779 results about "Nuclease" patented technology

Systems and methods for targeted gene modification, targeted insertion, perturbation of gene transcripts, and nucleic acid editing. Novel nucleic acid targeting systems comprise components of CRISPR systems and non-LTR retrotransposon elements.

The present invention relates to a nuclease polypeptide, such as a CRISPR nuclease polypeptide derived from a reference nuclease, which may be nuclease A, nuclease K or nuclease M, comprising a RuvC nuclease domain and a HNH nuclease domain. Also provided herein are gene editing systems comprising such nuclease polypeptides and gene editing methods using the gene editing systems.

The invention provides an engineered nuclease with high salt tolerance. The polypeptide comprises one or more mutations, so that the three-dimensional structure of the polypeptide has more surface areas with positive charges. Compared with the nuclease with the SEQ ID NO: 1 sequence, the polypeptide still has at least 60% nuclease activity under the condition that the solution ion strength exceeds 200 mM.

The invention discloses a construction method and application of an NMD escape model of an Alport syndrome mouse. A non-human animal model carrying Col4a5 gene c.4432delG frame shift mutation is prepared on the basis of a CRISPR / Cas9 gene editing technology. The method comprises the following steps: co-injecting gRNA of a 49 exon of a targeted Col4a5 gene, homologous recombinant donor oligonucleotide containing c.4432delG mutation and Cas9 nuclease into a mouse fertilized egg, and carrying out embryo transplantation to obtain an F0-generation mutant mouse; a mutation site is verified by combining PCR (Polymerase Chain Reaction) with sequencing, and a stably inherited mutation line is established through two generations of breeding. Through verification, the model accords with pathological characteristics of the Alport syndrome, can stably simulate typical clinical manifestation and pathological characteristics of the human X-linked Alport syndrome, and can be used as an important tool for research of the Alport syndrome.

Provided are variant Cas12f4 polypeptides, fusion proteins containing variant Cas12f4 polypeptides, nucleic acids encoding variant Cas12f4 polypeptides and fusion proteins, compositions and methods related to those variant Cas12f4 polypeptides and fusion proteins, and modified host cells comprising the variant Cas12f4 polypeptides and / or encoding nucleic acids. The variant Cas12f4 polypeptides disclosed herein exhibit improved functionality, stability, and / or other desirable properties and may be used in combination with guide Cas12f4 RNAs that can be used in a variety of applications ex vivo, in vitro, and in vivo.

The invention discloses a lipophilic liposome containing polydeoxyribonucleotide as well as a preparation method and application of the lipophilic liposome. The lipophilic liposome comprises the following components in parts by weight: 0.001-3 parts of polydeoxyribonucleotide; 0.5 to 3 parts of phospholipid; 0.1-2 parts of a cationic emulsifier; 0.1-3 parts of an anionic emulsifier; 40 to 70 parts of polyol; the total amount is 100 parts. According to the invention, an emulsifier system formed by combining lecithin with anionic and cationic compound surfactants is adopted, so that the transdermal absorption of PDRN is facilitated. The lipophilic liposome containing polydeoxyribonucleotide prepared by the invention can effectively resist nuclease degradation, prolong the acting time of PDRN in skin, and improve the skin anti-aging and repairing effects of the liposome. The anti-aging effect of the active ingredients is improved, and the bioavailability of the active ingredients is improved. The cosmetic composition can be widely applied to cosmetic formulas of various dosage forms.

The invention provides sgRNA for targeting and guiding nuclease to efficiently cut an HLA-DRA gene, a method for modifying a CAR-T cell by using the sgRNA, and a related gene editing system, reagent and kit. According to the CAR-T cell, the immunogenicity is greatly reduced, the risk of graft versus host disease and immunological rejection can be effectively reduced, and meanwhile the killing capacity of tumor cells of the CAR-T cell is not affected.

