43 results about "NHEJ Pathway" patented technology

The invention discloses a method for improving the efficiency of CRISPR mediated homologous recombination. The method includes the steps of: inhibiting the expression of Lig4, DNA-PK and XRCC6 by shRNA so as to inhibit the non-homologous end joining (NHEJ) repair pathway; conducting fusion expression on sgRNA and shRNA at targeting genome specific positions to form shRNA-sgRNA polycistron; and placing the polycistron at the RNA polymerase II or RNA polymerase III promoter downstream. The carrier constructed by the method provided by the invention has a concise structure and is convenient to use, also enhances the homologous recombination efficiency by dozens of times, greatly expands the function and application of the CRISPR carrier, and is a good tool for functional gene and signal pathway research, medical research, and recombinant protein expression.

The present invention relates to methods and compositions useful for targeted cleavage and alteration of a genomic sequence, targeted cleavage followed by homologous recombination between an exogenous polynucleotide and a genomic sequence, or targeted cleavage followed by non-homologous end joining.

Disclosed are methods and eukaryotic host cells for transgene expression. The cells may be treated and/or modified to increase homologous recombination (HR), decrease non homologous end joining (NHEJ) and/or to enhance a HR/NHEJ ratio in said cell. Such cells can be transfected with vectors comprising the transgene, which advantageously integrates into the genome of the cell to form a concatemeric structure which may comprise more than 200 transgene copies. Certain expression enhancing elements such as MARs are advantageously provided to further enhance and/or facilitate transgene expression. Disclosed is also a recombinant eukaryotic host cell, in particular a non-primate host cell, comprising a transgenic sequence encoding a protein and/or a RNA, in particular a primate protein and/or RNA, involved in translocation across the ER membrane and/or secretion across the cytoplasmic membrane.

The invention relates to a donor structure and a gene knockout method, system and kit. The gene knockout method comprises inserting a marker gene in the donor structure into a cell genome at a double-stranded cleavage site through non-homologous end joining. Through expressive cells without marker-enriched genes, the efficiency of gene knockout based on a sequence-specific nuclease is improved.

The invention relates to compositions and methods for interfering with the DNA repair of double strand breaks (DSBs). The invention discloses novel double-stranded nucleic acid molecules. that act as baits and hijack the holocomplex of enzymes responsible of DNA DSB sensing, signaling and/or repair pathways, in particular the non homologous end joining (NHEJ) pathway of DSB repair. The invention discloses the use of these molecules as adjuvant compositions to be used in association with a DNA breaking treatment, particularly radiotherapy or chemotherapy, in combination with a pharmaceutically acceptable carrier, in an efficient amount to be introduced in the tumor cell nuclei in order to neutralize transiently their DNA repair capacity and trigger their death.

The invention discloses application of a PARP1 inhibitor in the preparation of a medicine for reversing drug resistance of tumor cells to amethopterin and belongs to the technical field of tumour biotherapy. It is found by the inventor through researches that as for malignant cells with MTX resistance and DHFR gene high-amplification, by specifically inhibiting the key protein RARP1 of A-NHEJ pathway, DHFR gene copy number can be reduce and DMs in cells can be decreased so as to reverse drug resistance of tumors and enhance efficiency of tumor therapy. Therefore, on the basis of the above researches, the invention brings forward the application of the PARP1 inhibitor in the preparation of a medicine for reversing the drug resistance of tumor cells to MTX by reducing DHFR gene copy number. The invention provides a new targeting therapeutic scheme for the MTX as the main therapeutic drug and for the biotherapy of malignant tumors which are easy to resist drugs and also provides a scientific basis for effectively fighting against MTX drug resistance. In addition, the invention is also of great positive significance for deeply understanding the nature of chemotherapy resistance and finding resistance target for individualized treatment.

The invention discloses a sgRNA fragment. The DNA sequence of the sgRNA fragment comprises a length of a fixed sequence and a length of a DNA recognition sequence. The sgRNA can recognize different target sites on double strands of a target gene, bond with nuclease and guide nuclease to bond with the target sites of the target gene through recognition of PAM sequences at the target sites and to start random shearing; then nuclease falls off from a shearing opening, thereby forming a DSB gap; then cells repair the double strands of the target gene through a non-homologous end joining repair mechanism, which leads to frameshift mutation; and finally, the target gene is knocked out. The invention further discloses a gene fixed-point reconstruction method and a gene targeting kit. The invention elaborates a series of sgRNAs applicable to the mouse Myod1 gene, and the sgRNAs have high targeting efficiency, short construction time and low cost and can be highly efficiently used for mouse Myod1 gene targeting.

