1988 results about "CRISPR" patented technology

A federated distributed computational system enables secure biological data analysis and genomic medicine with enhanced oncological therapy capabilities. The system implements patient-specific tumor-on-a-chip analysis through microfluidic control systems and cellular heterogeneity preservation, while integrating fluorescence-enhanced diagnostics using CRISPR-LNP targeting and robotic surgical navigation. The architecture coordinates spatiotemporal analysis of gene therapy delivery through molecular imaging and immune response tracking, and implements bridge RNA integration with multi-target synchronization. Treatment selection is optimized through multi-criteria scoring and patient-specific simulation modeling. Each federated node contains a local processing unit for biological data analysis, privacy preservation protocols, and a hierarchical knowledge graph structure. The system implements cross-species genetic analysis, environmental response modeling, and multi-scale tensor-based data integration, enabling research institutions to collaborate on complex, large-scale biological analyses while maintaining strict data privacy controls.

The invention belongs to the technical field of pathogenic microorganism detection, and particularly relates to a sequence combination for detecting cochliobolus miyabeanus based on RPA-CRISPR / Cas12b and application of the sequence combination. The CRISPR / Cas system contained in the composition comprises Cas12b and sgRNA, and the CRISPR / Cas system can be cooperatively used with a specific reaction system. The rapid detection method comprises the following steps: sample pretreatment: treating a sample to extract nucleic acid; performing amplification reaction: performing RPA amplification reaction on the extracted nucleic acid by using the composition, and applying a nucleic acid amplification product to CRISPR / Cas system detection; result interpretation: reading and judging a detection result through a quality control line and a detection line. The composition and the kit provided by the invention are specially used for detecting the cochliobolus miyabeanus. The method is simple and convenient to operate, rapid in detection, short in time consumption, low in cost and suitable for on-site rapid detection.

The present disclosure provides compositions, methods, systems, and devices for polynucleotide processing and analyte characterization. Such polynucleotide processing may be useful for a variety of applications, including analyte characterization by polynucleotide sequencing. The compositions, methods, systems, and devices disclosed herein generally describe barcoded oligonucleotides, which can be bound to a bead, such as a gel bead, useful for characterizing one or more analytes including, for example, protein (e.g., cell surface or intracellular proteins), genomic DNA, and RNA (e.g., mRNA or CRISPR guide RNAs). Also described herein, are barcoded labelling agents and oligonucleotide molecules useful for “tagging” analytes for characterization.

The invention provides an enhancer RNA (Ribonucleic Acid) molecule MZGAe1 and application thereof, and relates to the technical field of biology. The invention provides an enhancer RNA (Ribonucleic Acid) molecule MZGAe1. The nucleotide sequence of the enhancer RNA molecule MZGAe1 is as shown in SEQ ID NO: 1; meanwhile, the invention further provides a specific sgRNA sequence for activating the molecule in a targeted manner and a recombinant vector of the specific sgRNA sequence. Expression of endogenous MZGAe1 of cells is specifically activated by adopting a CRISPR activation technology, and conversion of mouse embryonic stem cells to bicellular-like cells can be efficiently promoted. By providing a brand-new endogenous targeted MZGAe1 accurate activation tool, the efficient transformation of wild mouse embryonic stem cells to bicellular cells can be realized only under the condition of endogenous activation of single enhancer RNA molecule MZGAe1, the proportion is at least increased by 5%, the expression of a totipotent marker gene ZSCAN4 is activated, and the expression of the totipotent marker gene ZSCAN4 is promoted. And an efficient and specific brand-new research tool and scheme are provided for researching zygote genome activation, embryonic development early events and cell reprogramming.