The invention discloses a CRISPR / Cas12a-RPA (clustered regularly interspaced short palindromic repeats / CRISPR associated protein 12a-recombinase polymerase amplification)-based sequence combination for rapidly detecting mouse hepatitis virus and a kit thereof, and belongs to the technical field of biological detection The sequence combination comprises an RPA primer pair sequence, a crRNA sequence and an ssDNA probe sequence; the kit comprises an RPA primer pair sequence, a crRNA sequence and an ssDNA probe sequence, the kit further comprises a mouse hepatitis virus negative control standard substance, RNase-free water, an RPA reaction buffer solution, an NEB buffer solution, Lba Cas12a nuclease, dry powder RPA reaction microspheres containing recombinase polymerase and magnesium acetate with the concentration of 280 mM. The kit can be used for rapidly detecting the mouse hepatitis virus. The primer sequence combination provided by the invention is good in conservative property and specificity and relatively high in sensitivity, and rapid detection of the mouse hepatitis virus can be realized.

The invention provides sgRNA for targeting and guiding nuclease to efficiently cut a B2M gene, a method for modifying cells by using the sgRNA, and a related gene editing system, reagent and kit. The cells produced by the invention greatly reduce the immunogenicity, can effectively reduce the risk of graft versus host disease and immunological rejection, and does not affect the killing ability of the cells to tumor cells.

The invention relates to a method for carrying out gene editing on a TRAC gene locus. Specifically, the invention provides sgRNA targeting and guiding nuclease to efficiently cut a TRAC gene, a method for modifying a CAR-T cell by using the sgRNA, the CAR-T cell obtained by the method, and a related gene editing system, reagent and kit. According to the CAR-T cell, the immunogenicity is reduced, the risk of graft versus host disease and immunological rejection can be effectively reduced, and meanwhile the killing capacity of tumor cells of the CAR-T cell is not affected.

Provided are compositions for genome editing and site-directed integration in plants comprising microprojectile particles coated, treated or applied with a nuclease protein, guide RNA or RNP for delivery to a mature embryo explant from dry seeds. Further provided are methods for genome editing and site-directed integration in at least one cell of a plant using the disclosed compositions, and plants, plant parts and seeds comprising an edited genome or site-directed integration, which are produced by the disclosed methods.

Embodiments disclosed herein provide methods, including computer-implemented methods, for designing guide sequence which may be incorporated into custom, large scale guide sequence libraries. The methods require only a list of target genes as input and utilize on target and off target scores to generate an optimal set of guide sequences for a set of target genes. In certain embodiments, the methods may also utilize multi-tissue RNA-sequencing data and / or protein annotation to design targets to genes that are highly expressed and / or contain a functional protein domain. The invention further comprises guide libraries, cells comprising said guide libraries. Computer-implemented embodiments further improve computer system function by reducing excessive user wait time through the use of data structures that reduce search from linear to logarithmic time.

Methods to edit genes by administering a chimeric nuclease to a cell or organism without the use of a viral vector.

Compositions and methods comprising a deaminase for targeted editing of nucleic acids are provided. Also provided are compositions and methods for localizing a heterologous polypeptide to a target DNA molecule, and compositions and methods for targeted editing of nucleic acids. Fusion proteins comprising an RNA-guided nuclease (RGN) and at least one heterologous polypeptide inserted therein are provided, as well as fusion proteins comprising a DNA binding polypeptide and a deaminase. The heterologous polypeptide may be a pilot editing polypeptide or a base editing polypeptide. Compositions also include nucleic acid molecules encoding a deaminase or fusion protein. Vectors and host cells comprising the nucleic acid molecules encoding the deaminase or fusion protein are also provided.

Disclosed herein are methods to edit genes, for example by administering a chimeric nuclease to a cell or organism without the use of a viral vector. Some methods herein include editing DNA of a cell by contacting the cell with a nuclease comprising a Cas9 sequence, where the nuclease edits the DNA of the cell.

This invention relates to variants of Cas12a nucleases having altered protospacer adjacent motif recognition specificity. The invention further relates to methods of making CRISPR-CAS nuclease variants and methods of modifying nucleic acids using the variants.

