68 results about "Transcription Activator-Like Effectors" patented technology

The present invention relates to a method for the generation of compact Transcription Activator-Like Effector Nucleases (TALENs) that can efficiently target and process double-stranded DNA. More specifically, the present invention concerns a method for the creation of TALENs that consist of a single TALE DNA binding domain fused to at least one catalytic domain such that the active entity is composed of a single polypeptide chain for simple and efficient vectorization and does not require dimerization to target a specific single double-stranded DNA target sequence of interest and process DNA nearby said DNA target sequence. The present invention also relates to compact TALENs, vectors, compositions and kits used to implement the method.

This application provides transcription activator-like effector nucleases (TALENs), polynucleotide sequences encoding the TALENs, expression cassettes for producing TALENs to target cleavage of nucleic acids, and methods of producing and using the TALENs.

Engineered transcriptional activator-like effectors (TALEs) are versatile tools for genome manipulation with applications in research and clinical contexts. One current drawback of TALEs is their tendency to bind and cleave off-target sequence, which hampers their clinical application and renders applications requiring high-fidelity binding unfeasible. This disclosure provides engineered TALE domains and TALEs comprising such engineered domains, e.g., TALE nucleases (TALENs), TALE transcriptional activators, TALE transcriptional repressors, and TALE epigenetic modification enzymes, with improved specificity and methods for generating and using such TALEs.

Engineered transcriptional activator-like effectors (TALEs) are versatile tools for genome manipulation with applications in research and clinical contexts. One current drawback of TALEs is their tendency to bind and cleave off-target sequence, which hampers their clinical application and renders applications requiring high-fidelity binding unfeasible. This disclosure provides engineered TALE domains and TALEs comprising such engineered domains, e.g., TALE nucleases (TALENs), TALE transcriptional activators, TALE transcriptional repressors, and TALE epigenetic modification enzymes, with improved specificity and methods for generating and using such TALEs.

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.

The invention relates to a high-efficiency synthesis method of TALE (transcription activator like effectors) repeated segments. The high-efficiency synthesis method includes designing DNA (deoxyribonucleic acid) template and a primer for PCR (polymerase chain reaction) amplification, utilizing uracil-specific excision reagent (USER) to manufacture adhesive tail ends at two ends of the DNA segments by the uracil DNA excision method, connecting a plurality of DNA segments of a single, two or three TALE repeated units with carriers according to correct sequence to obtain recombinant carriers of TALE proteins capable of expressing specific target genes, and completing knockout, knock-in, overexpression and suppression expression of the target genes by means of the recombinant carriers. The high-efficiency synthesis method is timesaving, convenient, high in correctness and capable of realizing high throughput and meeting automatic requirements.

The invention discloses a side unit used for building TALE (transcription activator-like effector) repeated sequences. The side unit is a repeated unit DNA (deoxyribonucleic acid) segment containing isocaudarner or different flat terminase identification sites at two ends, and the preated unit DNA segment codes repeated units of RVD (repeated variable di-residue) containing NI, NG, HD, NK or NN or variants of the repeated units, wherein in the 5'end isocaudarner or flat terminase identification sites, the 3' end of the identification sites at least has one nucleotide participating in the amino acid coding the N end of the side unit; and in the 3' end isocaudarner or flat terminase identification sites, the 5' end of the identification sites at least has one nucleotide participating in the amino acid coding the C end of the side unit. The side unit has the advantages that the TALE repeated sequences containing any repeated unit number and any ranging sequences, plasmid vectors containing TALE repeated sequences, coding TALE protein DNA combined structural domains and various derived fusion protein plasmid vectors can be conveniently built.

The invention relates to biotechnology and provides novel methods for sequence-specific or sequence-directed transcription activator-like effector recombinase-mediated integration of DNA sequences of interest into host genomes. The invention also provides methods of use for novel plant transformation vectors and expression cassettes, which include novel combinations of chimeric recombinases with plant expression and transformation elements. Methods for gene-targeting, DNA sequence removal, genome modification, and molecular breeding are also provided.

