558 results about "Guide RNA" 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.

The present disclosure provides compositions and methods for the targeted modification of RNA molecules by RNA prime editing. The compositions and methods may be conducted invitro or in vivo within cells (e.g., human cells) for the therapeutic correction of disease-causing mutations and / or installation of motifs or mutations in RNA molecules of interest as a tool for scientific research. The disclosure provides compositions and methods for conducting RNA prime editing of a target RNA molecule (e.g., an RNA transcript) that enables the incorporation of one or more nucleotide changes and / or targeted mutagenesis of a target RNA molecule. The nucleotide change can include a single-nucleotide change, an insertion of one or more nucleotides, or a deletion of one or more nucleotides. More in particular, the disclosure provides a variety of configurations of the RNA prime editors each comprising a nucleic acid programmable RNA binding proteins (napRNAbp), such as Cas13, and an RNA-dependent RNA polymerase (RDRP), which are provided as fusion proteins or which can be separately provided in trans. The RNA prime editors are guided to a target RNA site by a guide RNA, which can be a rpegRNA that includes a template region for the synthesis of an RNA sequence to be installed on the RNA molecule attached to an available 3′ terminus. In others embodiments, the RNA template can be provided in trans.

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 relates to methods for culturing cells, and cultured animal cells for use in described method. Also provided is a modified animal cell and guide RNAs targeting genes to be modified in the modified and cultured cells of the invention.

The invention relates to the technical field of phytopathogen detection, and particularly discloses a primer, a kit and a method for detecting crop root rot fusarium solani and application, the primer comprises a specific RPA primer RPA-F and a specific RPA primer RPA-R; the specific RPA primer RPA-F is as shown in SEQ ID NO. 1, and the specific RPA primer RPA-R is as shown in SEQ ID NO. 2; the kit further comprises a guide RNA primer crRNA, and the guide RNA primer crRNA is as shown in SEQ ID NO. 3. The kit comprises a specific RPA primer RPA-F, a specific RPA primer RPA-R, a Cas12 protein, a T7 transcriptase, a crRNA DNA template, a DNA polymerase and a Cas12 / 13 nucleic acid detection test strip. The kit has the characteristics of good sensitivity, relatively good specificity and enough reliability.

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.

The present invention relates to a recombinant Mycobacterium bovis BCG strain comprising a plasmid having a short guide RNA (sgRNA) target sequence for knocking out the Mb3739 gene. Also provided are vaccine compositions for eliciting an immune response against Mycobacterium tuberculosis comprising a recombinant Mycobacterium bovis BCG strain engineered to activate the NOD-1 pathway. The recombinant Mycobacterium bovis BCG strain or vaccine composition can be used in a method against an immune response initiated by Mycobacterium tuberculosis. The invention also relates to a method for obtaining the recombinant bovine mycobacterium BCG strain.

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.

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

SpRYc is a grafted ScCas9++-SpRY chimeric Cas9 possessing minimal 5′-NNN-3′ PAM specificity. SpRYc comprises the N-terminus (residues 1-1119) of ScCas9++ (Sc++), including the flexible loop, followed by the region of SpRY (residues 1111-1368) spanning its PAM-interacting domain mutations. Methods of altering gene expression include use of SpRYc in complex with guide RNA in a CRISPR-Cas9 system.

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 discloses a double-CRISPR (clustered regularly interspaced short palindromic repeats) coupled isothermal amplification efficient detection technology, and a double-CRISPR coupled isothermal amplification reagent provided by the invention comprises a pair of efficient Cas12a variant-guide RNA (Ribonucleic Acid) complexes, an isothermal amplification system, bovine serum albumin, a fluorescence resonance energy transfer single-stranded DNA (Deoxyribose Nucleic Acid) probe, a reaction buffer solution and target nucleic acid to be detected, the double CRISPR coupling isothermal amplification detection technology disclosed by the invention has the remarkable advantages of high reaction speed, high signal-to-noise ratio, high sensitivity and strong specificity, can efficiently detect low-copy or low-quality target nucleic acid, and can be combined with a 3D printing chip to realize reagent freeze-drying and simultaneous detection of multiple types of target nucleic acid; the method shows a great application prospect, and particularly has a great potential in detection of bacteria deployed on site.

The invention discloses an efficient gene editing system for streptomyces as well as a construction method and application of the efficient gene editing system. The system comprises a TnpB nuclease which can be expressed in streptomyces and a guide RNA, wherein the guide RNA comprises an RNA skeleton, a gene targeting section and a gene sequence of hepatitis delta virus (HDV) ribozyme. Wherein the gene targeting segment is located at the 3'end of the RNA skeleton and is a nucleic acid fragment with the length of 12-40bp after a TAM sequence (5 'TTGAT) on a target gene; the hepatitis D virus (HDV) ribozyme is used for stabilizing the RNA skeleton-gene targeting section structure. According to the streptomyces mini-gene editing tool provided by the invention, in the presence of a homologous repair template, the gene editing efficiency can reach 70-100% in streptomyces, and the gene editing efficiency under the action of the guide RNA after rational design can reach 100%.

