85 results about "Gene knockin" patented technology

The invention discloses a method for producing transgenic mice through a homologous recombination technology. The method comprises the following steps: replacing mouse NGF genes on mouse chromosomes by human NGF genes through a Cas-9/CRISPR gene knock-in technology, and obtaining the genes with homozygous human NGF genes through breeding and knocking the genes in mice, thus obtaining filial generation mice with salivary glands capable of secreting human NGF. Compared with the conventional targeting technology utilizing positive and negative double screening homologous recombination genes, the method disclosed by the invention is simple to operate, capable of being realized only by transfecting mouse embryonic stem cells by three plasmid DNAs or carrying out pronucleus injection on the zygotes of mice, and high in success rate which is up to 2-5% and remarkably higher than the mouse embryonic stem cell positive rate of 0.1% of the common gene targeting technology.

The invention provides an exogenous gene knocking-in and integrating system on the basis of CRISPR/Cas9, a method for establishing the exogenous gene knocking-in and integrating system and application thereof. The exogenous gene knocking-in and integrating system comprises vectors with report/donor functions and Cas9 expression vectors. Each report/donor vector comprises two target gent homologous arms and an exogenous sequence fragment positioned between the two target gene homologous arms; homologous sequences, which are positioned on a target gene, of the two target gene homologous arms of each report/donor vector are respectively positioned on two sides of a target sequence of the target gene and are connected with the target sequence of the target gene; the exogenous sequence fragments comprise promoters, resistant genes, shorn peptide sequences, report genes and polyA tails which are sequentially arrayed, two SSA repair homologous sequences of each resistant gene are inserted into the resistant gene, and the target sequence of each target gene is inserted in a space between the two corresponding SSA repair homologous sequences. The exogenous gene knocking-in and integrating system, the method and the application have the advantages that exogenous genes can be integrated with endogenous gene sequences in an efficient site-directed and accurately targeted manner, and double-chromosome allelic gene double-knocking-in can be efficiently carried out.

The invention discloses single guide ribonucleic acid (sgRNA) capable of effectively editing a pig ROSA26 gene, and application of the sgRNA. Based on the sgRNA capable of specifically identifying the pig ROSA26 gene in a pig genome, a cell line of enhanced green fluorescent protein (EGFP) site-specific integrated porcine fetal fibroblasts is successfully established by using a CRISPR/Cas9-mediated gene knock-in technique; the results show that the cell line can stably and efficiently express an EGFP gene; furthermore, in the preparation process of the cell line, exogenous promoter genes and positive and negative screening marker genes are not introduced into the cell line. Therefore, the sgRNA greatly improves the safety of transgenic pigs, and has important significance for eliminating the biological and food potential safety hazards of agricultural products of the transgenic pigs.

The present invention relates to the technical field of biology, particularly to development and applications of a heat shock induced Cas9 enzyme transgene danio rerio. The present invention firstly provides a Cas9 enzyme expression vector, which utilizes a heat shock induced promoter HSP70 to drive the expression of a downstream Cas9 gene. According to the present invention, the Cas9 enzyme expression vector and Tol2 mRNA are co-injected into wild type danio rerio single cell fertilized egg, and selection is performed to obtain the heat shock induced Cas9 enzyme transgene danio rerio, such that the gene editing research of the CRISPR-Cas9 system in the danio rerio is successfully achieved, and the know-out of the MC4R gene in the transgene danio rerio is firstly achieved; and the Cas9 enzyme expression vector is further suitable for heat shock induced gene knockout, gene knock-in, gene expression modification and other applications of other fishes.