The invention discloses a Cg12n-v4.6 nuclease. The amino acid sequence of the nuclease is as shown in SEQ ID No. 12. The invention also discloses a preparation method of the nuclease. The Cg12n-v4.6 nuclease mutant with increased activity and improved editing efficiency is developed, and the editing efficiency of the Cg12n-v4.6 nuclease mutant is up to 70% or above. Meanwhile, through engineering modification of corresponding sgRNA of Cas12n, the size of the sgRNA skeleton is reduced. An enhanced Cas12n gene editing tool, namely an eCg12n gene editing system, is developed by combining an engineered Cg12n-v4.6 nuclease mutant and a simplified sgRNA skeleton. Meanwhile, by fusing deaminase, a compact cytosine base editor and a compact adenine base editor are constructed, and the cytosine base editor is applied to edit and induce disease genes to generate early termination codons, so that safe knockout of the genes is realized. Compared with an original wild type editing system, the Cg12n-v4.6 nuclease mutant and a compact eCg12n gene editing system derived from the Cg12n-v4.6 nuclease mutant have the advantages that the editing efficiency is improved, the system size is further simplified, and the Cg12n-v4.6 nuclease mutant and the compact eCg12n gene editing system derived from the Cg12n-v4.6 nuclease mutant are more suitable for gene editing application.

The invention provides a quantitative detection system combining a CRISPR-Cas system with a MetaSPR optical sensor, a detection method and application, and relates to the technical field of biosensing and optical detection. The system comprises a MetaSPR optical sensor, the surface of a gold substrate of the MetaSPR optical sensor is modified with single-stranded DNA H1, the 5'end of H1 is modified with sulfydryl, and H1 is fixed to the gold substrate of the MetaSPR optical sensor through an Au-S bond; h1 is combined with single-chain DNA H2 with gold balls through complementary base pairing to form a double-chain structure, the 5'end of H2 is also modified with sulfydryl, and the gold balls are fixed on the surface of a gold substrate; the CRISPR-Cas reaction system comprises a molecular solution to be detected, Cas protein, a buffer solution, guide RNA (Ribonucleic Acid), nuclease-free water and target DNA (Deoxyribose Nucleic Acid); the CRISPR-Cas reaction system is used for recognizing a target molecule, and when the target molecule is recognized, H1 is cut.

The invention belongs to the field of gene engineering, and relates to a plant genome directional editing tool based on transposon-coded nuclease. The invention aims to solve the technical problem that the application range of a plant genome editing tool based on CRISPR-Cas9 and Cas12a is limited to a great extent due to the fact that CRISPR-Cas9 and Cas12a are large in protein size and difficult to deliver. The invention provides transposon nuclease suitable for plant genome editing. The transposon nuclease is IsDge10, IsAam1 or enIscB subjected to codon preference optimization; and a gene editing system containing the three transposon nuclease is constructed. The gene editing system can be suitable for editing a coding region, a non-coding region and the like; the method can be widely applied to monocotyledonous plants, dicotyledonous plants, gymnosperm and the like.

A gene editing system comprising (a) a fusion polypeptide comprising an RNA-guided nuclease and a reverse transcriptase, or a nucleic acid encoding the fusion polypeptide, and (b) an RNA molecule comprising a guide RNA and a reverse transcription donor RNA, or a nucleic acid encoding the RNA molecule. Also provided herein are methods of using the gene editing system for modifying target genes of interest.

