768 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.

Methods and compositions for modulating a target genome and stable integration of a transgene of interest into the genome of a cell are disclosed. Specifically, provided herein comprises compositions comprising an RNA molecule comprising a reverse complement sequence comprising an exogenous sequence and homology arms, an RNA molecule comprising an endonuclease, and one or more guide RNAs for specific genomic integration of the exogenous sequence.

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 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 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.

The present disclosure relates to compositions, methods, and systems for genomic editing. In certain aspects, the compositions, methods, and systems provided herein can incorporate a SpyCas9 nickase, a DNA-dependent DNA polymerase, and / or a template guide RNA for template-based genome editing applications.

Methods and compositions for modulating a target genome and stable integration of a transgene of interest into the genome of a cell are disclosed. Specifically, provided herein comprises compositions comprising an RNA molecule comprising a reverse complement sequence comprising an exogenous sequence and homology arms, an RNA molecule comprising an endonuclease, and one or more guide RNAs for specific genomic integration of the exogenous sequence.

The invention discloses an adenine base editor for DFNB9 hearing loss repair, and belongs to the field of gene therapy of hearing repair. The fusion protein comprises nNme2Cas9, and adenine deaminase TadA-8e or TadA-8e-N108Q (adenine deaminase) or TadA-8e-N108Q-L145T (adenine deaminase). The invention further discloses a preparation method of the adenine deaminase. The amino acid sequences of the TadA-8e, the TadA-8e-N108Q and the TadA-8e-N108Q-L145T are respectively as shown in SEQ ID NO. 1 to SEQ ID NO. 3. The fusion protein and a guide RNA form a base editor. Compared with the prior art, the base editor is used as a treatment means, severe deafness can be recovered to a normal level, and the effect can be maintained for at least six months after treatment. In addition, the treatment window period is relatively long, and the method has clinical transformation significance.

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 invention belongs to the technical field of nucleic acid detection, and particularly relates to a CRISPR-Cas 9 nucleic acid detection method based on a single guide RNA splitting strategy and application of the CRISPR-Cas 9 nucleic acid detection method. According to the invention, splitting of single guide RNA into three parts, namely a tracrRNA sequence, a DR sequence and a spacer sequence is proposed for the first time, and it is found that the splitting mode can still effectively maintain the trans-cleavage function of CRISPR-Cas9, nucleotide sequences of the tracrRNA and the DR part can be kept unchanged, the universality is high, accurate detection for different nucleic acid targets can be realized only by replacing the spacer sequence, the application range is wide, and the method is suitable for large-scale popularization and application. The detection method has the advantages of being constant in temperature, rapid in detection, simple and convenient to operate, multiple in detection capacity, low in cost, capable of being combined with a test strip, wide in applicability and portability and the like, the detection convenience and efficiency are greatly improved, and the detection method is an innovative detection technology with high potential.

A composition for genetic editing of a hydroxyacid oxidase 1 (HAO1) gene, comprising: (a) a messenger RNA (mRNA) encoding a Cas12i2 polypeptide, (b) a guide RNA (gRNA) targeting the HAO1 gene; and (c) lipid excipients, which optionally form lipid nanoparticles. Also provided herein are methods for genetic modification of the HAO1 gene in host cells with the composition provided herein.

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.

Compositions and methods are provided herein for conducting prime editing of a target DNA molecule (e.g., a genome) that enables the incorporation of a nucleotide change and / or targeted mutagenesis. The compositions include fusion proteins comprising nucleic acid programmable DNA binding proteins (napDNAbp) and a polymerase (e.g., reverse transcriptase), which is guided to a specific DNA sequence by a modified guide RNA, named an PEgRNA. The PEgRNA has been altered (relative to a standard guide RNA) to comprise an extended portion that provides a DNA synthesis template sequence which encodes a single strand DNA flap which is synthesized by the polymerase of the fusion protein and which becomes incorporated into the target DNA molecule.