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 provides a novel TALE (Transcription Activator Like Effectors) protein and the application of the novel TALE protein. Through screening 400 different RVDs, a novel RVD of which the specificity is bound with a certain DNA basic group or combined with more than one DNA basic group at the same time is successfully identified, the joint efficiency of the novel RVD and the basic group or groups is detected, the novel RVD is used for synthesizing the TALE protein highly specifically and efficiently combined with a DNA target site, so that the choice of the target site of the TALE protein is more diversified, and the potential off-target effect is reduced.

The invention relates to the field of gene therapy and discloses a fixed-point knockout system for high-risk HPV (human papilloma virus) E6E7 gene, namely targeted cutting of TALENs (transcription activator-like effector nucleases) of HPV E6E7 gene, wherein a TALENs expression vector of the target specific site is designed according to the high-risk HPV E6E7 gene sequence, and the HPV E6E7 gene sequence in the cells are cut in a targeted manner so as to knock out the target gene and induce the apoptosis increase and proliferation inhibition of corresponding subtype cells while the TALENs do not act on the HPV positive or HPV negative cells of other subtypes. By transfecting the TALEN expression vector in a subcutaneous tumor model of HPV positive cervical cancer cells, the growth of subcutaneous tumor can be obviously inhibited, and the tumor weight is reduced. According to the method for targeted knockout of high-risk HPV E6E7 gene using TALEN provided by the invention, the virus oncogene can be efficiently broken on the DNA level so as to cause apoptosis and proliferation inhibition of the HPV infected cells, and the method is of tremendous therapeutic value on HPV infection related diseases and particularly precancerous lesions such as cervical intraepithelial neoplasias.

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 invention aims to provide modular construction of a synthetic gene route in a mammalian cell by a TALE (transcription activator-like effector) transcription suppressor. According to a protected method for realizing regulatable expression of two proteins involved in the invention, an expression cassette A includes: a coding sequence of a feedback element, a promoter A, protein A's coding gene and TALER protein A's coding gene that are connected through self-shearing polypeptide, and a target sequence A (including the target sequence of shRNA1); an expression cassette B includes: a coding sequence of a feedback element, a promoter B, protein B's coding gene and TALER protein B's coding gene that are connected through self-shearing polypeptide, and a target sequence B (including the target sequence of shRNA2); and an expression cassette C includes a constitutive promoter and a coding sequence of an activating element. A recombinant vector A with the expression cassette A, a recombinant vector B with the expression cassette B and a recombinant vector C with the expression cassette C are imported into a host cell, and shRNA1 or shRNA2 is added to regulate expression of protein A and protein B.

The invention relates to transcription activator like effectors (TALE). The TALE can identify and bind with DNA fragments in human CCR5 gene active regions or CXCR4 gene active regions. The human CCR5 gene active regions comprise three regions and the CXCR4 gene active regions comprise two regions. The TALE binds with endonuclease so that a fusion protein TALEN is formed. Through TALEN specific loci aiming at the CCR5 or CXCR4 gene, a shearing process is carried out so that after gene regulation and control, the cell has HIV resistance.

The invention relates to the field of genetic engineering and discloses a pair of polypeptides and encoding genes thereof, with sequences shown as SEQ ID NO. 1 to 4. The pair of polypeptides is used for establishing a pair of a recombinant transcription activator like effector (TALE) and a transcription activator like effector nuclease (TALEN); the TALEN is a fusion protein formed by fusing a pair of DNA recognizing proteins with a DNA severing protein respectively; and the TALEN can perform targeted severing on a target site of a human insulin gene so as to achieve targeted modification on the human insulin gene, with the characteristics of strong specificity, high efficiency and high accuracy.

The invention discloses an MCIR (MelanoCortin 1 Receptor) gene carrier and a construction method thereof. The MCIR gene carrier comprises MC1R-neo and MC1R-GFP (Green Fluorescent Protein), wherein the nucleotide sequence of the MC1R-neo is SEQ ID NO:1, and the nucleotide sequence of the MC1R-GFP is SEQ ID NO:2. A CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas9 system is adopted to construct an MC1R gene knock-out vector, and only a sgRNA (Ribonucleic Acid) of which the length is about 20bp needs to be designed by aiming at a gene knock-out point to be connected with a universal Cas9 gene. Therefore, compared with traditional gene knock-out technologies including ZFNs (Zinc finger nuclease), TALENs (Transcription Activator-Like Effector Nucleases) and the like, the construction method disclosed by the invention adopting the CRISPR / Cas9 to construct the gene knock-out vector is simpler and more convenient and is more convenient to be further popularized and applied to subsequent experiments.

