1060 results about "Nuclease" patented technology

The present application provides guide RNAs and genome-editing complexes or nanoparticles that are useful for specifically targeting a mutated KRAS. Exemplary genome-editing complexes or nanoparticles comprise cell-penetrating peptides, and optionally a DNA nuclease (such as Cas9) or a polynucleotide encoding the DNA nuclease.

Some aspects of this disclosure provide compositions, methods, systems, and kits for controlling the activity of RNA-programmable endonucleases, such as Cas9, or for controlling the activity of proteins comprising a Cas9 variant fused to a functional effector domain, such as a nuclease, nickase, recombinase, deaminase, transcriptional activator, transcriptional repressor, or epigenetic modifying domain. For example, the inventive proteins provided comprise a ligand-dependent intein, the presence of which inhibits one or more activities of the protein (e.g., gRNA binding, enzymatic activity, target DNA binding). The binding of a ligand to the intein results in self-excision of the intein, restoring the activity of the protein.

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 discloses a novel separated guide editor based on a Csy4 system and related protein. The invention belongs to the technical field of biology, and particularly relates to a novel separated guide editor based on a Csy4 system and related protein. The fusion protein is a protein obtained by fusing Csy4 nuclease of which the amino acid sequence is SEQ ID No: 1 to one end of RT protein, the Csy4 nuclease is fused to the N end or C end of the RT protein, and the amino acid sequence of the fusion protein is SEQ ID No: 2 or SEQ ID No: 3. The fusion of Csy4 improves the editing efficiency of separated guided editing, and compared with separated PE3max, the editing efficiency of 12 important agronomic trait targets of 11 genes of rice is greatly improved. The research lays a foundation for developing a Csy4-based separated guide editor with higher editing efficiency in the future.

Disclosed are compositions comprising a nucleic acid molecule. The nucleic acid molecule may encode an exonic sequence or portion thereof of a target ribonucleic acid (RNA) sequence. The nucleic acid molecule may further encode one or more stabilization domains. The one or more stabilization domains may be configured to reduce a cellular nuclease activity compared to a nucleic acid molecule that does not comprise the one or more stabilization domains.

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 present invention relates to a fusion protein that produces point mutations in cells, its preparation and use. Specifically, the fusion protein provided by the present invention contains a Cas enzyme that lacks cytosine deaminase and nuclease activity and retains helicase activity, or is formed by a Cas enzyme that lacks cytosine deaminase and nuclease activity and retains helicase activity. The present invention also relates to the coding sequence of the fusion protein, a polynucleotide sequence containing the coding sequence, a nucleic acid construct containing the polynucleotide sequence, a corresponding host cell, a method for producing point mutations in a cell, and a kit, etc. By using the present invention, it is possible to achieve site-directed mutagenesis while obtaining high mutation efficiency and a variety of mutation combinations in a specific gene region.

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 present invention provides a chimeric nucleic acid-guided nuclease including at least one domain substituted with distinct bacterial species. These chimeric nucleases improve function of modified guide polynucleotides, enhancing the genomic modification abilities of nucleic acid-guided nucleases. CRISPR-Cas systems such as those described herein are often used in genome editing, but the complexity of the polypeptides and polynucleotides leads to unpredictable outcomes. The chimeric CRISPR-Cas system disclosed herein was surprisingly able to rescue function of a previously non-functional guide nucleotide. The nucleases and methods described herein are useful for treating diseases associated with a gene or polynucleotide.

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.

Provided herein are methods of inserting a nucleic acid encoding a polypeptide of interest into a target genomic locus in a cell or a population of cells in a subject, methods of expressing a polypeptide of interest from a target genomic locus in a cell or a population of cells in a subject, methods of treating an enzyme deficiency in a subject in need thereof, and methods of preventing or reducing the onset of a sign or symptom of an enzyme deficiency in a subject in need thereof. The methods use CD40 inhibitors (e.g., CD40 antigen-binding molecules) to mitigate immune response and facilitate redosing of nucleic acid constructs encoding a polypeptide of interest and nuclease agents targeting a target genomic locus to achieve, for example, a step-wise increase in expression of a polypeptide of interest in a subject following insertion of the nucleic acid construct without overshooting.

