1455 results about "Nuclease" patented technology

The invention relates to the improvement of endonuclease-based antimicrobials by blocking DNA repair of double-strand break(s) (DSB(s)) in prokaryotic cells. In this respect, the invention especially concerns a method involving blocking DNA repair after a nucleic acid has been submitted to DSB, in particular by a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated programmable double-strand endonuclease. The invention particularly relates to the use of an exogenous molecule that inhibits DNA repair, preferably a protein that binds to the ends of the double-stranded break to block DSB repair. The invention also relates to vectors, particularly phagemids and plasmids, comprising nucleic acids encoding nucleases and Gam proteins, and a pharmaceutical composition and a product containing these vectors and their application.

Engineered CRISPR-Cas9 nucleases with altered and improved PAM specificities and their use in genomic engineering, epigenomic engineering, and genome targeting.

The invention discloses a polypeptide and application thereof in preparation of a medicine for preventing and / or treating cancers. The amino acid sequence of the polypeptide is shown as SEQ ID No. 1 or SEQ ID No. 2. The cancer is at least one of breast cancer, colorectal cancer, leukemia, esophageal cancer, pancreatic cancer, brain glioma, kidney cancer, skin cancer, osteosarcoma, ovarian cancer and prostate cancer. The invention designs and synthesizes the polypeptide of a targeted staphylococcus tumor protein compound SND1-MTDH containing a nuclease structural domain 1 (SND1 and a heteromucin (MTDH), finds that the polypeptide has excellent inhibitory activity on various different types of human cancer cells for the first time, and provides an optional scheme for broad-spectrum anti-cancer drugs. The polypeptide applied in the invention is simple in synthesis process and low in production cost.

To provide a method for detecting the presence of multiple specific nucleic acid sequences or single nucleotide polymorphisms in a temperature-dependent fashion.SOLUTION: Provided is a method, in which an invasive oligonucleotide and a primary probe oligo (a 5' flap oligonucleotide) hybridize to a target strand nucleic acid to form an invasive cleavage structure cleavable by a FEN-1 endonuclease to release a cleaved 5' flap (primary cleaved flap); in a secondary reaction, the primary cleaved flap from the primary reaction hybridizes to a FRET cassette 1, a hairpin oligonucleotide labeled with a fluorescent dye and a quencher molecule, to form a second invasive cleavage structure that is cleavable by a FEN-1 endonuclease at a site between the fluorophore and a quencher; and cleavage of FRET cassette 1 separates the quencher from the fluorophore, such that fluorescence signal from the fluorophore can be detected.SELECTED DRAWING: Figure 1

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.

The invention belongs to the technical field of biology, and particularly relates to an extraction method of polydeoxyribonucleotide, which comprises the following steps: carrying out homogenate treatment on pretreated in-vitro testis tissue, filtering the obtained homogenate, adding a pancreas extract into the filtrate, and stirring to obtain enzymatic hydrolysate; and precipitating protein in the enzymatic hydrolysate, removing the protein, adding sodium acetate for treatment, adding absolute ethyl alcohol for precipitation, centrifuging, washing the obtained precipitate, and drying to obtain the product. Common trypsin and lipase in fish pancreas are beneficial to release of DNA, the fish pancreas extract is applied to extraction of the polydeoxyribonucleotide, the extraction time can be remarkably shortened, the yield can be increased, meanwhile, nuclease in the fish pancreas extract can effectively degrade DNA, and small-fragment polydeoxyribonucleotide is obtained.

The invention relates to methods and compositions for treating or preventing an infection by E coli cells in human or animal subjects. The method comprises administering to the subject a plurality of transduction particles that encode a nuclease for targeting the genomes of B2 phylogroup E coli cells.

