766 results about "Gene targeting" patented technology

Gene targeting is a technique to introduce genetic change into one or more specific locations in the genome of a cell. For example, gene targeting can introduce genetic change by modifying, repairing, attenuating or inactivating a target gene or other chromosomal DNA. In one aspect, this disclosure relates to methods and compositions for gene targeting with high efficiency in a cell. This disclosure also relates to methods of treating or preventing a genetic disease in an individual in need thereof. Further disclosed are chimeric nucleases and vectors encoding chimeric nucleases.

Materials and methods for gene targeting using Clustered Regularly Interspersed Short Palindromic Repeats / CRISPR-associated (CRISPR / Cas) systems are provided herein.

The invention discloses a specie limitation-free eucaryote gene targeting method having no bio-safety influence and a helical-structure DNA sequence, and belongs to the field of gene engineering. The specie limitation-free eucaryote gene targeting method comprises the following steps of 1, designing and constructing CRISPR / Cas9 and chimeric RNA, and 2, carrying out Cas9mRNA internal translation so that Cas9 nuclease and the chimeric RNA are bonded, carrying out fixed point clipping so that DNA double-chain cleavage is realized after the clipping, and introducing an exogenous DNA by induction of a natural DNA restoration process which is a non-homologous end bonding process of cells so that cell endogenous gene modification is realized. The specie limitation-free eucaryote gene targeting method has simple processes, realizes flexible site recognition and has low energy consumption.

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 provides compositions and methods for specific gene targeting and precise editing of DNA sequences in plant genomes using the CRISPR (cluster regularly interspaced short palindromic repeats) associated nuclease. Non-transgenic, genetically modified crops can be produced using these compositions and methods.

It is intended to provide an efficient and sure gene targeting method embodied at an arbitrary position in the genome of an organism and a kit therefor. It is also intended to provide a method for targeted-disruption of an arbitrary gene in the genome of an organism which comprises: 1) the step of providing the whole sequencial data of the genome of the organism; 2) the step of selecting at least one arbitrary region in the sequence; 3) the step of providing a vector containing a sequence homologous with the region selected above and a marker gene; 4) the step of transforming the organism by the vector; and 5) the step of providing the organism under such conditions as allowing homologous recombination. Moreover, the genome of a hyperthermostable bacterium and its array are provided.

An Engineered Transgene Integration Platform (ETIP) is described that can be inserted randomly or at targeted locations in plant genomes to facilitate rapid selection and detection of a GOI that is perfectly targeted (both the 5′ and 3′ ends) at the ETIP genomic location. One element in the subject disclosure is the introduction of specific double stranded breaks within the ETIP. In some embodiments, an ETIP is described using zinc finger nuclease binding sites, but may utilize other targeting technologies such as meganucleases, CRISPRs, TALs, or leucine zippers. Also described are compositions of, and methods for producing, transgenic plants wherein the donor or payload DNA expresses one or more products of an exogenous nucleic acid sequence (e.g. protein or RNA) that has been stably-integrated into an ETIP in a plant cell. In embodiments, the ETIP facilitates testing of gene candidates and plant expression vectors from ideation through Development phases.

This invention describes gene targets for the development of therapeutics to treat drug addiction. Animal models of drug craving and relapse have been developed and used to find gene expression changes in key brain regions implicated in cocaine addiction. The genes whose expression levels are altered serve as pharmacological targets with the purpose of preventing or inhibiting cocaine craving and relapse in human cocaine addicts.

A method for producing a transgenic plant includes providing a nucleic acid molecule comprising at least two regions of nucleic acid sequence that lack sequence homology with genomic DNA of the plant cell, and at least two zinc finger nuclease recognition sites, wherein the at least two regions of nucleic acid sequence that lack sequence homology with genomic DNA of the plant cell flank the at least two zinc finger nuclease recognition sites. A plant cell or tissue having the nucleic acid molecule stably integrated into the genome of the plant cell is transformed. A plant is regenerated from the plant cell. Transgenic plants are produced by the method. Seeds are produced by the transgenic plants.