The invention discloses use of a transforming growth factor (TGF)-beta1 inhibitor in lung cancer treatment. The TGF-beta1 inhibitor inhibits a TGF-beta1 channel to increase the radiosensitivity of lung cancer cells. Through the manner, according to the use of the TGF-beta1 inhibitor in lung cancer treatment, the sensitivity enhancement effect of the TGF-beta1 inhibitor on the lung cancer cells is caused by inhibition on non-homologous end joining repair of deoxyribonucleic acid (DNA) double-strand break, change of cycle distribution of the lung cancer cells after irradiation, and obvious reduction of tumor growth by the TGF-beta1 inhibitor combined with X-ray treatment of 5Gy. The DNA repair capacity can be reduced by inhibition on the TGF-beta1 channel of tumor cells before irradiation, the cell cycle distribution is changed, the cell cloning ability is reduced, the tumor growth is delayed, and effective auxiliary means are provided by inhibiting the TGF-beta1 channel as radiotherapy of a patient with a lung cancer.

The invention discloses a method for increasing HR (Homologous Recombination) repairing frequency of ovine embryo fibroblasts after gene editing. The method provides application of a substance for restraining the expression of an Lig4 gene in in-vitro ovine embryo fibroblasts containing gene editing DNA (Deoxyribose Nucleic Acid) fragments in preparation of products for promoting HR repairing of the in-vitro ovine embryo fibroblasts containing the gene editing DNA fragments. An experiment of the invention shows that siRNA (Small Interfering Ribose nucleic Acid) which restrains the expression of the ovine Lig4 gene is screened, the expression of the ovine Lig4 gene is restrained through the siRNA, an intracellular HR repairing path is stimulated by restraining an NHEJ (Non-homologous End Joining) repairing path of the ovine embryo fibroblasts, the frequency of adopting HR repairing by cells is increased, and a basis is provided for increasing gene targeting efficiency of the ovine embryo fibroblasts and researching accurate editing of an ovine genome.

The invention relates to a universal donor construct, and a gene knockout method, system and kit. According to the gene knockout method in the invention, a marker gene included in the universal donorconstruct is inserted to a double-strand break position in a cell genome by non-homologous end joining, and cells in which genes are knocked out are enriched by an expressed marker, so the efficiencyof gene knockout via sequence-specific nucleases is improved. The universal donor construct of the invention comprises a universal linear donor DNA which contains a universal target sequence cleavableby the sequence-specific nucleases; the universal target sequence does not exist in the genome of a cell to undergo gene knockout; therefore, when performing gene knockout, it is not necessary to specifically construct a matching linear donor DNA, and a general linear donor DNA and a gRNA targeting to the universal linear donor DNA can be directly used.

This document provides methods and materials involved in assessing responsiveness to PARP inhibitors and platinating agents. For example, methods and materials for using levels of non-homologous end-joining pathway members (e.g., artemis mRNA or polypeptide levels, Ku80 mRNA or polypeptide levels, or DNA-PKcs mRNA or polypeptide levels) to determine if cancer cells that are homologous recombination-deficient are likely to be susceptible or resistant to PARP inhibitors and platinating agents are provided.

Compounds, methods of editing a target genomic region(s), methods of repairing of a DNA break via a HDR pathway, methods of inhibiting or suppressing repair of a DNA break via a NHEJ pathway, and methods of modifying expression of a gene(s) or protein(s) comprise administering to one or more cells that include one or more target genomic regions, a genome editing system and a DNA protein-kinase (DNA-PK) inhibitor disclosed herein. Kits and compositions for editing a target gene comprise a genome editing system and a DNA-PK inhibitor disclosed herein.