The invention discloses a novel intracellular scaffold structure based on TALE (transcription activator-like effectors) and a method. The intracellular scaffold structure comprises a DNA scaffold sequence constructed on a plasmid vector and two sections of adjacent TALE-enzyme infusion protein binding sequences. By virtue of a TALE-DNA scaffold technology, an intracellular heterogeneous metabolic pathway enzyme system is co-regionalized, so that a metabolism rate is improved and the yield of target metabolites is increased.

The present invention relates to polypeptides and more particularly to Transcription Activator-Like Effector derived proteins that allow to efficiently target and / or process nucleic acids. The present invention also concerns methods to use these proteins. The present invention also relates to vectors, compositions and kits in which RVD domains and Transcription Activator-Like Effector (TALE) proteins of the present invention are used.

The invention discloses an efficient assembling method of a transcription activator-like effectors (TALE) repeating region for editing a silkworm genome. The efficient assembling method specifically comprises the steps of firstly numbering random sequences containing X basic groups sequentially and dividing the random sequences into Y groups according to numbers, wherein each group contains 1-4 basic groups; designing a primer pair for amplifying TALE repeating units, and then utilizing the designed primer pair to perform polymerase chain reaction (PCR) amplification with four types of regulatory volume decrease (RVD) repeating units as templates so as to obtain a PCR product library, wherein the primer pair contains II type restriction enzyme recognition sites and specific joints connected with adjacent TALE repeating units in a seamless mode; numbering and grouping target sequences with X basic groups by means of the same method, taking out PCR products with the same numbers as the target sequences and identifying corresponding basic groups from the PCR product library, mixing the PCR products in the same groups, performing GoldenGate connection, and then performing the GoldenGate connection of connection products again to obtain the TALE repeating region containing X repeating units. By utilizing the method, a large amount of deoxyribonucleic acid (DNA) is not required to be synthesized, and the cost is low. The efficient assembling method can be used for preparing the TALE repeating region for editing the silkworm genome.

The invention discloses a goat MSTN (myostain) gene fixed-point modification system and an application method thereof. The goat MSTN gene fixed-point modification system can effectively identify and modify transcription activator like effector nucleases (TALENs) of a goat MSTN gene target sequence, the TALENs provided by the invention is composed of nucleases TALEN-R and TALEN-L which can respectively identify identification modules TALMEX-1F and TALMEX-1R; the nuclease TALEN-L is a protein coded by a nucleotide sequence shown in SEQ ID NO.2 or SEQ ID NO.3, and the nuclease TALEN-R is a protein coded by a nucleotide sequence shown in SEQ ID NO.4 or SEQ ID NO.5.The invention also discloses a method for obtaining a muted cell line by carrying out targeted modification on a goat cell MSTN gene by adopting the knock-out system. According to the goat MSTN gene fixed-point modification system, the goat MSTN gene is knocked out or modified for obtaining a new variety with a high high-quality meat factor, so that the goat MSTN gene fixed-point modification system has an important application value on mechanism study of an MSTN gene regulation and control mechanism.

The present invention relates to a method for the detection of a specific nucleic acid. Specifically, the invention provides a method and kits for detecting the presence of a specific nucleic acid using engineered DNA-binding domains such as Transcription Activator Like-Effector (TALE) domain or modular base-per-base binding domains (MBBBD). The method of the invention is particularly useful for in vitro diagnostic application.