Compositions and methods are provided for integrating one or more genes of interest into cellular DNA without substantially disrupting the expression of the gene at the locus of integration, i.e., the target locus. These compositions and methods are useful in any in vitro or in vivo application in which it is desirable to express a gene of interest in the same spatially and temporally restricted pattern as that of a gene at a target locus while maintaining the expression of the gene at the target locus, for example, to treat disease, in the production of genetically modified organisms in agriculture, in the large scale production of proteins by cells for therapeutic, diagnostic, or research purposes, in the induction of iPS cells for therapeutic, diagnostic, or research purposes, in biological research, etc. Reagents, devices and kits thereof that find use in practicing the subject methods are also provided.

Gene targeting allows the deletion (knock out), the repair (rescuing) and the modification (gene mutation) of a selected gene and the functional analysis of any gene of interest. Targeting of nuclear genes has been a very inefficient process in most eukaryotes including plants and animals due to the dominance of illegitimate integration of the applied DNA into non-homologous regions of the genome. The present invention provides a method for increasing the ratio of homologous to non-homologous recombination of a polynucleotide into a host cell's DNA by suppressing non-homologous recombination. Surprisingly, the number of non-homologous recombination events can be reduced if the polynucleotide is applied as a purified single-stranded DNA, preferably coated with a single strand binding protein.

The present invention provides bacterial vectors and fusion proteins containing a TTSS polypeptide, compositions of such fusion proteins including polynucleotides, and methods of delivering one or more genes into a target cell that involve contacting the cell with a composition that includes such a fusion protein. Compositions and methods of gene delivery that involve a bacterium and a TAT, Antp, or HSV VP22 polypeptide are also disclosed. The invention also concerns methods of delivering one or more genes into a target cell utilizing a bacterium capable of becoming internalized within the cell, wherein the bacterium includes one or more genes targeted for delivery to the cell, a gene encoding an RNA polymerase, and a gene that causes lysis of the bacterium.

The invention provides a breeding method for knocking out fish MC4R gene by using a CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system. The method comprises the following steps: determination of MC4R gene targeted site, preparation of gRNA, in-vitro microinjection and knock-out verification, screening of MC4R-gene-knock-out fish, culture of hybrid fish, and the like. The breeding method is suitable for all commercial fishes, and can implement quick growth and breeding of fishes by knocking out the fish MC4R gene. Compared with the traditional breeding process, the method provided by the invention has the characteristics of high accuracy, low cost and short pure line acquisition time. Compared with the transgenic breeding, the gene knock-out can cause gene malfunction of the fish, and can not bring in any external gene, thereby avoiding the problem of transgenic safety.

Compositions and methods for modifying genetic material are provided. One embodiment provides aptamers capable of binding to a site-specific DNA binding moiety to facilitate the exchange of homologous genetic information between a donor molecule and the desired target locus (aptamer-guided gene targeting or AGT). One embodiment provides an oligonucleotide containing a aptamer, preferably a DNA aptamer at the 5′ end. The oligonucleotide also contains a region of homology, also referred to as donor DNA, to a desired nucleic acid, locus, or gene. The DNA binding moiety can be a nucleic acid, a protein, or a complex of proteins. In a preferred embodiment the DNA binding moiety is a homing endonuclease that cuts DNA to facilitate the modification of the DNA by the donor DNA.

The invention discloses directional knockout on sheep MSTN gene by applying the CRISPR / Cas9 technology, and a method for verifying the impact effect of directional knockout on skeletal muscle satellite cell differentiation. The concrete contents are as follows: firstly, target gene cloning; secondly, gRBA design and synthesis; thirdly, CRISPR / Cas9 gene knockout vector construction; fourthly, CRISPR / Cas9 gene knockout vector exogenous activity detection; fifthly, CRISPR / Cas9 gene knockout vector endogenous activity detection; sixthly, CRISPR / Cas9 gene knockout vector knockout effect detection. The method has the following advantages: firstly, the experimental period is shorter; secondly, the method is simple and practicable, has high repeatability, and can be performed in common laboratories; thirdly, active vector constructed through the method is small in toxic and side effects, and can be applied to the preparation and production of transgenic animals; fourthly, the method is small in non-specific shear, and obviously improves the gene targeting knockout efficiency; fifthly, the method is high in applicability.