The invention discloses a method for rapidly establishing an ovarian cancer model based on SauriCas9, and particularly discloses a recombinant plasmid for targeted knockout of Pten and Trp53 genes, and the recombinant plasmid comprises an EPI vector system. The recombinant plasmid takes an ori element as a replication start site, and sequentially comprises an sgRNA sequence of a targeted Trp53 gene and Pten controlled by double U6 promoters, a CAG promoter, a SauriCas9 nuclease expression unit, a fluorescent protein expression element, a resistance gene, an orip element and an EBNA1 protein expression element. The invention also discloses a method for rapidly establishing an ovarian cancer model based on SauriCas9, and the established ovarian cancer cell model. By adopting the method to construct the ovarian cancer cell model, the period from cell editing to animal tumor formation is shortened, the stability and immune integrity of the genetic background of the model are ensured, and large-scale drug screening and high-throughput experiments are facilitated.

The invention relates to the field of molecular biology, and discloses a nucleic acid releasing agent for isothermal amplification and a use method of the nucleic acid releasing agent. Comprising the following steps: step 1, preparing a polyoxypropylene polyoxyethylene copolymer diluent, a defoaming agent SE-15 diluent, an ethyl phenyl polyethylene glycol NP-40 diluent, a sodium polyethylene sulfonate diluent and a Proclin300 diluent in sequence through RNA enzyme-free water; and 2, sequentially preparing an ethylenediamine tetraacetic acid disodium salt mother solution, a sodium dodecyl sulfate mother solution and a glycine mother solution through RNA enzyme-free water. Sodium polyethylene sulfonate can be specifically combined with protein amplification inhibitors in a sample to remove mucoprotein, hemoglobin, polysaccharide and other inhibitors in the swab sample, and disodium ethylene diamine tetraacetate can chelate metal ions to reduce the influence of nuclease on target nucleic acid.

The invention discloses a primer and a method for quantitatively monitoring the biomass of sargassum hemiphyllum based on an environmental DNA technology, and belongs to the technical field of molecular ecology. The primer probe group comprises an upstream primer, a downstream primer and a fluorescent probe which are specifically targeted to the sargassum hemiphyllum mitochondria COX1 gene, and the sequence is shown as SEQ ID NO.1-3. The kit comprises the primer probe group, a qPCR (quantitative polymerase chain reaction) premixed solution, nuclease-free water and a sargassum hemiphyllum plasmid positive control. The method comprises the following steps: collecting a water sample, enriching eDNA, performing qPCR detection by using the primer probe group after extraction and purification, and realizing qualitative detection and quantitative evaluation of sargassum hemiphyllum through a Cq value or a standard curve. The method disclosed by the invention has the advantages of high sensitivity, strong specificity, no damage to the environment and target organisms, capability of realizing large-scale rapid general survey and the like, and is suitable for early warning of gulfweed blooms, investigation of population distribution and evaluation of ecological influence.

The invention relates to a Cas13 protein, a CRISPR (clustered regularly interspaced short palindromic repeats)-Cas system and application of the CRISPR-Cas system. The amino acid sequences of the Cas13 protein are shown as SEQ ID NO.1-SEQ ID NO.9, and the Cas13 protein is respectively named as Cas13a10, Cas13a11, Cas13b3, Cas13b4, Cas13b5, Cas13T9, Cas13T15, Cas13T16 and Cas13Z2; the 9 Cas13 proteins have low homology with reported Cas13 proteins, show RNA nuclease activity, and have great application prospects in gene editing.

The invention discloses an engineered artificial vesicle and application of the engineered artificial vesicle in multiple in-situ detection of urine exosome miRNA, and belongs to the field of biosensors. The method comprises the following steps: constructing planar framework nucleic acid simultaneously modified with cholesterol and an aptamer through annealing reaction; a molecular beacon with a fluorophore is designed according to a target gene sequence, and the molecular beacon and a double-strand specific nuclease (DSN) system are jointly encapsulated in an artificial vesicle; the engineered artificial vesicles and a sample to be detected are incubated, the fluorescence modified molecular beacons can specifically recognize target genes, cyclic cutting and signal amplification are achieved under DSN mediation, and the broken molecular beacons release fluorescence signals; all fluorescence signals in the vesicles are collected through a fluorescence imaging technology, and a characteristic fluorescence spectrum of the to-be-detected sample is obtained. According to the present invention, the miRNA heterogeneity analysis at the single vesicle level and the precise multi-target detection of bladder cancer and other diseases can be achieved, and the problems of low throughput, target number limitation and the like of the existing vesicle in-situ detection are effectively overcome.