The invention provides characterization and application of novel programmable endonuclease Argonaute (Ago), and particularly provides a detection system for directly detecting target nucleic acid molecules, the system comprises: (a) guide DNA (gDNA) and / or guide RNA (gRNA); (b) a programmable endonuclease Argonaute (Ago), wherein the programmable endonuclease Argonaute (Ago) is a programmable endonuclease Argonaute (Ago) which is used for guiding the shearing of the target DNA and / or the target RNA by taking the DNA and / or the RNA as a guide; (c) a reporter nucleic acid; wherein the target nucleic acid molecule is a target DNA and / or a target RNA, the invention also accidentally finds that the nuclease MaAgo provided by the invention can be used for independently detecting the target DNA or the target RNA and also can be used for simultaneously detecting the target DNA and the target RNA, and has a shearing characteristic different from that of the shearing DNA when the RNA is sheared, so that a new enzyme resource is expected to be provided for nucleic acid detection and in-vivo editing.

The invention discloses a CRISPR (clustered regularly interspaced short palindromic repeats)-Cas gene editing system for preparing a universal CAR-T. The invention discloses a CRISPR-Cas gene editing system for preparing a universal CAR-T. The CRISPR-Cas gene editing system comprises a Cas12i editor and an engineered guide RNA, a nucleic acid composition for encoding the Cas12i editor and the engineered guide RNA, a composition, a kit and a method for modifying nucleic acid by utilizing the components.

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.

Compositions and methods for binding to a target sequence of interest are provided. The compositions find use in cleaving or modifying a target sequence of interest, visualizing a target sequence of interest, and modifying the expression of a sequence of interest. Compositions comprise RNA-guided nuclease (RGN) polypeptides, polymerase editors (PEs), guide RNAs, and polynucleotides encoding the same. Vectors and host cells comprising the polynucleotides are also provided. Further provided are RGN systems for binding and modifying a target sequence of interest, wherein the RGN system comprises an RNA-guided nuclease polypeptide and one or more guide RNAs, as well as PE systems for modifying a target sequence of interest, wherein the PE system comprises a polymerase, a RGN polypeptide, and one or more polymerase editing guide RNAs.

The present invention relates to a prime editing system with enhanced correction precision and a use thereof. More specifically, the present invention relates to a recombinant vector for prime editing with improved precision, comprising: a prime editor expression cassette including a mutant Cas9 (R221K, N394K, D840A), a nucleocapsid protein, and an RNaseH domain-deleted mutant MMLV reverse transcriptase (D200N, T306K, W313F, T330P, L603W); and a pegRNA (prime editing guide RNA) expression cassette transcriptionally regulated by a U6 composite promoter.

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.

Provided are guide RNAs targeting the HSD17B13 gene. Also provided are vectors,compositions and methods for treating subjects having liver disease.

The present disclosure relates to CRISPR-related genome editing systems having a gRNA molecule with one or more modifications for editing and / or modulating the expression of a target nucleic acid sequence of interest. The present disclosure is also directed to methods and applications thereof in connection with the treatment and / or management of disease using the described genome editing systems and gRNA molecules.

Provided in some aspects are epigenetic-modifying DNA-targeting systems, such as CRISPR-Cas / guide RNA systems, that bind to or target a target site in a gene or regulatory element thereof in a T cell. In some aspects, the provided epigenetic modifying DNA-targeting systems provided herein modulate a T cell phenotype or activity. In particular, the provided embodiments relate to the transcriptional repression of genes to promote a stem cell-like memory T (TSCM) cell phenotype. In some aspects, also provided are compositions, polynucleotides, vectors, cells, and pluralities and combinations thereof, and methods and uses related to the provided epigenetic-modifying DNA-targeting systems, for example in modulating the phenotype in T cells including in connection with adoptive T cell therapy.

The present invention addresses the problem of providing a lipid nanoparticle in which a nucleic acid, etc., required in genome editing is encapsulated and which can be produced by an alcohol dilution method using flow channels and contributes to high genome editing efficiency. The present invention pertains to a lipid nanoparticle which comprises a lipid component, a DNA nuclease, a guide RNA and a single-stranded oligonucleotide, wherein: the lipid component comprises a pH-sensitive cationic lipid, a neutral phospholipid and a polyalkylene glycol-modified lipid; the ratio of the pH-sensitive cationic lipid relative to the total lipids constituting the lipid nanoparticle is 30-50 mol %; the ratio of the neutral phospholipid relative to the total lipids constituting the lipid nanoparticle is 20-50 mol %; and the ratio of the polyalkylene glycol-modified lipid relative to the total lipids constituting the lipid nanoparticle is 1-4 mol %.

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.

Provided herein are modified prime editing guide RNA (PEgRNA) and nicking guide RNA (ngRNA) sequences and compositions comprising the same, as well as using the same in methods for gene editing.

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%.