The invention discloses an integrated fluorescence detection kit and a testing method thereof.The integrated fluorescence detection kit comprises an upper cover assembly, a lysate tube assembly, a main body assembly, a diluent tube assembly and a decorative cover, and the lower end of the upper cover assembly is connected with the lysate tube assembly and the diluent tube assembly; the side wall of the upper cover assembly is connected with the upper part of the main body assembly, and the main body assembly is nested in the decorative cover; a reagent system in the kit comprises an RT-RAA reaction system and a CRISPR reaction system which are stored in the detection kit at normal temperature in the form of freeze-dried microspheres and closed liquid, and a lysis solution and a diluent are released through the lysis solution tube assembly and the diluent tube assembly and react in the main body assembly. According to the invention, the problems that the existing fluorescence detection operation process is complicated, the instrument is not portable and depends on professional analysis and the like are solved, and the rapid, portable and user-friendly detection on the virus is realized based on the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) detection system.

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.

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 invention discloses a kit for visually detecting an H5 subtype avian influenza virus based on an RPA-CRISPR / Cas12b system and application of the kit, and belongs to the technical field of molecular detection. The kit comprises an RT-RPA (Reverse Transcription-Recombinase Polymerase Amplification) primer pair and sgRNA1 The RT-RPA primer pair comprises a forward primer with a nucleotide sequence as shown in SEQ ID NO.1 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO.2; and the nucleotide sequence of the sgRNA1 is as shown in SEQ ID NO. 7. According to the kit, nucleic acid amplification and detection are synchronously completed through a tubular reaction system, the detection sensitivity reaches 1 copy / mu L, the coincidence rate with fluorescent RT-PCR is 100%, and no cross reaction exists on H7, H9, NDV and IBV. The kit does not need a complex instrument, is suitable for rapid screening on a basic level site, and provides an efficient solution for prevention and control of avian influenza.

Disclosed are lipid nanoparticle (LNP) delivery systems that specifically target T cells. The LNP delivery system comprises antibodies conjugated to the surface of the LNP, e.g., via maleimide chemistry, that target at least two T cell surface proteins, e.g., CD3 and CD28. The LNP delivery system can have a single population of LNP conjugated to either a bispecific antiCD3 / antiCD28 antibody, or two monospecific antiCD3 and antiCD28 antibodies, or two populations of LNP wherein each population comprises a monospecific antibody. The payload of the LNP delivery system can be, e.g., mRNA encoding chimeric antigen receptors (CAR), a linear DNA fragment or a plasmid encoding chimeric antigen receptors (CAR) or therapeutic proteins such as antibodies, components of a gene editing systems (e.g., CRISPR-Cas), small molecules, antibody-drug conjugates (ADC), and any combination thereof, either encapsulated in the LNP or attached to its surface (e.g., conjugated). Also provided are lipids, pharmaceutical compositions, kits, and methods of treatment.

The invention provides a CRISPR (clustered regularly interspaced short palindromic repeats) / Cas-based kit for isothermal rapid visual detection of H1N1 influenza virus and mycoplasma pneumoniae and a detection method thereof. The kit and the detection method provided by the invention can be used for timely, sensitively and specifically detecting a nucleic acid target in a sample. The invention provides a lateral flow analysis (LFA) detection platform of a three-stage amplification system integrating recombinase polymerase amplification (RPA), CRISPR / Cas12a and catalytic hairpin assembly (CHA) circulation, the detection platform is used for sensitively and accurately detecting mycoplasma pneumoniae DNA (MP DNA) and influenza A virus subtype H1N1RNA (H1N1RNA), and compared with other researches based on an LFA-CRISPR / Cas12a system, the detection platform has the advantages that the detection efficiency is greatly improved, and the detection cost is reduced. According to the kit and the detection method provided by the invention, nucleic acids of two pathogens can be visually detected on a single LFA at the same time, and the sensitivity is high.