The present application relates to a modified guide agRNA (ADAR guide RNA, agRNA), which has a phosphorothioate modification at an agRNA site where off-target editing is present, and further has other modifications. The present application further provides a composition containing the modified agRNA composition, a method for editing RNA, and the use.

Aspects of the disclosure relate to recombinant gene editing complexes comprising a recombinant gene editing protein and guide RNA (gRNA) that specifically hybridizes to a region of a C90RF72 gene (e.g., a region flanking a G4C2 repeat or within a exonic region of the gene).

This vector for nucleic acid introduction contains a nucleotide sequence that encodes a Cas protein, a nucleotide sequence that encodes a guide RNA, and a nucleotide sequence of a nucleic acid to be introduced. The guide RNA targets an intron adjacent to the 5' side, with reference to a sense strand, of an exon that includes a stop codon. Also provided is a nucleic acid introduction method comprising a step for introducing a nucleic acid to be introduced through non-homologous end joining by targeting an intron that is adjacent to the 5' side, with reference to a sense strand, of an exon that includes a stop codon.

The invention provides a method for cultivating anti-aging glutinous highland barley, highland barley starch as well as a preparation method and application of the highland barley starch, and particularly relates to a method for improving the starch quality character of an existing high-quality glutinous highland barley variety, in particular to a method for cultivating anti-aging glutinous highland barley by utilizing a gene editing technology without exogenous DNA (Deoxyribose Nucleic Acid) integration. A method for fixed-point knockout of an SSIIa gene to cultivate an anti-aging glutinous highland barley new strain under the background of a "Zhengnongglutinous 232" variety comprises the following steps: providing an acceptor material derived from a highland barley variety "Zhengnongglutinous 232" with a glutinous character, and deleting a Wx gene function of the "Zhengnongglutinous 232"; the method comprises the following steps: assembling guide RNA (Ribonucleic Acid) designed aiming at an SSIIa gene and Cas9 protein in vitro to form a ribonucleoprotein RNP complex; introducing the RNP complex into cells of an acceptor material so as to enable Cas9 protein to generate double-chain breakage at a target site of the SSIIa gene; carrying out culture and plant regeneration on the introduced cells to obtain a T0-generation plant of which the SSIIa gene is edited; and screening plants with the function loss of the SSIIa gene, wherein the plants keep the glutinous character of adult agricultural glutinous 232.

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.

Disclosed herein are compositions and methods that allow for the efficient integration of a gene of interest into a safe harbor locus in the genome. Such compositions and methods include a small V-type nuclease, a guide RNA, preferably targeting the albumin locus.

Compositions and methods are provided for variant Cas systems and elements comprising such systems, including, but not limiting to, Cas endonuclease variants, guide polynucleotide / Cas endonuclease complexes comprising Cas endonuclease variants, as well as guide polynucleotides and guide RNA elements that can interact with Cas endonuclease variants. Compositions and methods are provided for genome modification of a target sequence in the genome of a cell. The methods and compositions employ a guide polynucleotide / Cas endonuclease system comprising a Cas9 endonuclease variant to provide an effective system for modifying or altering target sequences within the genome of a cell or organism.

The present invention provides novel RNA base editing compositions, systems, methods and uses. Guide RNAs for site-specific RNA editing of RNA encoding transcriptional coactivators YAP1 or TAZ are provided, and compositions and systems comprising the same with a programmable RNA binding protein (e.g. a Cas protein) and / or a base editor. Methods for RNA editing of YAP1 or TAZ are also provided. RNA editing of YAP1 or TAZ is used for targeting phosphorylation sites, and activating transcription of proteins in regenerative therapy for treating cardiac disease.

The invention relates to the technical field of animal husbandry low-carbon breeding and precise breeding, in particular to a beef cattle carbon emission data intelligent analysis and breeding decision making system based on artificial intelligence, which comprises a phenotype and carbon efficiency fusion unit, obtains dynamic methane release waveform and rumination rhythm data through an implantable rumen capsule, calculates a carbon efficiency ratio correction value, and calculates a carbon efficiency ratio correction value; the gene editing target prediction unit is used for analyzing waveform key mutation characteristics by using a deep convolution-attention hybrid model, matching an epigenetic marker database, and positioning and hierarchically regulating target genes of carbon effect; a guide ribonucleic acid sequence, a homologous recombination repair template and a base editing scheme are respectively designed aiming at high, medium and low regulation potency targets and are synchronized to a cattle gene editing platform and a phenotype feedback optimization unit, methane release waveforms of offspring are collected in real time, target potency parameters are corrected, accurate positioning and differential editing of carbon effect related genes are realized, and a cattle gene editing result is obtained. The beef cattle low-carbon breeding efficiency is improved.