The invention provides a yarrowia lipolytica genetic engineering bacterium for producing beta-carotene. The yarrowia lipolytica genetic engineering bacterium for producing beta-carotene is prepared bythe following steps: knocking a carRP gene and a carB gene into chromosomes of uracil and leucine auxotrophic yarrowia lipolytica, then knocking a GGGS1 gene, a HMG gene, two copied carRP gene, one copied of carB gene, and an ERG13 gene into the chromosomes, and finally performing backfilling by two auxotrophic screening markers of uracil and leucine. Continuous feeding fermentation is performed,and the yield of beta-carotene can reach 4.5g/L. The yarrowia lipolytica genetic engineering bacteria constructed according to the invention has the advantages of simple and convenient operation andstable and reliable performance, and can be applied to large-scale commercial production, and the obtained beta-carotene can be safely used, so that a good prospect is achieved.

The invention discloses a recombinant vector for gene knock-in. The recombinant vector comprises a DNA (deoxyribonucleic acid) fragment structure IL17A-IRES-luc, wherein IL17A is an encoding gene of interleukin 17A, IRES is internal ribosome entry site, and luc is an encoding gene of secreting luciferase. The invention also provides a method for preparing the gene knock-in recombinant vector and a mouse model prepared by using the recombinant vector, wherein the mouse model can be used for detecting the expression of the IL17A gene by detecting the expression of the secreting luciferase in blood or urine; a fluorescent microscope or other complicated equipment is not needed; the secreting luciferase emits light by virtue of an oxidizing reaction of substrate coelenterazine in the catalysis of luciferase, and laser light is not needed, so that the fluorescent background is far lower than GFP (green fluorescent protein) fluorescence, the detection sensitivity is higher, the background is cleaner, and the experiment results are more accurate.

The invention provides a method for realizing genome editing and accurate and targeted knock-in of genes in fishes. A technology of producing DNA single-chain gap based on nicking enzyme is utilized,assisted with a set of homologous recombination repair factor RecOFAR, so as to realize high-efficient, accurate and targeted integration of genome editing and gene knock-in in the fishes. The problems that a current existing method is low in efficiency, and random mutation efficiency of target points is higher are overcome.

The invention discloses a PirB gene-knock-in mouse animal model, which comprises gRNA1 and gRNA2 for determining specific target sites to be knocked in by PirB gene, wherein the PirB gene is knocked into an intron 1 of ROSA26 gene of a C57BL/6J mouse; the gene sequence of the gRNA1 is shown in SEQ ID NO.1; and the gene sequence of the gRNA2 is shown in SEQ ID NO.2. The invention further specifically discloses a construction method of the mouse animal model, the mouse animal model provides a good foundation for research on PirB gene function and in-vivo verification. Hybridization of PirB-knock-in animal model with different types of Cre mice can be used to study the functions of PirB in different organs or different cell types or different disease models.

This invention provides an HLA class I-expressing non-human animal that can be prepared efficiently in a simple manner within a short period of time, in which the transgene copy number to be introduced thereinto and the integration site of transgene are regulated, and in which the expression level of the transgene and the site of expression thereof are regulated. The invention also provides a method for preparing such non-human animal. In such HLA class I gene knock-in non-human animal, an artificial chimeric gene encoding an artificial chimeric protein comprising a protein composed of β2 microglobulin, HLA class I α1 and α2 regions, and an MHC class I α3 region of a non-human animal ligated in such order from the N terminus is expressed under the control of a transcription regulatory region of β2 microglobulin gene of the non-human animal.

The invention provides a gene knock-in composition containing a single incision enzyme or polynucleotide coding the same, recombinase or polynucleotide coding the same, and an optional donor DNA containing an exogenous gene. The invention also provides a corresponding gene knock-in method and a method for preparing a transgenic non-human mammal model. The composition and the methods provided by the invention can be used for realizing efficient homologous recombination integration, and meanwhile various defects of insertion or deletion, genome instability and the like which are generated easily in the prior art can be avoided.

The invention provides a composition used in insect targeted gene knock-in, which includes a single nickase or an encoding polynucleotide thereof; a recombinant factor or an encoding polynucleotide thereof; and optionally, a donor DNA of an exogenous gene. The invention also provides a corresponding gene knock-in method and a method for preparing transgenic insects. The composition and the method in the invention can achieve high-effective homologous recombination integration in insect cells and meanwhile can avoid the defects in gene knock-in technologies in the prior art. The composition also can stably and high-effectively achieve homologous recombination integration of a fragment being more than 10 kb in length in the insect cells, so that the composition can be used for transferring a genetic selection marker into the insect cells, preferably a visible genetic selection marker, which is convenient to determine strains of transgenic insects.