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 invention belongs to the technical field of living cell imaging, and discloses a living cell nucleic acid imaging system and method based on CRISPR (clustered regularly interspaced short palindromic repeats) and a nano antibody and application, the system comprises: a modified dCas protein expression vector containing n antigen polypeptide tags; the modified sgRNA and / or crRNA expression vector comprises a specific guide sequence; the fluorescent nano-antibody expression vector contains m fluorescent nano-antibody coding sequences which are connected in an operable manner, and fluorescent protein serving as an internal tag is inserted between nano-antibody splitting sites, can be combined with a polypeptide tag carried by dCas protein and can stably emit light; and the fluorescent nano antibody in a free state can be self-degraded, so that the fluorescent nano antibody does not emit fluorescence. According to the invention, the single-copy nucleic acid sequence, especially the imaging marker of the single-copy nucleic acid sequence in a living cell, can be visualized with single-molecule resolution and high signal-to-noise ratio.

The invention provides an RPA-CRISPR-Cas12a system-based detection reagent for the drug resistance of a chilo suppressalis organophosphorus insecticide, a detection method and application, and belongs to the technical field of molecular detection. The invention provides a detection reagent for chilo suppressalis organophosphorus insecticide resistance, the reagent comprises a specific RPA2 primer pair designed for chilo suppressalis acetylcholin esterase ace-1 gene A314S resistance mutation site and crRNA, a double screening mechanism of RPA amplification and CRISPR-Cas12a is combined, wild type and mutant type genes can be accurately distinguished, and the detection reagent can be used for detecting the insecticide resistance of the chilo suppressalis organophosphorus insecticide resistance to the chilo suppressalis organophosphorus insecticide resistance to the chilo suppressalis organophosphorus insecticide resistance to the chilo suppressalis organophosphorus insecticide resistance to the chilo suppressalis. The rapid, sensitive and visual nucleic acid detection of the chilo suppressalis ace-1 gene A314S resistance mutation site is realized, and a visual detection means which is high in specificity, simple and convenient to operate, economical and efficient is provided for field on-site determination of chilo suppressalis resistance populations.

The invention discloses a rhodosporidium toruloides strain with high homologous recombination efficiency as well as a construction method and application thereof, and belongs to the field of oil-producing yeast engineering bacteria. According to the rhodosporidium toruloides strain, the rhodosporidium toruloides strain NP11-SaCas9-1 is used as an original strain, and an RAD52 gene is over-expressed. The modified yeast engineering bacterium provided by the invention can realize high homologous recombination through CRISPR system mediation, the homologous recombination efficiency reaches 90-100%, a multi-gene editing platform can be constructed on the basis, and the modified yeast engineering bacterium can be applied to the fields of synthetic biology, metabolic engineering, genetic engineering and the like, so that the modified yeast engineering bacterium is simpler, quicker, more convenient and more accurate in the genetic modification process. And the homologous recombination activity of the rhodosporidium toruloides can be improved by expressing the homologous recombination related protein Rad52, and the genetic engineering strategy can be used for modifying other unconventional yeasts.

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 present invention relates to CRISPR / Cas based biosensing materials, assays, and methods. Specifically, the present technology relates to CRISPR / Cas-based ultra-sensitive detection methods for nucleic acid assays using specific molecular constructs, including constructs referred to as nucleic acid mediators, comprising single-stranded and double-stranded nucleic acid sequences of a cyclic conformation, and palindromic oligonucleotides. The materials and methods according to the invention can also be used to enhance the sensitivity of existing bioassays.

The invention discloses a small CRISPR (clustered regularly interspaced short palindromic repeats)-Cas gene editing system and application thereof. According to the invention, based on microbial genome and metagenome data, a class of CRISPR-Cas family protein is mined through a biological information method, and is named as Cas12r. A CRISPR-Cas12r editing tool constructed on the basis of the gene can realize gene editing in prokaryotic or eukaryotic cells. The CRISPR-Cas12r gene editing system obtained by the invention has the characteristics of miniaturization and various PAM types.