The invention discloses an RAA-CRISPR / Cas12a detection system of a circle virus avian origin type 1 and application of the RAA-CRISPR / Cas12a detection system, and relates to the technical field of microbiological detection. The detection system comprises an RAA amplification primer pair, crRNA, an FQ-ssDNA probe, an FB-ssDNA probe and an LbCas12a nuclease, and is characterized in that the detection system comprises an RAA amplification primer pair; the RAA amplification primer pair comprises an upstream primer of which the nucleotide sequence is shown as SEQ ID NO.5 and a downstream primer of which the nucleotide sequence is shown as SEQ ID NO.8; the nucleotide sequence of the crRNA is as shown in SEQ ID NO. 11. The RAA-CRISPR / Cas12a detection kit established by adopting the detection system developed by the invention has the advantages of high efficiency, high specificity and high sensitivity, can be used for on-site rapid detection of GyVg1, and provides a powerful diagnostic tool with a cost advantage for a basic-level working environment.

The technology described herein relates to novel CRISPR / Cas systems and components thereof, vectors, proteins, fusion proteins, ribonucleoprotein complexes and methods of using the novel systems and components. The new system can be used in CRIS PRi applications and can be fused with any number of effector domain patterns, such as nuclease, base editor, leader editor, and the like, to conduct a variety of DNA modifications.

The invention relates to the technical field of biological sample preservation, in particular to a sample preservation reagent for a giant spiny frog InDel molecular marker and a preparation method of the sample preservation reagent, degradation of DNA in the preservation process can be remarkably reduced through multiple action mechanisms of maintaining the pH value stable through a buffer solution system, inhibiting nuclease activity through a nucleic acid protective agent, scavenging free radicals through an antioxidant and the like, and the preservation effect is good. The method has the advantages that the cell stabilizer is added, DNA completeness is guaranteed, a reliable template is provided for follow-up accurate detection of InDel molecular markers, protective films can be formed around cells by the aid of the cell stabilizer, the cells are prevented from being damaged by ice crystals, osmotic pressure balance of the cells is maintained, high activity of the cells can be kept in the storage process, and follow-up extraction of high-quality DNA from the cells is benefited; the addition of the antibacterial agent can effectively inhibit possible microbial contamination of the sample in the preservation process, prolong the preservation time of the sample, and ensure the quality and safety of the sample.

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 an engineered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system comprising engineered dual guide nucleic acids (e.g., RNAs) capable of activating a CRISPR-Associated (Cas) nuclease, such as a type V-A Cas nuclease. Also provided are methods of targeting, editing, and / or modifying a nucleic acid using the engineered CRISPR system, and compositions and cells comprising the engineered CRISPR system.

The invention discloses a detection system for simultaneously detecting circle virus poultry source type 1 and circle virus human source type 1 and application of the detection system, and relates to the technical field of biological detection. The detection system comprises an RAA amplification primer pair of GyVg1, an RAA amplification primer pair of GyH1, a 12-crRNA-3 (12-crRNA-3), a 13-crRNA-2 (13-crRNA-2), an ssDNA (single-stranded deoxyribonucleic acid) probe, an ssRNA (single-stranded ribonucleic acid) probe, an LbCas12a nuclease, an LbuCas13a nuclease and a T7 transcriptase. According to the present invention, based on GyVg1 and GyH1 conserved genes, the specific crRNA and the RAA primer are respectively designed, and the RAA amplification technology and the CRISPR-Cas fluorescence detection system are combined so as to establish the detection system for simultaneously detecting GyVg1 and GyH1; the detection system can detect two different subtypes of circle viruses at the same time, the detection efficiency is improved, and a new technical means is provided for dynamic research of virus infection.

The invention discloses a novel tsRNA high-sensitivity detection kit based on CRISPR (clustered regularly interspaced short palindromic repeats) / Cas13a-Cas14a, and belongs to the field of tumor marker detection. The tsRNA (tRNA-derived small RNA) is a diagnostic marker which is found recently and has great potential. In order to realize the clinical transformation of tsRNA as soon as possible, the tsRNA detection kit is developed on the basis of nuclease P1 combined with CRISPR / Cas13a-Cas14a linkage dual systems, and comprehensive, ultra-sensitive, high-specificity, low-cost and rapid tsRNA detection is realized through multiple signal amplification. Compared with the traditional technology, the sensitivity of the method is improved by 105-107 times, 1% of tsRNA in the mixture can be detected, and the method has more advantages in clinical application. Therefore, a new scheme is provided for application of the tsRNA in clinical diagnosis of diseases, and meanwhile, a reliable technology is provided for mechanism research of the tsRNA in occurrence and development of the diseases.