The invention relates to the technical field of microorganism application, in particular to low-nucleic-acid yeast protein as well as a preparation method and application thereof. The preparation method of the low-nucleic-acid yeast protein comprises the following steps: (1) carrying out inactivation treatment on high-protein yeast; (2) carrying out enzymolysis on the inactivated yeast by adopting neutral protease, glucanase, mannase and first nuclease, and after enzyme deactivation, separating and taking a heavy phase to obtain crude yeast protein; (3) performing high-pressure homogenization treatment on the crude yeast protein; and (4) carrying out enzymolysis on the crude yeast protein subjected to high-pressure homogenization treatment by adopting second nuclease and lipase, and after enzyme deactivation, separating and taking a heavy phase to obtain the low-nucleic-acid yeast protein. The yeast protein with the nucleic acid content of 1.0% or below is obtained by taking the high-protein yeast as a raw material through inactivation, compound enzyme enzymolysis, high-pressure homogenization treatment and secondary enzymolysis, is neutral and pure in taste, and can be widely applied to the fields of nutrition, health and food such as protein supplementation and protein substitution.

Systems, methods and compositions for targeting polynucleotides are detailed herein. In particular, engineered DNA-targeting systems comprising IscB polypeptides, novel IscB nucleases and reprogrammable targeting nucleic acid components and methods and application of use are rovided.

The invention discloses a novel nuclease editing system, and belongs to the field of gene engineering. The invention provides sequence-specific nuclease which has targeted cleavage activity and can be combined with target DNA in a targeted manner under the guidance of reRNA or sgRNA. In addition, the invention also provides a gene editing system containing the sequence-specific nuclease and a modified nuclease system, and the gene editing system and the modified nuclease system can effectively target a target spot and cut or modify a target site.

The application discloses a kind of compositions, reagents and its detection method for high-throughput gene sequencing, belong to gene sequencing technical field, the composition includes Tris-HCl buffer 80-120 parts, potassium chloride 40-60 parts, ammonium sulfate 10-20 parts, magnesium chloride 15-25 parts, dNTPs mixed solution 30-50 parts, heat-stable DNA polymerase mutant 8-15 parts, PCR promoter complex 5-12 parts, modified iridium quinoline ketone complex 1-5 parts, glycerol 150-250 parts, Tween-20 1-3 parts, EDTA disodium salt 0.5-2 parts and nuclease-free ultrapure water, by introducing the modified iridium quinoline ketone complex of innovation, effectively solve the technical problems of low amplification efficiency, poor multiplex PCR reaction uniformity and high non-specific amplification background for high GC content, complex secondary structure genomic template, significantly improve the specificity of amplification, uniformity and the quality and reliability of final sequencing data.

The invention relates to the technical field of water pollution and water body odor substance detection, in particular to a primer group, a kit and a method for detecting cyanobacteria generating 2-methylisoborneol in a water body based on a recombinase-mediated isothermal nucleic acid amplification (RAA) technology. The primer group is designed based on a mic gene conserved region of the 2-methylisoborneol-producing cyanobacteria, and comprises a forward primer, a reverse primer and a specific probe; the 5'end of the reverse primer is modified with a fluorescent marker, the 5 'end of the probe is modified with biotin, the 3' end of the probe is modified with a phosphorylation group, and nuclease cleavage sites are contained in the probe. The primer group disclosed by the invention can effectively amplify the mic gene of the 2-methylisoborneol, has relatively high specificity and sensitivity, has no cross reaction with cyanobacteria which does not generate the 2-methylisoborneol, and can be combined with lateral flow immunochromatography to be used for on-site rapid detection of the 2-methylisoborneol; the method is of great significance in early warning of excessive content of 2-methylisoborneol in the water body.