The invention belongs to the field of biology, and discloses a CRISPR / Cas12a freeze-dried microsphere, which is characterized in that the CRISPR / Cas12a freeze-dried microsphere is obtained by freeze-drying the following raw materials: enzyme-free water, 10 * HOLMES Buffer for Cas12a, an RNA enzyme inhibitor, CRISPR / Cas12a protein, crRNA, an upstream primer F, a downstream primer R, a fluorescent probe Reporter and a freeze-drying protective additive; the CRISPR / Cas12a protein is an LbaCas12a protein, and the CRISPR / Cas12a protein is a Cas12a protein; the freeze-drying protective agent is prepared from mannitol, trehalose, polyethylene glycol, glucan and BSA (Bovine Serum Albumin). By further optimizing a freeze-drying protective agent, freeze-drying microballing of a CRISPR / Cas12a system is achieved, the freeze-drying microballing can be matched with RPA constant-temperature amplification double microballing, and detection is conducted through a one-step method. Meanwhile, the invention further discloses a three-microsphere detection system, application and a kit.

The invention relates to the technical field of nucleic acid detection, in particular to a method for regulating and controlling CRISPR-Cas protein activity through temperature and application of the method, a CRISPR reaction system contains Cas protein, guide RNA, a nucleic acid reporter probe and a buffer solution, and then the cis-cleavage activity or trans-cleavage activity of the Cas protein is activated or inactivated by regulating and controlling the temperature of the reaction system; the guide RNA includes a guide RNA targeting a target nucleic acid sequence that does not contain a PAM site. Multiple detection can be realized only by using the same type of Cas protein, the design of PAM-free sgRNA or crRNA facilitates the selection of sgRNA or crRNA, meanwhile, the activity of PAM-free sgRNA or crRNA can be regulated and controlled by utilizing temperature rise, and the Cas protein with lower reaction temperature can be inactivated, so that the Cas protein cannot cause interference to the detection of the second stage.

The invention discloses a portable nucleic acid detection micro-fluidic chip, a nucleic acid detection device and a nucleic acid detection method, and belongs to the field of micro-fluidic chips, a detection module comprises a quantitative chamber, a micro-channel, a reaction chamber, a first connecting channel and a color developing module; the reaction chamber comprises an RPA reaction chamber in which an RPA amplification reagent is pre-embedded, a porous filter membrane and a CRISPR reaction chamber in which a CRISPR detection component is pre-embedded, and the RPA reaction chamber, the porous filter membrane and the CRISPR reaction chamber are sequentially positioned at the same position of the three adjacent chip layers; the aperture of the porous filter membrane is larger than the particle size of an RPA amplification product and smaller than the particle size of a core enzyme in a CRISPR detection component; two ends of the micro-channel are respectively communicated with the quantitative chamber and the RPA reaction chamber; two ends of the first connecting channel are respectively communicated with the CRISPR reaction chamber and the color developing module; the flow resistance of the first connecting channel is smaller than that of the micro-channel. According to the invention, the sequential reaction of RPA amplification and CRISPR cleavage can be completed without depending on artificial transfer of amplification products.

The invention discloses a primer group, a kit and a method for detecting brucella, and belongs to the technical field of gene detection. The primer group comprises two pairs of specific RPA (recombinase polymerase amplification) primers: a primer pair B1F1 (SEQ ID No.3) and a primer pair B1R1 (SEQ ID No.6) targeting a B1 sequence, and a primer pair B2F1 (SEQ ID No.8) and a primer pair B2R1 (SEQ ID No.11) targeting a B2 sequence. The invention also provides a kit containing the primer group, and a detection method based on the RPA-CRISPR-Cas12a. The invention also provides a kit containing the primer group and a detection method based on the RPA-CRISPR-Cas12a. According to the application, the high efficiency of RPA amplification is combined with the ultrahigh specificity of CRISPR-Cas12a detection, the defects of long time consumption, low sensitivity and easy generation of false positive in the traditional method are overcome, rapid, sensitive and specific detection of Brucella is realized, and the application has important application value in early screening and monitoring of Brucella.