Provided herein is a nucleic acid detection system comprising a detection CRISPR system having an effector protein and one or more guide RNAs each designed to bind to corresponding target molecules that are diagnostic for a tick-borne disease state; and an RNA-based masking construct. In some embodiments, the detection system of may comprise i) two or more CRISPR systems, each CRISPR system comprising an effector protein and a guide RNA designed to bind to a corresponding target molecule that is diagnostic for a tick-borne disease state; and ii) a set of detection constructs, each detection construct comprising a cutting motif sequence that is preferentially cut by one of the activated CRISPR effector proteins. Exemplary tick-borne detectable microbes include Babesia microti, Anaplasma phagocytophilum, and Borrelia miyamotoi.

The present application relates to an agRNA (antisense oligonucleotide guide RNA, Antisense Oligonucleotide guide RNA) for editing RNA, which is characterized in that one agRNA molecule can bind to a plurality of non-continuous target regions of the target RNA. The invention also provides a composition containing the agRNA, a method for editing RNA, and application of the agRNA and the composition.

The present invention relates generally to compositions, systems and methods for modulating hepatitis B virus (HBV) by targeting gene repression. In particular, the present invention provides an epigenetically modified DNA targeting system, such as a CRISPR-Cas / guide RNA (gRNA) system, for transcriptional repression of hepatitis B virus genes to promote cell phenotypes that lead to reduced HBV infection. In some embodiments, the epigenetically modified DNA targeting systems of the present invention bind to or target a target site of at least one gene in the hepatitis B virus DNA sequence or a regulatory element thereof in a cell. In some aspects, the systems provided herein relate to transcriptional repression of one or more hepatitis B virus genes and / or regulatory elements thereof. In some aspects, the invention also provides methods and uses associated with the provided compositions, such as in repression of hepatitis B virus replication and expression associated with hepatitis B infection.

Systems and methods for highly-effective CRISPR-Cas based genomic editing within human chromosome 15q11-q13 have been developed as therapeutic interventions for Angelman Syndrome (AS). Selective single guide RNA molecules (sgRNAs) that impart enhanced CRISPR-Cas editing of genomic mutations associated with Angelman Syndrome in human cells are described. The engineered sgRNAs induce activity of non-pathogenic, paternal UBE3A alleles to reduce, reverse and / or prevent the causative neurodevelopmental defects of AS in a subject in need thereof. Compositions and methods of engineered crRNAs, sgRNAs thereof and ribonucleoprotein (RNP) complexes thereof are provided for enhanced genomic engineering with increased on-off target specificity and on-target editing efficacy for treatment of AS.

This invention discloses a gene editing method and application for *Trichoderma reesei* based on a ribonucleoprotein complex. It aims to overcome the problems of low editing efficiency, easy generation of heterozygotes, and risk of exogenous DNA integration in *Trichoderma reesei* using plasmid-based CRISPR / Cas9 systems. This invention combines a dual-site cleavage strategy guided by double sgRNAs with direct RNP delivery. The method involves: designing two guide RNAs spaced 50-700 bp apart for the target gene, pre-assembling them with Cas9 protein in vitro to form an active RNP complex; delivering this complex along with exogenous donor DNA to *Trichoderma reesei* protoplasts. The exogenous donor DNA contains a left homologous arm, an editing sequence, and a right homologous arm, wherein the left and right homologous arms are homologous to the genomic sequences outside the dual cleavage sites on the target gene determined by the double sgRNAs, respectively; upon entering the cell, the RNP complex introduces dual-site cleavage at the target site, achieving precise editing under the guidance of the exogenous donor DNA.

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. Aspects of the invention in particular relate to Cpf1 mutants having altered PAM specificity.

Disclosed are methods of identifying a regulatory factor that regulates the abundance of a target RNA comprising introducing to a population of cells a plurality of single guide RNAs (sgRNAs), wherein the sgRNAs are specific for one or more nucleic acid sequences capable of encoding the regulatory factor in the population of cells; performing fluorescent in situ hybridization (FISH) on the population cells, using a probe specific to a target RNA in the population of cells; identifying cells in the population of cells that have altered abundance of the target RNA; sequencing DNA from the population of cells that have altered abundance of the target RNA; and identifying, based on the sequencing, sgRNAs that are specific to a nucleic acid sequence capable of encoding the regulatory factor that regulates abundance of the target RNA; thereby identifying a regulatory factor that regulates abundance of the target RNA.