callus to improve the winter resistance of Japanese lawngrass, which can include the following steps: 1) disseminating the Japanese lawngrass embryonal callus with agricillin liquor carrying CBF1 / DREB1b; 2) placing the disseminated Japanese lawngrass embryonal callus onto the coculture medium to perform coculture; 3) placing the cocultured embryonal callus onto the screening culture medium for screening; 4) placing the screened embryonal callus onto the differentiated culture medium for differentiation culture; 5) placing the differentiated and germinated young plant onto the rooted culture medium for radication; 6) placing the plant which can produce root onto strong-plant screening culture medium for screening the Japanese lawngrass strong-plant which has improved winter resistance. The invention has important theoretical and operation significance for culturing new species of Japanese lawngrass with improved cold resistance and extended green period and has a wide application foreground.

The invention discloses a vector for efficiently labeling zebra fish PGC, and a preparation method and a use of transgenic fish. A Gal4 / UAS transcription activation system is utilized and mRFP is used as a report gene to realize an inductive gene expression regulation technology in the primordial germ cells of zebra fish. The preparation method of the transgenic fish comprises the following steps: 1, respectively constructing activation transgenic line Tg (kop:KalTA4) and an effect transgenic line Tg (UAS:mRFP); and 2, hybridizing Tg (kop:KalTA4) male fish with Tg (UAS:mRFP) female fish to obtain the transgenic fish for efficiently labeling the primordial germ cells of zebra fish. The transgenic fish for efficiently labeling the primordial germ cells of zebra fish can be widely applied to the fish bioengineering.

The invention discloses a wheat protein TaMYB1 and a coding gene and application thereof. The protein provided by the invention is shown in (a) or (b), wherein (a) refers to a protein composed of an amino acid sequence shown in a sequence 2 in a sequence table, and (b) refers to a protein which is related to disease resistance or has transcription activation activity and is derived from the sequence 2 through substitution and / or deletion and / or addition of one or more amino acid residues of the amino acid sequence shown in the sequence 2 in the sequence table. Experimental results in the invention show that the protein TaMYB1 can resist diseases caused by Blumeria graminis f.sp.tritici physiological race E09 or Blumeria graminis f.sp.hordei after transient expression of the protein, has transcription activation activity and can be used as a transcription activating factor. Research in the invention shows that the protein lays a foundation for research on cultivation of a transgenic plant with disease resistance.

The present invention relates to an improved method for the production of recombinant human blood clotting factors, in particular of factor VIII and factor IX, utilizing an immortalized human cell line stably expressing viral transcription activator proteins and carrying a vector having a promoter functionally linked to a DNA sequence coding for a blood coagulating factor, provided that said promoter is not a viral promoter which is stimulated by said viral transcription activator proteins; an immortalized human cell line carrying said vector, factor VIII muteins particularly suitable for the above production method; pharmaceutical compositions comprising such factor VIII muteins and the use of such factor VIII muteins for preparing a medicament for treating hemophilia.

The present invention provides compositions and methods for regulating expression of nucleotide sequences in a plant. Compositions comprise novel nucleic acid sequences encoding a transcriptional activator involved in modulating gene expression in response to abiotic stress such as cold or drought. Methods for expressing the nucleic acid sequence in a plant and improving cold and / or drought tolerance of plants are also provided.

A method and a kit for detecting interactions between three or more proteins, in vivo, using reconstitution of the activity of a transcriptional activator is provided. Reconstitution of the transcriptional activator makes use of chimeric genes which express hybrid proteins. In one embodiment, three types of hybrid proteins are prepared. The first hybrid contains the DNA-binding domain of a transcriptional activator fused to the first test protein. The second hybrid protein contains a transcriptional activation domain fused to the second test protein. The third hybrid protein contains a nuclear localization peptide fused to a third test protein and mediates assembly of the three-protein complex involving the three hybrids. If the three test proteins are able to interact, they bring into close proximity the two domains of the transcriptional activator. This proximity is sufficient to cause transcription, which can be detected by the activity of a marker gene that contains a binding site for the DNA-binding domain.

A method for treating a disease associated with transcription activation by NF-κB by administering a composition for inhibiting transcription activation by NF-κB to a patient in need of treatment of the disease. The composition contains a fraction having a molecular weight of 3,000 or less obtained by purifying a water extract of Sargassum horneri as an active ingredient.

A binary expression carrier of Agrobacterium for culturing the seeds of plant by genetic engineering contains the closely linked corm ubiquitin promoter Pubi regulated Arabidopsisí» cold response gene transcription activation factor gene cbf1 and the Arabidopsis specific senescence gene promote SAG12 regulated iso-pentenyl transferase ipt geneí»s two-valence stress-resistant gene. Its preparing process includes such steps as inserting the T-Nos of sag12-ipt fusion gene into a binary carrier, inserting ubi-cbf1 fusion gene and inserting sag12-ipt fusion gene.

