1002 results about "CRISPR" 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 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 belongs to the technical field of plant genetic engineering, and discloses application of an OsSRO1c gene in controlling high-temperature resistance of rice. The OsSRO1c gene capable of improving the drought tolerance of rice is obtained through separation, cloning and functional verification, the nucleotide sequence of the OsSRO1c gene is as shown in SEQ ID NO.1, and the sequence of protein coded by the gene is as shown in SEQ ID NO.2. The gene OsSRO1c for controlling rice high-temperature response is cloned, CRISPR mutant phenotype identification is carried out on candidate genes, high-temperature stress phenotype identification in a seedling stage and an adult-plant stage shows that when a gene segment is deleted, the high-temperature resistance of rice is reduced, and when the expression quantity of the gene is increased, the high-temperature resistance of rice is improved, and the function and the application way of the gene are proved.

The invention discloses a method for detecting drug resistance of botrytis cinerea to succinate dehydrogenase (SDHI) bactericides. On the basis of an RPA-CRISPR / Cas12a system, a specific primer pair amplification target fragment and crRNA for activating Cas12a trans-cleavage activity are designed aiming at key mutation sites related to drug resistance in a bactericide action target SdhB gene, so that fluorescent light visible to naked eyes can be generated only when a resistant strain sequence is matched with the crRNA, and the fluorescent light is opposite to the fluorescent light generated when a sensitive strain sequence is matched with the crRNA. The method provided by the invention overcomes the dependence on laboratories and large instruments in the traditional drug resistance detection method, provides key technical support for accurate prevention and control and drug resistance management of grape gray mold, and provides direct drug use basis for scientific prevention and control in the field.

The invention is applicable to the technical field of virus detection, and provides a norovirus detection kit based on a photo-thermal isothermal amplification technology and a detection method, the norovirus detection kit comprises a nucleic acid amplification chamber, the nucleic acid amplification chamber is provided with reaction micropores for nucleic acid amplification; an RT-PRA system, a CRISPR-cas12a system, a blank control and a positive control are placed in the reaction micropore, the RT-PRA system comprises RT-PRA solutions R1-R5, and the CRISPR-cas12a system comprises CRISPR-Cas12a solutions C1-C3; and the photo-thermal film covers the nucleic acid amplification chamber and is used for converting sunlight into heat energy and radiating the heat energy to liquid in the reaction micropores so as to provide reaction temperature for nucleic acid amplification. According to the invention, the portable, rapid and accurate detection of norovirus infection can be realized, the detection cost can be effectively reduced, the application scene is wide, workers can be helped to deduce the virus transmission path, and key support is provided for investigation and analysis of norovirus epidemic situations.

The present disclosure provides one or more engineered gene effectors and systems, compositions, and methods of use thereof, wherein the one or more engineered gene effectors can be used to effect regulation of a target gene in a cell (e.g., an endogenous target gene in a cell). The one or more engineered gene effectors can be operatively coupled to a heterologous endonuclease, such as a CRISPR / Cas protein.

The invention relates to the field of microbiological detection, in particular to a method for detecting vibrio parahaemolyticus by combining RPA-CRISPR / Cas12a with a microfluidic biosensor. According to the method, the preloaded freeze-drying reagent is combined with centrifugally driven liquid operation, so that rapid and closed system detection is realized, and the pollution risk is effectively reduced. According to the invention, quantitative detection of vibrio parahaemolyticus is realized in a centrifugal micro-fluidic chip by systematically optimizing an RPA amplification primer and crRNA. The detection method shows high specificity and high sensitivity to non-target vibrio and other food-borne bacteria, and the lower detection limit is 6.08 copies / mu L. In a word, the invention provides a rapid, portable and sensitive platform, can realize quantitative detection in a wide linear range, provides a practical tool for real-time monitoring of pathogens, and has application prospects in monitoring of aquaculture biosafety and marine product safety.

