996 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 invention relates to the technical field of virus detection, and discloses a method for detecting infectious salmon anemia virus in salmon based on RAA-CRISPRCas12a and application, and the method comprises the following steps: dispersing a Cas12a protein solution, a guide RNA solution, an ssDNA reporter probe solution, a ribonuclease inhibitor, nuclease-free water and an RAA reactant in a buffer solution, incubating, and detecting the infectious salmon anemia virus in salmon based on RAA-CRISPRCas12a. Carrying out fluorescence intensity detection; the amino acid sequence of the Cas12a protein is as shown in SEQ ID NO. 6. The novel Cas12a protease with better cutting activity is used in the method, and can be successfully applied to detection of the infectious salmon anemia virus in salmons.

The invention relates to the technical field of virus detection, and discloses a method for detecting salmon alphavirus in salmon based on RAA-CRISPRCas12a and application, the method comprises the following steps: dispersing a Cas12 protein solution, a guide RNA solution, an ssDNA reporter probe solution, a ribonuclease inhibitor, nuclease-free water and an RAA reactant in a buffer solution, incubating, and carrying out fluorescence intensity detection; the amino acid sequence of the Cas12 protein is as shown in SEQ ID NO. 5. The novel Cas12a protease with better cutting activity is used in the method, and can be successfully applied to detection of salmon alphavirus in salmons.

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.

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 present disclosure provides Cas9 variants, and base editors comprising these variants, that recognize non-canonical protospacer adjacent motifs (PAMs) and have less restrictive PAM requirements for editing. The present disclosure provides Cas9 protein variants comprising one or more amino acid substitutions relative to wild-type Nme2Cas9. Fusion proteins comprising the Cas protein variants described herein are also provided by the present disclosure. Further provided herein are methods for editing a target nucleic acid using the Cas variants and fusion proteins provided herein. The present disclosure also provides guide RNAs, complexes, polynucleotides, cells, kits, and pharmaceutical compositions. Further described herein are phage-assisted continuous evolution (PACE) systems, vectors, methods, and devices.

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 a method of modulating the expression of an endogenous gene in a somatic cell derived from an induced pluripotent stem cell (iPSC), comprising differentiating an engineered iPSC capable of stable or inducible expression of a CRISPR-related protein into a somatic cell prior to introduction of a guide RNA (gRNA) sequence. The invention also relates to the engineered iPSC, cells obtained by the method and uses thereof.

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.

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

In one aspect, the disclosure relates to a gene modification tool that fuses a Cas protein with a polymerase. This DSB-independent approach utilizes a DNA-RNA chimeric prime-editor-like guide RNAs (chimeric pegRNAs or cpegRNAs) to enable programmable DNA sequence replacement or excision at endogenous human genomic sites. The Cas9 and RNA portion of cpegRNA helps with targeting a gene, while the DNA portion of cpegRNA is used as a template by the polymerase to repair. Furthermore, the disclosed method can be used to introduce large DNA modifications in the genome.

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 invention belongs to the field of gene engineering. Specifically, the invention relates to a base editing system for optimizing engineered T cells and application of the base editing system. The present invention provides a base editing system for modifying engineered T cells, comprising: i) a base editing polypeptide, and / or an expression construct encoding a base editing polypeptide, the base editing polypeptide comprising a DNA binding protein and at least one deaminase domain; ii) a guide RNA, and / or an expression construct encoding a guide RNA, the guide RNA targeting any one of TRAC, TGFBR2, PD-1, CISH, and / or FAS genomic regions, or any combination thereof. The base editing system disclosed by the invention has high efficiency of a gene editing tool and low Indel frequency, can realize accurate editing of T cell function enhancement related genes, and has important value for expanding application scenes of a base editing technology in the fields of preparation of safe and efficient engineered T cells and the like.

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 herein are epigenetic-modifying DNA-targeting systems, such as CRISPR-Cas / guide RNA (gRNA) systems, for the transcriptional repression of Hepatitis B viral (HBV) genes to promote a cellular phenotype that leads to the reduction of HBV infection. In some embodiments, the epigenetic-modifying DNA-targeting systems bind to or target a target site of at least one gene or regulatory element thereof in a Hepatitis B viral DNA sequence in cell. In some aspects, the provided systems relate to the transcriptional repression of one or more Hepatitis B viral gene and / or regulatory element thereof. In some aspects, also provided herein are methods and uses related to the provided compositions, for example in repressing Hepatitis B viral replication and expression in connection with Hepatitis B infections.

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.

The invention belongs to the field of biology, relates to a salmonella gene detection technology, and discloses a one-pot detection method combining LAMP (loop-mediated isothermal amplification) and CRISPR / BrCas12b mediated nucleic acid molecule detection. The method comprises the following steps: designing an LAMP primer, a small guide RNA (Ribonucleic Acid) and a transcription template overlapping PCR (Polymerase Chain Reaction) primer, performing loop-mediated isothermal amplification, constructing CRISPR / Cas shearing recognition (Cas-LAMP), constructing a single-tube one-pot reaction system of the CRISPR / Cas shearing recognition, detecting the specificity and sensitivity of Cas-LAMP amplification, and the like. In order to solve the technical problem that amplification and shearing recognition cannot be carried out synchronously, the invention provides a Cas-LAMP technology capable of linking two-step reaction of amplification and shearing recognition, and accurate, sensitive and rapid detection of nucleic acid molecules is realized in an in-vitro single reaction tube system. Compared with a traditional nucleic acid molecule detection method, the method has the following advantages that one-pot detection can be directly carried out without step-by-step operation, high sensitivity and strong specificity are achieved, and rapid field detection can be achieved. The method provides technical support for conventional nucleic acid detection, and has a wide field instant detection application prospect.

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.

To provide lipid nanoparticle-based compositions and methods useful for delivery of CRISPR / Cas gene editing components.SOLUTION: A lipid nanoparticle (LNP) composition comprises: mRNA encoding a Cas nuclease; guide RNA nucleic acid; and a plurality of component lipids comprising the following: 35 mol% to 55 mol% of lipid A, which is (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate; 35 mol% to 55 mol% of a helper lipid; 5 mol% to 15 mol% of a neutral lipid; and 1 mol% to 5 mol% of a stealth lipid.SELECTED DRAWING: None

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.