690 results about "Gene Modification" patented technology

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 includes compositions comprising at least one chimeric autoantibody receptor (CAAR) specific for an autoantibody, vectors comprising the same, compositions comprising CAAR vectors packaged in viral particles, and recombinant T cells comprising the CAAR. The invention also includes methods of making a genetically modified T cell expressing a CAAR (CAART) wherein the expressed CAAR comprises a desmoglein extracellular domain.

The invention provides a non-naturally occurring microorganism having one or more gene disruptions, the one or more gene disruptions occurring in genes encoding an enzyme obligatory coupling 3-hydroxypropionic acid production to growth of the microorganism when the gene disruption reduces an activity of the enzyme, whereby the one or more gene disruptions confers stable growth-coupled production of 3-hydroxypropionic acid onto the non-naturally occurring microorganism. Also provided is a non-naturally occurring microorganism comprising a set of metabolic modifications obligatory coupling 3-hydroxypropionic acid production to growth of the microorganism, the set of metabolic modifications having disruption of one or more genes including: (a) the set of genes selected from: (1) adhE, ldhA, pta-ackA; (2) adhE, ldhA, frdABCD; (3) adhE, ldhA, frdABCD, ptsG; (4) adhE, ldhA, frdABCD, pntAB; (5) adhE, ldhA, fumA, fumB, fumC; (6) adhE, ldhA, fumA, fumB, fumC, pntAB; (7) pflAB, ldhA, or (8) adhE, ldhA, pgi in a microorganism utilizing an anaerobic β-alanine 3-HP precursor pathway; (b) the set of genes selected from: (1) tpi, zwf; (2) tpi, ybhE; (3) tpi, gnd; (4) fpb, gapA; (5) pgi, edd, or (6) pgi, eda in a microorganism utilizing an aerobic glycerol 3-HP precursor pathway; (c) the set of genes selected from: (1) eno; (2) yibO; (3) eno, atpH, or other atp subunit, or (4) yibO, atpH, or other atp subunit, in a microorganism utilizing a glycerate 3-HP precursor pathway, or an ortholog thereof, wherein the microorganism exhibits stable growth-coupled production of 3-hydroxypropionic acid. The disruptions can be complete gene disruptions and the non-naturally occurring organisms can include a variety of prokaryotic or eukaryotic microorganisms. A method of producing a non-naturally occurring microorganism having stable growth-coupled production of 3-hydroxypropionic acid is further provided. The method includes: (a) identifying in silico a set of metabolic modifications requiring 3-hydroxypropionic acid production during exponential growth, and (b) genetically modifying a microorganism to contain the set of metabolic modifications requiring 3-hydroxypropionic acid production.

A gene activation/inactivation and site-specific integration system has been developed for mammalian cells. The invention system is based on the recombination of transfected sequences by FLP, a recombinase derived from Saccharomyces. In several cell lines, FLP has been shown to rapidly and precisely recombine copies of its specific target sequence. For example, a chromosomally integrated, silent β-galactosidase reporter gene was activated for expression by FLP-mediated removal of intervening sequences to generate clones of marked cells. Alternatively, the reverse reaction can be used to target transfected DNA to specific chromosomal sites. These results demonstrate that FLP can be used, for example, to mosaically activate or inactivate transgenes for a variety of therapeutic purposes, as well as for analysis of vertebrate development. The FLP recombination system of the present invention can be incorporated in transgenic, non-human mammals to achieve site-specific integration of transgenes, to construct functional genes or to disrupt existing genes.

The invention discloses a CAS9-carrying recombinant adenovirus and application thereof. The invention protects a recombinant adenovirus carrying coding gene of CAS9 protein; and the CAS9 protein is disclosed as Sequence 7 in the sequence table. The coding gene of the CAS9 protein is disclosed as 789th-5045th nucleotides from the 5' terminal of Sequence 6 in the sequence table. The invention also protects application of the recombinant adenovirus in preparing a kit for knocking out target gene. The CRISPR (clustered regularly interspaced short palindromic repeat) method has the characteristics of high efficiency, high speed and low cost. The CAS9 recombinant adenovirus disclosed by the invention can be used in a protein function cell platform (1) for quick gene modification and high flux screening, a liver protein function platform (2) for implementing quick and specific gene knock-out at the liver, and a mouse gene modification platform (3) for preparing a gene knock-out/knock-in mouse.

Provided herein are methods for a robust production of isoprenoids via one or more biosynthetic pathways. Also provided herein are nucleic acids, enzymes, expression vectors, and genetically modified host cells for carrying out the subject methods. Also provided herein are fermentation methods for high productivity of isoprenoids from genetically modified host cells.

The invention relates to construction of a soybean CRISPR/Cas9 system. The construction disclosed by the invention is mainly characterized in that a core sequence is designed according to a target gene, and the core sequence is constructed into an expression carrier with cas9. The soybean CRISPR/Cas9 system utilizes the core sequence to identify the target gene, the cas9 shears the target gene, and various mutations can be generated in the process of repairing a broken chain by an organism. The invention also provides application of the soybean CRISPR/Cas9 system in soybean gene modification. With the adoption of the construction and the application provided by the invention, the constructed soybean CRISPR/Cas9 system is simple, the soybean gene can be rapidly and efficiently modified, and the problems of the traditional ZFNs system is too complex, wastes time and is low in efficiency are overcome.