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.

The present invention relates generally to the field of medicine. More particularly, the present invention relates to polypeptide conjugates, compositions and methods for promoting immune tolerance to CRISPR-Cas (clustered regularly spaced short palindromic repeats-CRISPR associated proteins) systems for use in genomic engineered gene therapy.

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 invention provides a non-disease diagnosis purpose 5-hydroxymethylcytosine 5hmC detection method and a non-disease diagnosis purpose 5-hydroxymethylcytosine 5hmC detection kit. The detection method comprises the following steps: S1, glycosylation treatment; s2, carrying out enzyme digestion treatment on the GSREs; s3, carrying out APOBEC deamination treatment; s4, carrying out hydroxymethylation detection; the kit comprises a glycosylation reaction kit, a GSREs enzyme digestion reaction kit, an APOBEC deamination reaction kit and a PCR (Polymerase Chain Reaction) kit. The glycosylation reaction kit comprises a CutSmart buffer solution, UDP-Glucose, a T4-BGT enzyme and nuclease-free water, wherein the CutSmart buffer solution, the UDP-Glucose and the T4-BGT enzyme are used as raw materials; the GSREs enzyme digestion reaction kit comprises a GSREs enzyme, an enzyme digestion reaction buffer solution and nuclease-free water, the APOBEC deamination reaction kit comprises APOBEC, BSA (Bovine Serum Albumin), an APOBEC Reactive buffer solution and nuclease-free water; the PCR kit comprises a PCR reaction solution, a primer and a probe. According to the 5-hydroxymethylcytosine 5hmC detection method and kit for non-disease diagnosis purpose provided by the invention, precise quantification of extremely high specificity of 5hmC is realized, and the detection sensitivity is also improved while the specificity of a detection signal is improved.

The present disclosure provides nucleases and compositions, methods, and systems thereof for nucleic acid modification. More particularly, the present disclosure provides compositions and system comprising a nuclease comprising an amino acid sequence having at least 70% identity to any of SEQ ID NOs: 1-1096 and at least one gRNA.

The invention discloses application of programmable TnpB nuclease gene editing, and relates to the technical field of gene editing. The TnpB nuclease is any one of (A1), (A2) and (A3): (A1) TnpB nuclease, the amino acid sequence of which is as shown in SEQ ID NO.1; (A2) carrying out substitution, deletion and / or addition of one or more amino acid residues on the amino acid sequence of the TnpB nuclease to obtain a protein which has 80% or more of identity with the TnpB nuclease and has the same biological function with the TnpB nuclease; and (A3) a fusion protein obtained by connecting a protein tag to the N terminal or / and C terminal of (A1) or (A2). The TnpB nuclease provided by the invention has cis-cleavage activity and trans-cleavage activity, and has a wide application prospect in the fields of nucleic acid detection and gene editing.

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.

Disclosed herein is a composition for nuclease treatment of hybridized probes in experiments to profile and analyze biological samples. Also disclosed herein, is a kit for nuclease treatment of hybridized probes in experiments to profile and analyze biological samples. Also disclosed herein, is a method for nuclease treatment of hybridized probes in experiments to profile and analyze biological samples.

Provided herein are dual cleaving chimeric nucleases comprising an I-TevI domain and a RNA-guided domain such as Cas9 for use in gene editing methods, as well as chimeric nuclease systems comprising a single nucleic acid that encodes said chimeric nuclease, one or more guide RNAs (gRNAs), one or more tRNAs, and optionally one or more donor polynucleotides. Also provided herein are methods of making and using such chimeric nucleases, chimeric nuclease systems, and nucleic acids encoding such chimeric nucleases and chimeric nuclease systems.