The invention provides a hybrid crop transgenic safety control method. An transgenic plant automatic deletion double-unit system, comprising a recombinase system, a transcription activation system and an exogenous gene expression control system, is established; and a plant flower primordium cell specific promoter is utilized as a promoter in the transgenic plant automatic deletion double-unit system to control the transcription activation system, and the transcription activation system controls the start of the recombinase system, so that the exogenous gene introduced into the plant stably exists in the F1-generation plant hybrid as well as root, stem, leaf and other non-deletion tissues, but is deleted in the pollen and seed of the F1-generation plant, thereby implementing the hybrid crop transgenic safety control.

The invention relates to a chimeric antigen receptor and an expression gene thereof, a double-antigen regulated type T cell modified by the chimeric antigen receptor, and application thereof. The expression gene of the chimeric antigen receptor comprises a first fusion protein expression gene and a second fusion protein expression gene, wherein the first fusion protein expression gene comprises a mucoprotein-1 antibody expression gene, a notch receptor expression gene and a Gal4-VP64 transcription activating protein expression gene which are sequentially connected; the second fusion protein expression gene comprises a Gal4-UAS promoter expression gene and an anti-mesothelin expression gene which are sequentially connected. By adopting the innovative design, the chimeric antigen receptor has the advantages that the chimeric antigen receptor can be successfully imported into the T cell to form the T cell modified by the chimeric antigen receptor; the immune reaction cannot be produced until the mucoprotein-1 and the anti-mesothelin signals simultaneously exist, so as to reach the controllable immune reaction of the chimeric antigen receptor, fewer side effects in treatment, and high specificity.

The invention relates to a solid-phase synthesis method of transcription activator-like effector (TALE). The method comprises the following steps of: immobilizing a segment of nucleic acid adaptation sequence (DNA (Deoxyribonucleic Acid) linker) to a solid-phase interface, and shearing with a restriction endonuclease to generate 3'-cohesive end; contacting a product with TALE linkage unit which is treated by the restriction endonuclease and is provided with 5'-cohesive end, connecting under the action of a DNA ligase, and eluting unconnected matrix; treating the product obtained in the previous step with restriction endonuclease to shear and generate 3'-cohesive end on DNA again, contacting a product with TALE linkage unit which is treated by the restriction endonuclease and is provided with 5'-cohesive end, connecting under the action of a DNA ligase, and eluting unconnected matrix; repeatedly operating the steps for at least one time; shearing the connected DNA molecule from the solid-phase interface with restriction endonuclease, and separating and purifying to remove the unconnected matrix; and expressing the DNA molecule into protein molecule, namely fusion protein (such as TALEN (TALE nuclease) or TALEA (TALE activator)) containing target TALE.

The invention discloses a pair of transcription activator like effector nucleases, a coding gene and an application thereof. The pair of transcription activator like effector nucleases (TALEN) is obtained by respective fusion of a pair of DNA recognins with two heterogenous subunits of a Fok1 DNA endonuclease, and can specifically recognize two adjacent loci on human RHD or RHCE gene exon1. When the pair of transcription activator like effector nucleases is simultaneously transferred to a host cell, targeting of the exon1 loci of the host cell gene can be realized; the targeted loci are subject to gene mutation; therefore, targeting modification of the human RHCE or RHD gene is realized, and advantages of strong specificity, high targeting efficiency, high accuracy and the like are provided.

The present disclosure provides a combinatorial metabolic engineering system based on an orthogonal tri-functional CRISPR system that combines transcriptional activation, transcriptional interference, and gene deletion (CRISPR-AID). This strategy enables perturbation of the metabolic and regulatory networks in a modular, parallel, and high throughput manner. The present disclosure further provides a multi-functional genome-wide CRISPR (MAGIC) system for high throughput genotype-phenotype mapping.

The present invention discloses a method for splitting Cas9 and an application. The present invention provides a method for splitting Cas9 protein at a plurality of sites of the Cas9 protein into different segments of amino acid sequences. The Cas9 protein is spCas9(D10A) protein. The split Cas9 protein can be transported into target cells / vectors more conveniently, and can be recombined by meansof intein. Accordingly, the present invention provides proteome and fusion proteome obtained by the method and nucleic acid constructs and vectors for expressing the fusion proteome, and engineering cells. The fusion proteome and the vectors can be used for editing cytogenes, or in targeted positioning or gene expression transcription activation or gene expression transcription inhibition, and used in preparation of pharmaceutical preparations for gene editing.