To improve allele discrimination, the inventors adapted the system combining Cas13a detection sensitivity with allele-specific PCR amplification to propose CASPER (Cas13a Allele-Specific PCR Enzyme Recognition) as a new versatile, easy-to-implement, and highly sensitive method to detect low-frequency of sequence variant. CASPER enabled specific and sensitive detection of KRASG12D with low DNA input such as DNA extracted from patient's pancreatic ultrasound-guided fine-needle aspiration fluids. CASPER is easy to implement and a versatile reliable method virtually adaptable to any point mutation.

The invention relates to the technical field of molecular biology and pathogenic microorganism detection, and discloses a Klebsiella pneumoniae and Escherichia coli synchronous detection method based on RPA-CRISPR / Cas12a and application, and the method comprises the following steps: extracting DNA from a sample, carrying out isothermal amplification by adopting an RPA primer designed aiming at a Klebsiella pneumoniae rcsA gene and an Escherichia coli uriA gene, and carrying out amplification by adopting an RPA primer designed aiming at a Klebsiella pneumoniae RcsA gene and an Escherichia coli uriA gene; a detection system containing Cas12a enzyme, crRNA and a fluorescence report substrate is constructed, and a result is interpreted through a fluorescence or lateral flow chromatography test strip. Detection can be completed within 70 minutes at the constant temperature of 37 DEG C, the sensitivity reaches 5 * 10 copies / mu L, the specificity is high, no cross reaction exists, the kit is suitable for clinical early screening, hospital infection monitoring and on-site POCT application, and an efficient and reliable new tool is provided for pathogen diagnosis.

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.

Described herein are methods and compositions that utilize a CRISPR / Cas system. In some cases, the utilization includes the use of such systems for gene activation or interference.

The invention discloses a method for visually detecting grape black root bacteria Ilyonectria sp. Based on RPA-CRISPR / Cas12a and application of the method for visually detecting the grape black root bacteria Ilyonectria sp. Based on the RPA-CRISPR / Cas12a. According to the method, an RPA specific amplification primer, a CrRNA probe and an ssDNA probe are adopted; according to the RPA specific amplification primer, an HIS gene is used as a specific recognition target, and specific amplification can be carried out on the grape black root pathogen Ilyonectria. The method for detecting the grape black root pathogenic bacteria has high specificity and sensitivity, is simple, convenient and rapid in operation flow, and is suitable for rapid screening and preliminary diagnosis of target pathogens in a field environment or a basic detection scene.

The invention discloses a kit for detecting a muscovy duck reovirus based on a RAA-CRISPR / Cas12a technology. Belongs to the technical field of Muscovy duck reovirus detection. The invention aims to detect the Muscovy duck reovirus. The invention provides a kit for detecting a muscovy duck reovirus based on an RAA-CRISPR / Cas12a technology. The detection kit comprises an RAA isothermal amplification system and a CRISPR / Cas12a detection system, the method effectively solves the problems that a traditional detection technology is long in time consumption, depends on precise instruments and is complex in operation to detect the pain points on the breeding site.

The present disclosure provides methods of processing or analyzing a sample. A method for processing a sample may comprise hybridizing a probe molecule to a target region of a nucleic acid molecule (e.g., a ribonucleic acid (RNA) molecule), barcoding the probe-nucleic acid molecule complex, and performing extension, denaturation, and amplification processes. The nucleic acid molecule may be a nucleic acid molecule associated with CRISPR, (e.g., a guide RNA). A method for processing a sample may comprise hybridizing first and second probes to adjacent or non-adjacent target regions of a nucleic acid molecule (e.g., an RNA molecule such as a guide RNA molecule), linking the first and second probes to provide a probe-linked nucleic acid molecule, and barcoding the probe-linked nucleic acid molecule. One or more processes of the methods described herein may be performed within a partition, such as a droplet or well. One or more processes of the methods described herein may be performed on a cell, such as a permeabilized cell.