The invention relates to a method for preparing general type CAR-T cells by using CRISPR/Cas9+AAV, and belongs to the field of immunocytotherapy. The method utilizes CRISPR/Cas9+AAV to knock out an immunological checkpoint PD-1, a human leukocyte antigen (HLA) and TCR; multi-gene knockout is employed to prepare the general type CAR-T cells; and a CRISPR/Cas9 gene editing technology is used to perform precise gene knockout, and is used in combination with adeno-associated virus (AAV) to replace lentiviruses for precise gene knock-in and to avoid the potential risk of random integration of the lentiviruses. The method of the invention can realize multi-target gene knock-in and knock-out, construct the general type CAR-T cells for allogeneic treatment, and solve the problems existing in traditional CAR-T cell therapy such as risk of random insertion mutation of lentiviruses, high production cost and long preparation period for autologous CAR-T design and culture, and insufficient cell number and expansion ability.

The invention relates to a negative selection marker for escherichia coli gene knock-in, namely, the virulent gene ccdB driven by a plac promoter, and plac-ccdB and the gentamicin resistance gene aacC1 form a gene cassette for gene knock-in. In the first step, DNA fragments containing homologous arms plac-ccdB and aacC1 are obtained through amplification, and electro-transformed into escherichia coli for expression of recombinase. Under gentamicin screening, recombinase catalyzes homologous recombination and the DNA fragments are integrated into escherichia coli genome. In the second step, same homologous arms are utilized for amplification of foreign DNA fragments, ccdB is expressed in the bacterial strains obtained in the first step during the electrotransformation for expression of recombinase under the screening of isopropyl-beta-d-isopropylthiogalactoside, the toxicity serves as the feature of the negative selection marker, recombinase catalyzes homologous recombination among homologous arms, and foreign DNA fragments replace a plac-ccdB-aacC1 gene cassette to realize gene knock-in.

Provided herein are methods and systems for targeted gene disruption (knock-out, missense mutation) and targeted gene knock-in in mammalian cells using base editors and guide RNAs (gRNAs) designed to target splice acceptor-splice donor sites. Also provided herein are universally acceptable genetically engineered cells comprising targeted disruptions in immunotherapy-related genes and comprising a CAR/TCR for therapeutic applications.

The present invention provides a non-human animal in which a DNA comprising an hp7 sequence-encoding DNA and a poly A addition signal-encoding DNA added on the 3′ side of a DNA encoding an arbitrary foreign gene is inserted in the same reading frame as that of an arbitrary target gene present on the genome of the non-human animal.

The invention relates to a construction method of an STAT3 mitochondrial localization conditional gene knock-in mouse model. The method comprises the following steps: firstly, constructing an STAT3 mitochondrial localization conditional gene knock-in recombinant targeting vector, transfecting the recombinant targeting vector to fertilized ova of a donor mouse, and then transplanting the donor mouse fertilized ova into the uterus of a fake pregnant mother mouse to produce a chimeric mouse; mating the chimeric mouse with a C57BL/6J mouse to obtain an F1 generation; mating the F1 generation witha mouse of tissue-specific expression Cre recombinase to obtain a double-hybrid mouse; and are intercrossing the double-hybrid mouse and an STAT3<f/+> or STAT3<f/f> mouse, and inducing the obtained offspring by tamoxifen to obtain the STAT3 mitochondrial localization conditional gene knock-in mouse model. According to the mouse model disclosed by the invention, the STAT3 gene is expressed in specific tissues and cell types, and an ideal model is provided for researching the action mechanism of STAT3 in specific cell development and differentiation, body metabolism and tumor occurrence and development.