The invention discloses a method for knocking out a microRNA gene family by utilizing CRISPR-Cas9 specificity, wherein a target gene is knocked out by mainly adopting a CRISPR/Cas9 system. The microRNA family is knocked out by utilizing the specificity of the CRISPR/Cas9 system for the first time; the disadvantage that only one gene can be knocked out for one time in traditional transgenosis can be overcome by utilizing the novel method; the time for establishing a model organism is reduced to three weeks; furthermore, the construction step is simple; an expensive molecular reagent is also reduced; the period for constructing a genetically modified mouse is greatly shortened to four months; multiple genes constructed for one time can be knocked out simultaneously; the fund investment is obviously reduced; F1 generations of mice can be obtained only in need of 50000 Yuan; the gene modification efficiency can be above 90%; the unreliability of the traditional technology is reduced; the operation technology is simple; and a series of complex steps including constructing a targeting vector, screening ES (Embryonic Stem) cells, selectively breeding chimeric mice and the like are unnecessary.

A genetically modified non-human animal is provided, wherein the non-human animal expresses an antibody repertoire capable of pH dependent binding to antigens upon immunization. A genetically modified non-human animal is provided that expresses a single light chain variable domain derived from a single rearranged light chain variable region gene in the germline of the non-human animal, wherein the single rearranged light chain variable region gene comprises a substitution of at least one non-histidine encoding codon with a histidine encoding codon. Methods of making non-human animals that express antibodies comprising a histidine-containing universal light chain are provided.

The invention provides a targeting sgRNA for pig CD163 gene editing, a modification vector, and a preparation method and application thereof, which relate to the technical field of gene engineering. The sgRNA and the gene modification vector provided by the invention have high specificity, and can be used for efficiently editing a pig CD163 gene on a cell level through a CRISPR/Cas9n system. According to the preparation method of a blue-eared disease resistant pig provided by the invention, a blue-eared disease virus receptor is damaged, the target gene CD163 is edited, no other exogenous gene is intruded, no non-specific editing is carried out on a non-CD163 gene area on a genome, a genetic background is clean and clear, and the work of later transgene safety evaluation is greatly reduced. The blue-eared disease resistant pig obtained by utilizing the invention can be ensured to survive normally, and resists the infection of a blue-eared disease virus, so that the economic loss caused by the blue-eared disease in pig husbandry is greatly reduced.

The invention discloses a simple, convenient and efficient fixed-point gene editing method relating to the fields of transgenic animal preparation, gene functional study and biomedicines. A testis injection method is a sperm-mediated gene transferring method developed by utilizing the sperm capacity of actively combining, transferring and integrating an exogenous DNA. By using the method disclosed by the invention, an animal testis injection method is optimized, a carrier constructed by utilizing a clustered regularly interspaced short palindromic repeat system (CRISPR/Cas9) is integrated with a sperm chromosome through multi-point testis injection or seminiferous tubule microinjection, so that the aims of deleting, replacing and inserting genes are achieved. Then, a transgenic animal descendant is obtained through various approaches such as natural mating and artificial insemination. According to the method, the advantages of a CRISPR/Cas9 gene editing system are sufficiently utilized, and the existing transgenic animal system is combined, so that the complexity of in-vitro cell operation and requirement on expensive precise instruments/equipment are avoided while the gene modification animal obtaining efficiency is greatly increased.

The invention relates to the technical fields of molecular biology and cellular immunology and in particular relates to a preparation method of tumour cell specific polyclonal T cells. The preparation method of the tumour cell specific polyclonal T cells comprises the following steps: (1) obtaining peripheral blood mononuclear cells; (2) mixing the obtained peripheral blood mononuclear cells with gene modifying tumour cells; and (3) resuspending the obtained mixed cells in a culture medium containing cell factors which can maintain cell growth, proliferation and differentiation. The prepared tumour cell specific polyclonal T cells are mainly applied to medicines which are sued for preventing or treating cancers. The preparation method of the tumour cell specific polyclonal T cells has the advantages that the prepared tumour cell specific polyclonal T cells have the characteristics of large quantity, high specificity and strong killing capability; besides, a technology is simple, and the preparation cost is obviously reduced compared with the prior art.

The invention relates to a humanized gene genetically modified non-human animal, particularly a genetically modified rodent, especially a genetically modified mouse, and in particular relates to a construction method of a humanized OX-40 gene animal model and application of the model in the biomedicine field.

The invention relates to a humanized gene genetically modified non-human animal, particularly a genetically modified rodent, especially a genetically modified mouse, and in particular relates to a construction method of a humanized PD-L1 gene animal model and application of the model in the biomedicine field.

The invention discloses application of novel ScCas12a protein in nucleic acid detection. It is found through research that the novel ScCas12a protein with DNA cleavage activity can be used for nucleicacid detection, gene editing and gene modification as a novel CRISPR/ScCas12a system and provide new necessary tool selection for Cas12a-based gene editing, modification and molecular detection. Theinvention also provides a novel nucleic acid detection system comprising the ScCas12a protein and gRNA and a kit. The molecular detection with high sensitivity and high precision can be achieved at room temperature of 25-37 DEG C, the detection specificity is good, the sensitivity is high, the cost is low, the operation is convenient and fast, the application range is wide, and the application prospect in nucleic acid detection is good.