The invention relates to the technical field of bacterial detection, in particular to a nucleic acid colorimetric / photo-thermal dual-mode instant detection kit and system based on CRISPR / Cas12a and Au (at) PtNPs. One purpose of the invention is to provide a nucleic acid colorimetric / photo-thermal dual-mode instant detection kit based on CRISPR / Cas12a and Au (at) PtNPs. The nucleic acid colorimetric / photo-thermal dual-mode instant detection kit based on CRISPR / Cas12a and Au (at) PtNPs comprises the following components: Au (at) PtNPs coupled with Biotin-ssDNA-SH, a specific primer targeting a GBS conserved gene, a CRISPR / Cas12a cleavage reaction mixed solution and a reaction mixed solution. According to the technical scheme, the high robustness and reliability of the detection result in bedside, community clinics and other basic-level application scenes are jointly ensured through the simplified pretreatment process and the internal cross validation mechanism of the colorimetric / photo-thermal dual-mode signals, and a solid foundation is laid for wide popularization of the technology.

The invention discloses a method for visually detecting Dactylonectria spp. On the basis of RPA (recombinase polymerase amplification)-CRISPR (complementary reactive interspaced short palindromic repeats) / Cas12a and application of the method. According to the detection method, an RPA specific amplification primer, crRNA and an ssDNA probe are adopted; according to the RPA specific amplification primer, a TUB gene is used as a specific recognition target, and specific amplification can be carried out on the grape black root pathogen Dactylonectria. The method for detecting the grape black root pathogenic bacteria has high specificity and sensitivity, is simple, convenient and rapid in operation flow, and is suitable for rapid screening and preliminary diagnosis of target pathogens in a field environment or a basic detection scene.

The invention belongs to the technical field of bioengineering, and discloses a high-yield hyaluronic acid bacillus subtilis strain for expressing a hyaluronic acid synthase gene, a construction method and application, and the construction method comprises the following steps: inserting a codon-optimized sphaasA gene into a lytH site in a bacillus subtilis 168 strain in a traceless manner by utilizing a CRISPR / dCas9 system; the preparation method comprises the following steps: synthesizing hyaluronic acid oligosaccharides, simultaneously integrating genes for expressing a synthetic pathway of hyaluronic acid oligosaccharides, reducing the transcriptional level of zwf and pfkA genes, weakening the metabolic flux of a competitive pathway, and finally increasing the expression of sphaasA and tuaD genes according to a ratio of 2: 1 by using plasmids and the interaction of protein scaffolds RIAD and RIDD. When the strain is fermented for 48 hours in a 5L fermentation tank, the yield of the hyaluronic acid reaches 38.02 g / L, and the average molecular weight HA Mw of the low-molecular-weight hyaluronic acid is about (5-13) * 10 < 4 > Da.

This invention provides a CRISPR / Cas12a fluorescence detection method for FocTR4 based on magnetic bead-ssDNA-HRP probes, belonging to the field of nucleic acid detection technology. This invention involves isothermal amplification of the FocTR4 target sequence using RPA primers; then, a magnetic bead-ssDNA-HRP probe is prepared; and the Cas12a / crRNA complex, RPA amplification product, and probe are co-incubated. The activated Cas12a cleaves the probe, releasing HRP. After magnetic separation, a colorimetric solution containing OPD is added, and the presence and concentration of FocTR4 are determined by detecting the fluorescence signal. This invention combines the high efficiency of CRISPR / Cas12a cleavage with the high sensitivity and strong specificity of RPA technology, achieving highly sensitive and specific detection of FocTR4, with a linear detection range of 10. 2 ~10 7 With a detection limit as low as 70 CFU / mL, it can quickly and accurately detect Foc TR4 in soil samples, enabling rapid on-site detection and providing reliable technical support for the early diagnosis and control of banana wilt disease.

The invention relates to a normal temperature amplification-CRISPR Cas12a one-pot method detection system and a nucleic acid detection method based on the detection system. The detection system consists of a normal-temperature nucleic acid amplification system (unit I) which focuses on target signal amplification and a CRISPR Cas12a system (unit II) which focuses on target recognition and signal output. By means of a self-driven and deoxyribozyme-mediated reaction equilibrium transfer strategy, the equilibrium in the initial stage of the reaction tends to the unit I, and the equilibrium in the middle and later stages of the reaction tends to the unit II, so that the compatibility of the two units is remarkably improved. By means of the system, high-sensitivity and high-specificity portable one-pot detection of nucleic acid targets can be achieved.