The invention discloses an all-potent nuclease mutant and application thereof, and relates to the technical field of biology. The invention provides an all-potent nuclease mutant. Compared with wild type all-potent nuclease as shown in SEQ ID NO: 1, the all-potent nuclease mutant comprises mutations at the following two positions: S22 and S115. The all-purpose nuclease mutant disclosed by the invention still has relatively high specific activity in a high-salt environment, and has a wide application prospect in preparation of medium-salt all-purpose nuclease products with high specific activity and good stability.

The invention discloses a gastric signet-ring cell carcinoma biomarker and a detection kit thereof, belongs to the technical field of biological medicines, and particularly relates to the detection kit of the gastric signet-ring cell carcinoma biomarker, which comprises a total RNA (Ribonucleic Acid) extraction reagent, an RNA purification reagent, a cDNA (Complementary Deoxyribonucleic Acid) first strand synthesis premixing reagent, a rapid fluorescent quantitative premixing reagent (SYBR Green), nuclease-free double distilled water and a specific primer. The RNA purification reagent at least comprises isopropanol, chloroform and absolute ethyl alcohol; the specific primer comprises an upstream specific primer and a downstream specific primer. The detection kit for the gastric signet-ring cell carcinoma biomarker is high in RNA purification and recovery efficiency and excellent in specificity.

Provided herein are methods of inserting a nucleic acid encoding a polypeptide of interest into a target genomic locus in a cell or a population of cells in a subject, methods of expressing a polypeptide of interest from a target genomic locus in a cell or a population of cells in a subject, methods of treating an enzyme deficiency in a subject in need thereof, and methods of preventing or reducing the onset of a sign or symptom of an enzyme deficiency in a subject in need thereof. Some methods, such as when a subject has preexisting against an immunogen to be administered, use plasma cell depleting agents or combinations comprising plasma cell depleting agents to mitigate immune response and facilitate redosing of nucleic acid constructs encoding a polypeptide of interest and nuclease agents targeting a target genomic locus to achieve, for example, a step-wise increase in expression of a polypeptide of interest in a subject following insertion of the nucleic acid construct without overshooting. Other methods, such as when a subject has no preexisting immunity against an immunogen to be administered, use B cell depleting agents (e.g., anti-CD20xCD3 antibody or functional fragment thereof) to mitigate immune response and facilitate redosing of nucleic acid constructs encoding a polypeptide of interest and nuclease agents targeting a target genomic locus to achieve, for example, a step-wise increase in expression of a polypeptide of interest in a subject following insertion of the nucleic acid construct without overshooting.

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.

The invention relates to the technical field of nucleic acid on-site rapid visual detection, in particular to a one-pot rapid nucleic acid detection system based on an SfaTnpB combined isothermal amplification technology and application of the one-pot rapid nucleic acid detection system. According to the invention, an RAA isothermal amplification technology and an LAMP isothermal amplification technology are respectively combined with SfaTnpB nuclease, and finally, an RAA-SfaTnpB one-pot method system and an LAMP-SfaTnpB one-pot method system are successfully established. The one-pot system established by the invention has the advantages of high sensitivity, high specificity, fast reaction, strong accuracy and no need of complex equipment, the detection result can be visually interpreted by naked eyes, the system has high convenience and practicability, the adaptability and practicability of the system are further improved, and the rapid detection requirements in various scenes can be flexibly met. The system has a wide application prospect in the fields of rapid pathogen detection, genotype detection, early diagnosis of infectious diseases and the like.

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 provides a post-treatment method of single-cell protein fermentation liquor and a fermentation method of single-cell protein thalli. Nuclease and cell wall lytic enzyme are respectively added into fermentation liquor containing single-cell protein thalli to treat the thalli, so that nucleic acid and cell wall residues can be reduced, and the problems that in a traditional fermentation process, the application of the thalli in food is limited due to the high nucleic acid content of the thalli, and the protein extraction efficiency is reduced due to the existence of the cell walls are solved. In addition, by optimizing fermentation conditions, the protein content of the single-cell protein is remarkably increased. The process is simple, low in cost, suitable for industrial production and capable of being widely applied to the fields of feed, food and medicine.