The invention discloses construction and application of a plasmid-free and auxotrophy-free L-tryptophan production strain, and belongs to the technical field of bioengineering. According to the invention, a plurality of key anti-feedback inhibition enzymes in a tryptophan synthesis route are optimized and combined, and the optimal combination is integrated into an escherichia coli genome by utilizing a CRISPR (clustered regularly interspaced short palindromic repeats)-ASPR (clustered regularly interspaced short palindromic repeats)-trans-spansses (clustered regularly interspaced short palindromic repeats) system. According to the invention, the plasmid-free and nutritional deficiency-free engineering strain ZH-3 for producing the L-tryptophan is successfully constructed, and under the conditions of no use of antibiotics, use of inducers and no addition of additional substances, the total yield of the L-tryptophan reaches 43g / L and the sugar-acid conversion rate is increased to 0.180 g / g after the strain is cultured for 35h in a 3L fermentation tank. The strategy used by the invention is not only suitable for construction of tryptophan genome integration expression strains, but also provides reference for construction of other plasmid-free amino acid production strains.

The invention discloses a primer for rapidly detecting nucleic acid of schistosoma japonicum katsurada based on an Sj28S-LAMP-CRISPR / Cas12a system, crRNA and application of the primer and the crRNA. A specific LAMP primer is designed aiming at a specific target sequence in a schistosoma japonicum Sj28S gene, and a target fragment is amplified; specific crRNA is designed to guide Cas12a to recognize a target fragment and activate the trans-cleavage activity of Cas12a, a fluorescence signal report group FQ probe is cleaved, and a result can be observed in real time through a luminoscope. On the basis, the invention further provides a kit and a detection method for rapidly detecting the nucleic acid of the schistosoma japonicum katsurada. The kit and the detection method are simple to operate, high in sensitivity and good in specificity, detection can be completed only through constant-temperature heating equipment, the kit and the detection method can be suitable for multi-scene on-site screening, and a new technology and a new method are provided for detection of schistosoma japonicum katsurada.

The invention relates to the technical field of rapid detection methods, in particular to a one-step detection method for urinary tract infection escherichia coli with a non-disease diagnosis or non-treatment purpose. According to recombinase polymerase amplification provided by the invention, target DNA is specifically amplified through primers, CRISPR / Cas12a specifically recognizes a target sequence through crRNA, and through double screening of RPA and CRISPR / Cas12a, the target sequence is specifically recognized through crRNA; the rapid, sensitive and specific detection on the Escherichia coli infected with the urinary tract can be realized. Meanwhile, an RPA primer, crRNA and reaction conditions are optimized, so that an RPA reaction system and a CRISPR / Cas12a reaction system can be premixed in a tube, and the reaction can be completed only by a one-step method. The method can specifically detect urinary tract infection escherichia coli and has no cross reaction with common pathogenic bacteria, optimized conditions can avoid the competitive effect of CRISPR / Cas12a and RPA, the reaction rate and sensitivity are guaranteed, and meanwhile, the risk of nucleic acid aerosol pollution is also avoided due to the fact that a cover does not need to be opened.

The invention provides a composition, a kit and a detection method for detecting staphylococcus aureus or a drug-resistant gene thereof based on RPA-CRISPR / Cas12a and application of the composition, the kit and the detection method, and belongs to the technical field of gene detection. A CRISPR / Cas12a system is combined with recombinase polymerase amplification (RPA), an RPA-CRISPR / Cas12a detection method for rapidly detecting the staphylococcus aureus in the wastewater and the drug-resistant gene of the staphylococcus aureus is established, a specific primer and crRNA are designed, the high-specificity nucleic acid cutting capability of a gene editing enzyme CRISPR / CAS system and the isothermal rapid amplification advantage of a nucleic acid isothermal amplification technology are fully fused, and the detection method has the advantages of high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity The fluorescent probe has high-specificity sequence recognition capability, realizes efficient detection of target nucleic acid, is strong in detection specificity, high in sensitivity and simple and convenient to operate, does not need complex equipment and professionals, and is suitable for rapid detection in an on-site environment with limited resources.