This invention discloses an amplification-free electrochemical biosensor and method based on CRISPR-Cas13a. The sensor includes an electrochemical biosensor substrate, a dual-channel RNA reporter molecule, a CRISPR-Cas13a reaction system, and a dual-channel differential signal processing module. The working electrode surface is immobilized with a stem-loop structured first reporter molecule and a linear second reporter molecule loaded with gold nanoparticles, labeled with methylene blue and ferrocene signal molecules respectively, and immobilized with a ferrocene-specific capture probe. When target RNA is present, Cas13a is activated and non-specifically cleaves the reporter molecule, reducing the methylene blue signal and enriching and enhancing the ferrocene signal. Background self-calibration is achieved through dual-channel differential ratio calculation. This invention integrates signal attenuation and signal enhancement modes, offering advantages such as nucleic acid amplification-free operation, fast detection speed, high sensitivity, and strong anti-interference capability.

The invention belongs to the technical field of biology, and relates to a pathogen detection technology based on a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system, in particular to a tubercle bacillus detection and activity determination method based on fluorescent T7-CRISPR. The first purpose is to provide a reagent combination for detecting viable tubercle bacillus, and the reagent combination comprises a) a T7 reverse transcription reaction reagent and b) a CRISPR-Cas13a reaction system; a) the T7 reverse transcription reaction reagent comprises a T7 reverse transcription primer and a T7 reverse transcriptase; and b) the CRISPR-Cas13a reaction system comprises specific crRNA, a fluorescence labeled ssRNA reporter, a reaction buffer solution, a Cas13a protein, RNase-Free H2O and a T7 reverse transcriptase. The invention also provides application of the reagent combination in detection of viable tubercle bacillus. The primer combination disclosed by the invention has the advantages of strong specificity and high sensitivity in detection application, can realize rapid detection and visual detection, and is low in detection cost, simple and convenient to operate and suitable for large-scale clinical application.

The present invention relates to specific natural or artificial RNA or DNA / RNA substrates for cleaving by a Cas nuclease. The invention furthermore relates to a complex comprising the specific artificial RNA or DNA / RNA substrate, at least one of a Cas nuclease enzyme and at least one preselected guide RNA binding to at least one target RNA. The present invention also relates to methods for cleaving the natural or artificial RNA or DNA / RNA substrate and methods for detecting at least one target RNA in a cell, tissue, cellular nucleus, and / or sample using the substrate or for eliminating a cell expressing the at least one target RNA.

The invention relates to a nucleic acid molecule composition, a reagent and a kit for visually and rapidly detecting pigeon group A rotavirus based on RT-RAA-CRISPR / Cas12a and application, the nucleic acid molecule composition comprises an upstream primer PiRVA-RAA-F2 and a downstream primer PiRVA-RAA-R3, the nucleotide sequences of the upstream primer PiRVA-RAA-F2 and the downstream primer PiRVA-RAA-R3 are respectively shown as SEQ ID No: 4-5, the nucleic acid molecule composition further comprises crRNA, the crRNA is PiRVA-crRNA, the nucleotide sequence of the crRNA is shown as SEQ ID No: 10, and the nucleotide sequence of the crRNA is shown as SEQ ID No: 6. The reagent and the kit comprise the nucleic acid molecule composition. The detection method established by using the nucleic acid molecule composition has the advantages of rapid detection, high efficiency, high sensitivity, strong specificity and good repeatability.

Described herein are compositions and methods for reducing adaptered-tag sequencing reads during the identification and nomination of on- and off-target CRISPR edited sites. One embodiment is a method for reducing adaptered-tag sequencing reads during the identification and nomination of on- and off-target CRISPR edited sites, the method comprising: contacting in an amplification reaction one or more adaptered-tag blocking oligonucleotides with an isolated genomic DNA having one or more tag sequences and adapter sequences; wherein the adaptered-tag blocking oligonucleotides comprise one or more blocking moieties and hybridize to adaptered-tag sequences at a junction region between the adapter and tag sequences to reduce amplification of the adaptered-tag sequences.