The invention relates to an application of an up-regulating agent of a homologous recombination repair (HR) or non-homologous end joining (NHEJ) pathway in preparation of a medicament for treating diabetes and preventing diabetic individual tumorigenesis. Inventors find that DSB and DSB repair mechanism damage exist in diabetic human cells and tissue, and confirm that EWS/Fbxw7 repairs damaged DNAby activating the HR pathway and the NHEJ pathway for the first time, and the cell cycle is blocked at G0/G1 phase to facilitate DNA repair; the EWS/Fbxw7 can significantly reduce the excessive activation of PARP in diabetic large blood vessels, microvascular endothelium and retinal nerve tissue; the mechanism that inhibits the excessive activation of the PARP can be related to the activation ofthe HR and NHEJ pathways; and results suggest that the up-regulating agent of the HR or NHEJ pathway can be used to prepare the medicament for alleviating a diabetes mellitus vascular endothelial dysfunction, or strengthening the DNA damage repair, or treating the diabetes mellitus, or preventing diabetic complications, or preventing the diabetic individual tumorigenesis.

The invention discloses a method for determining the repair activity of NHEJ. In this method, HPRT gene is mutated by using a site-directed gene mutation technology, and plasmid transfection, and 6-TG treatment are performed. By means of the method of the invention, the NHEJ repair activity level of cells can be observed by measuring the cell viability. The method can be used for screening the effects of different drugs and different genes on the NHEJ repair activity.

The invention discloses a molecular marker and a kit for cancer diagnosis, and particularly relates to application of BEND4 gene promoter region DNA methylation as a molecular marker in preparation of cancer diagnosis products. According to the application disclosed by the invention, frequent high methylation of a BEND4 promoter region in pancreatic cancer, breast cancer and esophageal cancer is found through an MSP method for the first time, BEND4 participates in an NHEJ pathway of DNA damage repair, and BEND4 methylation is a synergistic lethal marker for killing tumor cells by combining an ATM inhibitor with chemotherapeutic drugs such as cis-platinum and the like. The invention also provides a primer and a kit for detecting the methylation state of the promoter region of the BEND4 gene. The application of the primer and the kit to detect the hypermethylation state of the BEND4 gene promoter region can be used as a powerful means for diagnosis, curative effect observation, prognosis judgment, minimal residual disease detection and the like of pancreatic cancer, breast cancer and esophageal cancer, and the primer and the kit are simple and convenient to operate and good in stability and have profound clinical significance and generalization performance.

Compositions and methods to modify at least one target locus in a plant cell are provided, which comprises providing a plant cell, a plant, or a plant part with one or more target loci and one or more donor loci, providing at least one cleaving site specific nuclease to produce a double strand break within the target loci, followed by non-homologous end joining of at least one donor locus within at least one target locus. Target loci, donor loci and nuclease loci used in these methods, and plant cells, plants and plant parts comprising these target loci, donor loci, nuclease loci and/or the recombined loci are also provided.

The invention discloses a preparation method of Cas9-RNAi RNP with efficient homologous directional repair activity, which comprises the following steps: transiently inhibiting key protein Ligase 4 of an NHEJ pathway, constructing a similar structure of shRNA by prolonging the 3'end of gRNA of Cas9RNP, and expressing Cas9RNP by using escherichia coli cells in a one-step method to obtain Cas-RNAi RNP carrying specific sgRNA-shRNA. According to the Cas-RNAi RNP disclosed by the invention, the function combination of gene editing and RNA interference is realized, and the shRNA base sequence aiming at the Ligase 4 gene is designed so as to temporarily inhibit the NHEJ pathway, so that efficient HDR repair is realized.

The invention relates to a preparation method of a cell line with a high homologous recombination rate, which comprises the following steps: stably inserting a donor vector into an AAVS1 position by using a siRNA technology and a gene editing technology, knocking down important genes LIG4 and Ku70 in an NHEJ pathway, and simultaneously overexpressing important proteins SWIT5 and SFR1 in a homologous recombination pathway, so that the cell line with the high homologous recombination rate is obtained. And screening by a monoclonal sorting method to obtain the improved 293T cell line with high homologous recombination efficiency. The proliferation effect and transfection effect of the cell line obtained by the method are similar to those of a wild type, and the homologous recombination efficiency is 20 times that of the wild type, so that the subsequent accurate gene editing operation is facilitated, and the scientific research can be more efficiently carried out at low cost.