The invention discloses a rapid detection method for fusarium wilt in corn based on an RPA-CRISPR / Cas12b technology, and belongs to the technical field of nucleic acid detection. The invention provides a specific reagent combination, a kit and a detection method in order to solve the problems of high amplicon pollution risk, complicated operation and the like in the existing technologies such as RPA (recombinase polymerase amplification). The reagent combination comprises an RPA (recombinase polymerase amplification) primer pair with sequences as shown in SEQ ID NO.1 and SEQ ID NO.2, and sgRNA (single guide ribonucleic acid) with a sequence as shown in SEQ ID NO.3. The method comprises the following steps: carrying out RPA amplification by taking nucleic acid of a sample to be detected as a template, carrying out mixed incubation on an amplification product, sgRNA, Cas12b protein and fluorescent reporter molecules, and judging a result by detecting a fluorescent signal. The optimized RPA and CRISPR systems are integrated on the centrifugal micro-fluidic chip, so that the full-flow closed tube and automatic detection is realized, the detection can be completed within 30 minutes, the detection sensitivity reaches 1E2 copies / T, the risk of aerosol pollution is greatly reduced, and the centrifugal micro-fluidic chip is suitable for port quarantine and field on-site rapid detection.

The invention relates to an extracellular vesicle surface protein detection paper-based kit and application thereof, and belongs to the technical field of biology. The technical problems that an existing sEVs detection technology is insufficient in specificity, depends on professional instruments and cannot meet the instant detection requirement are solved. According to the method, sEVs are synergistically captured through double aptamers, the target specificity is improved, signals are amplified in a cascaded mode through hyper-branched rolling circle amplification (HRCA) and a CRISPR / Cas12a system, and visual detection is achieved by detecting FAM-biotin probe separation before and after cutting through a transverse flow test strip. According to the method, interference of free protein is effectively avoided through double-target recognition, and the detection limit is as low as 406 parts / mL; cRISPR and a paper-based technology are combined to realize high sensitivity and instant detection; the system is simple and convenient to operate, does not need professional instruments, is suitable for tumor diagnosis and treatment monitoring of clinical samples, and provides a portable solution for precise medical treatment.

The invention provides for systems, methods, and compositions for targeting nucleic acids. In particular, the invention provides non-naturally occurring or engineered DNA-targeting systems comprising a novel DNA-targeting CRISPR effector protein and at least one targeting nucleic acid component like a guide RNA.

The invention provides a method for detecting salmonella enteritidis, which comprises the following steps of: 1, expressing a nano antibody for resisting salmonella enteritidis FliC by using escherichia coli, and verifying the nano antibody; step 2, preparing immunomagnetic beads for resisting FliC protein, and verifying the immunomagnetic beads; step 3, carrying out specific enrichment on the target bacteria; 4, performing rapid splitting decomposition to release nucleic acid; step 5, carrying out isothermal amplification on the target DNA fragment; and step 6, CRISPR / Cas12a mediated signal amplification and detection are carried out. The kit has the effects of ultra-fast detection, high sensitivity, high specificity, portability, easiness in operation, no need of bacterial culture and the like.

The invention discloses application of kaempferol in promotion of gene editing, and belongs to the field of biological medicine. The application specifically comprises kaempferol as an accelerant for CRISPR (clustered regularly interspaced short palindromic repeats) gene editing and application of kaempferol in preparation of non-viral site-specific integrated CAR T cells. The invention finds that the kaempferol can significantly increase the CRISPR gene editing efficiency, and cell experiments find that the kaempferol has a significant promotion effect on the positive rate of CAR T cells after electrotransfection, and the tumor killing function of the T cells is greatly improved.