The invention relates to a liver cell membrane bionic liposome drug carrier, a preparation method of the liver cell membrane bionic liposome drug carrier, and application of the liver cell membrane bionic liposome drug carrier. The liver cell membrane bionic liposome drug carrier is characterized in that (1) the liposome drug carrier has a cell membrane protein component; (2) the liposome drug carrier is capable of loading drug in vitro, and is used for cell targeting fusion release; (3) the cell membrane protein component of the liposome comes from immortalized human liver cells, and is used for conveying targeted shear plasmids of a hepatitis virus genome; (4) the cell membrane protein component of the liposome comes from an immortalized liver tumor cell line, and is used for targeted drug delivery of liver tumor. The liver cell membrane bionic liposome drug carrier can be used for in vivo targeted hepatic cell transmission and drug delivery of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) gene targeting plasmids.

The invention provides a method for precisely editing a genome DNA sequence. A TALEN, CRISPR / Cas9 and other gene targeting technologies and homologous recombination technologies are utilized in a combined mode for precisely editing the genome DNA sequence. According to the method, the technical problem that in the prior art, a specific sequence cannot be deleted is solved, the specific base sequence can be seamlessly inserted or deleted in a fixed point mode, and seamless free modification of the genome DNA sequence of eukaryocyte is achieved for the first time.

The invention belongs to the field of gene engineering and discloses a target sequence identified by a CRISPR-Cas9 system from neisseria meningitidis. The target sequence is as shown in the nth-24th sites of optional one of SEQ ID No. 1-20, and n=1-9. The invention further discloses sgRNA and coding DNA molecules thereof, the sequence of the sgRNA is 5'-identificiation sequence-sequence recruiting Cas9 protein-3', and the DNA sequence corresponding to the identification sequence is identical with the target sequence. The invention also discloses the CRISPR-Cas9 system which comprises the Cas9 protein, the sgRNA and / or a coding sequence carrying the Cas9 protein and a vector of the coding sequence of the sgRNA. The invention also discloses application of the CRISPR-Cas9 system to editing of CCR5 genes and preparation of medicine for HIV infection. By the CRISPR-Cas9 system, the CCR5 genes can be edited, and cells cannot be infected by HIV.

The novel germ-line transformation systems disclosed in this patent application allow the physical deletion of transposon DNA following the transformation process, and the targeting of transgene integrations into predefined target sites. In this way, transposase-mediated mobilization of genes-of-interest is excluded mechanistically and random genomic integrations eliminated. In contrast to conventional germ-line transformation technology, our systems provide enhanced stability to the transgene insertion. Furthermore, DNA sequences required for the transgene modification (e.g. transformation marker genes, transposase or recombinase target sites), are largely removed from the genome after the final transgene insertion, thereby eliminating the possibility for instability generated by these processes. The RMCE technology, which is disclosed in this patent application for invertebrate organisms (exemplified in Drosophila melanogaster) represents an extremely versatile tool with application potential far beyond the goal of transgene immobilization. RMCE makes possible the targeted integration of DNA cassettes into a specific genomic loci that are pre-defined by the integration of the RMCE acceptor plasmid. The loci can be characterized prior to a targeting experiment allowing optimal integration sites to be pre-selected for specific applications, and allowing selection of host strains with optimal fitness. In addition, multiple cassette exchange reactions can be performed in a repetitive way where an acceptor cassette can be repetitively exchanged by multiple donor cassettes. In this way several different transgenes can be placed precisely at the same genomic locus, allowing, for the first time, the ability to eliminate genomic positional effects and to comparatively study the biological effects of different transgenes.

The invention discloses a method for knocking out MSTN (myostatin) genes in a targeted manner by utilizing CRISPR-Cas9. According to the method, multiple species are subjected to homology comparison, two pairs of MSTN gene target sequences are selected, two pairs of DNA double strands which have the sequences different from the target sequences, the same expression protein and the same BsmBi sticky ends are synthesized according to the design principle of PAM of gRNA, the double strands are connected with pPDNA330 plasmids, a recombinant vector carrying MSTN homologous genes of multiple species is obtained and is used for detecting the gene knockout efficiency with a fluorescent protein expression method, carrier-transfected receptor cells, having higher knockout efficiency, of multiple species are selected, gene knockout and verification are performed, and simple, efficient and accurate gene knockout on the MSTN genes of the multiple species is completed.