The invention discloses a method used for discovering and identifying liver cancer serum differential expression proteins and verifying marker proteins. According to the method, iTRAQ labeling MALDI-TOF MS/MS technology is adopted for discovering and identifying liver cancer serum differential expression proteins and MRM verifying of marker proteins. According to the method, searching on effectivetumor markers possesses important meaning on canceration mechanism study, disease early stage diagnosis, and prognosis. Selected novel liver cancer protein markers are subjected to in vivo and in vitro experiments such as gene knockin and knockout, and nude mice transplanted cancer, influences of blocking or up-regulating of expression of candidate genes on tumor biological behavior are observed,and key proteins capable of influencing human liver cancer occurrence and development are positioned, the effects of the key proteins in a system signal network is analyzed based on bioinformatics analysis, and the feasibility, the liver cancer diagnosis value, and the clinical significance of the key proteins in clinical applications including liver cancer prevention, early stage diagnosis, andprognosis is evaluated based on MRM absolute quantitative verification.

The invention provides an efficient and stable fixed-point integrated gene knock-in method based on a pCAG-flox-neo vector. The method comprises the following specific steps: (1) carrying out double enzyme digestion on a pCAG-STOP2 vector; the synthesized fragment is connected to a pCAG-STOP2 carrier; (2) carrying out enzyme digestion on the synthesized carrier again, and then connecting the carrier into polyA-loxP to obtain a pCAA-flox-neo carrier; (3) inserting the sgRNA sequence of the pig pH 11 site capable of being identified by the sgRNA and the PAM sequence into the T3 promoter, so as to obtain a final gene knock-in vector skeleton pCAG-floxP-neo-pH 11, wherein the gene knock-in vector skeleton pCAG-floxP-neo-pH 11 is obtained; and (4) carrying out PCR (Polymerase Chain Reaction) amplification on the required hKCNH2-T618I and hmuPKD2 fragments, and inserting the fragments into a pCAG-floxP-neo-pH11 vector skeleton in a one-step cloning manner. The invention establishes a method which is efficient, accurate, simple to operate and capable of knocking out the target gene at the same time, and has important scientific significance for researching the effect of the target gene in mammals or disease models.

The invention relates to a microorganism method for detecting metallic-arsenic-like in water, in particular to an escherichia coli report bacterial strain culturing method improved through genetic engineering, and an escherichia coli culturing method for detecting arsenic in water. The detection bacterial strain is obtained by adopting a Red recombination system to knockout arsenic resistant arsB genes of wild type escherichia coli MC4100, and then adopting the gene knockin technology to substitute pars-arsR-gfpmut2 report genes to the positions of araB coding genes. The biological detection bacterial strain is named as E.coli WMC-011p. The lowest arsenic detection range meets the Integrated Wastewater Discharge Standard of GB8978-1996. The bacterial strain overcomes the shortcomings of high fluorescence background value, unstable detection signals, inaccurate results and the like of a biological detection bacterial strain based on a plasmid vector, and has the advantages of high specificity, high sensitivity, low cost and the like.

A gene knockin method and a kit for gene knockin are provided. The method comprises (a) introducing a RNA-guided endonuclease that cleaves the chromosome at the insertion site into the cell; (b) introducing a guide RNA into the cell; and (c) introducing a donor plasmid into the cell, wherein the donor plasmid comprises the donor sequence flanked with a 5′ homology arm and a 3′ homology arm, a 5′ flanking sequence upstream of the 5′ homology arm, and a 3′ flanking sequence downstream of the 3′ homology arm, wherein the 5′ homology arm is homologous to a 5′ target sequence upstream of the insertion site on the genome and the 3′ homology arm is homologous to a 3′ target sequence downstream of the insertion site on the genome, wherein the guide RNA recognizes the insertion site, the 5′ flanking sequence, and the 3′ flanking sequence, wherein the RNA-guided endonuclease cleaves the donor plasmid at the 5′ flanking sequence and the 3′ flanking sequence, thereby producing a linear nucleic acid, wherein the donor sequence is inserted in to the genome at the insertion site through homology-directed repair.