The invention discloses an efficient transgenosis method with mediation of a transcription activator-like effector (TALE) protein. The method comprises the steps that a helper plasmid containing a transcription activator-like effector protein DNA (Deoxyribonucleic Acid) sequence and piggyBac transposon (PBase) DNA sequence is constructed by adopting a molecular biological technique, and transcribed to mRNA (Messenger Ribonucleic Acid) in vitro; mRNA of the helper plasmid and DNA of a piggyBac transposon plasmid containing an exogenous gene are imported to a fertilized egg of an animal by adopting a microinjection method; a transgenic animal of which the exogenous gene can be stably inherited and expresses is obtained; and a transgenosis positive individual is screened out according to a marker gene or the expression characteristic of the gene. With the adoption of the method, a piggyBac transposon can be efficiently inserted into a genome of a host organism by the action of a TALE-PBase fusion protein; the transgenosis efficiency mediated by the piggyBac transposon can be improved remarkably; and helps are offered to researches such as transgenic animal acquirement and insertion mutagenesis.

The invention relates to a transcription activator like effector (TALE)-functional group-estrogen receptor (ER) function protein and an application thereof, wherein the TALE-functional group-ER function protein is formed by sequentially linking TALE, ER and a functional group or sequentially linking TALE, a functional group and ER, wherein the functional group is a combination comprising one or more than two materials selected from activation domain (AD), nuclease, methylase, demethylase and recombinase. During practical application, tamoxifen (TAM) or 4-OH-tamoxifen (4-OH-TAM) are introduced into cytoplasm so as to control the function protein to enter nucleus at the specific time.

The invention relates to a site-directed mutagenesis system of a mustard genome and an application of the site-directed mutagenesis system' and belongs to the technical field of biology. The site-directed mutagenesis system of the mustard genome is characterized by comprising two transcription activator-like effector nucleases (TALEN) in two target sequences of a specifically-recognized target gene, wherein the target sequences include a first target sequence and a second target sequence, the first target sequence is located at a 5'-terminal of the target gene, and the second target sequence is located at a 3'-terminal of the target gene; the two transcription activator-like effector nucleases include transcription activator-like effector nucleases TALEN-L and TALEN-R which can specifically recognize the first target sequence and the second target sequence. The invention further discloses an application of a deletion system. According to the application, rich breeding materials can be provided, and the site-directed mutagenesis system has a wide application prospect.

The present invention relates to polypeptides and more particularly to Transcription Activator-Like Effector derived proteins that allow to efficiently target and / or process nucleic acids. Particularly, the present invention reports the characterization of TALE derived proteins that can efficiently target methylated DNA. The present invention more specifically relates to TALE derived proteins that allow activation of methylated promoters responsible for gene silencing.

The invention relates to a construction method of a TALE (Transcription Activator-Like Effector) aggregate library. The method is capable of establishing the TALE aggregate library through TALE monomer amplification, construction of an enzyme-cut and link-up product, amplification of the enzyme-cut and link-up product and BP reaction. With the TALE aggregate library, 20-25 bpTales modules can be assembled and identified, and the identification region is expanded. The method is high in accuracy rate, flexible and high in throughput, and also capable of establishing carriers accurately at high throughput and greatly saving the cost. The method is capable of expanding the identification region, accurately establishing the carriers at high throughput and greatly saving the cost.

The invention discloses a pair of transcription activator-like effector nucleases (TALENs) and coding genes and application thereof. The pair of TALENs are obtained through a pair of deoxyribonucleic acid (DNA) recognition proteins which are respectively merged with two heterogenous subunit of a Fokl DNA incision enzyme, and can specifically recognize two adjacent loci of a goat or sheep prion protein (PRNP) gene exon 2. When the pair of TALENs enters a host cell, the pair of TALENs can target at exon 2 loci of the PRNP gene of the host cell and can enable the targeted loci to be genetically mutated to realize the targeted modification of the goat or sheep PRNP gene. The pair of TALENs has the advantages of high specificity, high targeting efficiency, high accuracy and the like.

Disclosed herein are chimeric polypeptides, including compositions thereof, expression vectors, and methods of use thereof, for the generation of transgenic cells, tissues, plants, and animals. The compositions, vectors, and methods of the present invention are also useful in gene therapy techniques. The invention provides a chimeric polypeptide. The polypeptide includes: a) a recombinase, nuclease or transcription factor, or fragment thereof; and b) a transcription activator-like effector (TALE) protein.