To provide a method for determining the presence or absence of a target nucleic acid molecule in a sample.SOLUTION: CLAIMS What is claimed is: 1. A method of amplifying a target nucleic acid molecule in a sample, comprising: a) an effector complex comprising a Type III CRISPR-associated effector protein (Cas) and at least one CRISPRRNA (crRNA) that binds to the target nucleic acid molecule; and b) means for directly or indirectly determining the level of cyclic oligoadenylate (cOA). Providing a sample with a Regularly Interspaced Short Palindromic Repeats (CRISPR) - based ribonucleic acid detection system; incubating the sample under conditions that allow the crRNA to bind to its target nucleic acid molecule; and directly or indirectly determining the level of cyclic oligoadenylate (cOA), wherein an increase in the determined cOA level relative to a control is indicative of the presence of the target molecule in the sample.SELECTED DRAWING: None

The present disclosure relates generally to (CRISPR) RNA (crRNA) for the precision silencing of transcripts. In some embodiments, the crRNA are enriched for guanosine (G) nucleotides at key spacer positions, which is useful in enhancing the silencing efficacy of otherwise inefficient crRNA, thereby expanding the targeting spectrum of Cas13 endonucleases, e.g., Cas13b and Cas13d. In other embodiments, the crRNA comprise a spacer sequence having at least one nucleotide mismatch relative to the target RNA sequence, wherein the target RNA sequence is a wild-type transcript and / or a variant transcript (e.g., a transcript comprising a single nucleotide variant (SNV)). The present disclosure also provides RNA editing systems comprising the crRNA described herein in complex a Cas13 effector protein and a target RNA sequence, methods for the selective targeting of transcripts encoding proteins that are difficult to target, or are not amenable to pharmacological targeting, e.g., oncogenic fusion transcripts or oncogenic transcripts comprising single nucleotide variant(s), and methods for the design and selection of potent crRNA.

The invention relates to the technical field of virus detection, and discloses a method and a kit for detecting Chinese sacbrood virus. The method comprises: firstly, carrying out isothermal amplification on a CSBV specific target sequence in a sample nucleic acid by using an RPA technology, the RPA technology adopting a specific primer pair, the sequences of which are SEQ ID NO: 4 and SEQ ID NO: 5; then, an amplification product is mixed with a CRISPR / Cas12b detection system, and the system comprises Cas12b protein, specific sgRNA with the sequence shown as SEQ ID NO: 2 and a single-chain DNA reporter probe marked by fluorescence; when a CSBV target exists, the activated Cas12b protein trans-cleavage reporter probe generates a detectable fluorescence signal, so that rapid and high-sensitivity detection of the CSBV is realized. The invention further provides a detection kit suitable for the method. The detection kit comprises the primer pair, sgRNA and Cas12b protein. When the CSBV is detected based on the method and the kit, complex instruments are not needed, the operation is simple and convenient, the detection time is shorter than 1 hour, the sensitivity and the specificity are high, and the method and the kit are suitable for onsite rapid detection in a bee field.

The invention belongs to the technical field of biology, and particularly relates to a construction method of epigenome edited zebrafish, sgRNA of a targeted zebrafish runx2b gene promoter region and application of the sgRNA. The invention provides a construction method of epigenome edited zebrafish, which comprises the step of carrying out methylation editing by using a CRISPR / dCas9-methyltransferase system targeting runx2b gene promoter region, thereby inhibiting runx2b gene expression and reducing intermuscular bone formation. According to the method, the expression level of the runx2b gene is accurately regulated and controlled through an epigenetic editing technology on the premise that a DNA sequence is not changed, and formation of fish intermuscular bones is effectively reduced. The zebra fish F0 treated by the method represents that the runx2b mRNA expression level is obviously reduced, and the intermuscular bones of the eleventh to sixteenth sarcomedo regions of adult F0 generation fish are obviously shorter than those of a control group, and the shortening range reaches 23.3% to 38.8%. Besides, the method provides theoretical basis and technical support for improving fish economic traits by accurately regulating and controlling the expression mode of the key economic trait gene and simultaneously keeping the integrity of genome DNA (Deoxyribose Nucleic Acid).