The invention provides a method for improving the gene targeting efficiency and a method for carrying out in-situ repair on the base on a beta-globulin gene locus. The method for improving the gene targeting efficiency comprises the following steps of: S10, determining a gene locus to undergo in-situ repair; and S20, introducing a CRISPR / Ca system to cleave editing lotus DNA to cause DNA damage and simultaneously providing a repair template segment. Based on the deficiency of existing gene modification, the gene modification efficiency, the timeliness and the modification quality are improved after single-stranded oligonucleotides (ss ODNs) and a small molecule compound participate in a CRISPR / Cas9 gene modification system.

A simple method for modifying genes in a recombination deficient host cell is disclosed. Such modifications include generating insertion, deletions, substitutions, and / or point mutations at any chosen site in the independent origin based cloning vector. The modified gene can be contained in an independent origin based cloning vector that is used to introduce a modified heterologous gene into a cell. Such a modified vector may be used in the production of a germline transmitted transgenic animal, or in gene targeting protocols in eukaryotic cells.

The invention belongs to the field of gene engineering and discloses a target sequence identified by a CRISPR-Cas9 system from neisseria meningitidis. The target sequence is as shown in the nth-24th sites of optional one of SEQ ID No. 1-15, and n=1-9. The invention further discloses sgRNA and coding DNA molecules thereof, the sequence of the sgRNA is 5'-identificiation sequence-sequence recruiting Cas9 protein-3', and the DNA sequence corresponding to the identification sequence is identical with the target sequence. The invention also discloses the CRISPR-Cas9 system which comprises the Cas9 protein, the sgRNA and / or a coding sequence carrying the Cas9 protein and a vector of the coding sequence of the sgRNA. The invention also discloses application of the CRISPR-Cas9 system to editing of CXCR4 genes and preparation of medicine for HIV infection. By the CRISPR-Cas9 system, the CXCR4 genes can be edited, and cells cannot be infected by HIV.

The invention provides sgRNA and a gene vector for inhibiting bladder cancer by targeting human lncRNA-UCA1 and an application of the sgRNA; specifically, sgRNA sequences of an lncRNA-UCA1 gene and a PD-1 gene suitable for CRISPR-Cas9 targeted splicing are designed; by transforming plasmids of specific splicing lncRNA-UCA1 gene and CRISPR-Cas9 nuclease gene into human bladder cancer cells, the expression of the lncRNA-UCA1 gene drops so as to inhibit the growth of tumor cells; and the plasmid, together with a human PD-1 gene targeted knockout vector, is transformed into a humanized mouse model of bladder cancer transplanted tumor, so as to obviously inhibit the growth of tumors. According to the invention, the vector is simple in preparing steps, the sgRNA is good in targeting property and the CRISPR-Cas9 is high in knockout efficiency.

Compositions and methods for modifying genetic material are provided. One embodiment provides aptamers capable of binding to a site-specific DNA binding moiety to facilitate the exchange of homologous genetic information between a donor molecule and the desired target locus (aptamer-guided gene targeting or AGT). One embodiment provides an oligonucleotide containing a aptamer, preferably a DNA aptamer at the 5′ end. The oligonucleotide also contains a region of homology, also referred to as donor DNA, to a desired nucleic acid, locus, or gene. The DNA binding moiety can be a nucleic acid, a protein, or a complex of proteins. In a preferred embodiment the DNA binding moiety is a homing endonuclease that cuts DNA to facilitate the modification of the DNA by the donor DNA.

The invention provides methods and compositions for inserting a DNA sequence in the genome of a cell by homologous recombination. In particular, the method utilizes a selection scheme in which a selection marker gene that encodes a fluorescence protein, such as a green fluorescence protein, is used for selection against random, non homologous insertions.