17 results about "Direct repeat" patented technology

A Class 2, Type VI clustered regularly interspaced short palindromic repeat (CRISPR) RNA (gRNA) which comprises a direct repeat (DR) stem loop sequence and a guide or spacer sequence, is provided. Also described are methods for generating, selecting, characterizing and optimizing a clustered regularly interspaced short palindromic repeats (CRISPR) RNA (gRNA) for use in the CRISPR-CaslSd system described herein. Also provided is a screening method to identify gRNA particularly suited for use with specified targets.

The invention provides a method for improving the efficiency and detection sensitivity of establishing a CRISPR nucleic acid detection one-step method, and particularly provides a direct Repeat (DR) sequence for forming gRNA and guide RNA containing the DR sequence, the invention finds for the first time, and provides a method for improving the efficiency and detection sensitivity of establishing a CRISPR nucleic acid detection one-step method and a method for establishing the CRISPR nucleic acid detection one-step method for establishing the CRISPR nucleic acid detection one-step method for establishing the CRISPR nucleic acid detection one-step method. When the guide RNA adjusted by the invention is used for detecting target nucleic acid in a one-step detection system, the establishment efficiency and the detection sensitivity of the CRISPR nucleic acid detection one-step method can be remarkably improved.

The present invention relates to a method for recording a transcriptome of a cell by: providing a test cell that includes a first transgene nucleic acid sequence encoding a fusion protein that is a reverse transcriptase polypeptide and a Cas1 polypeptide and a second transgene nucleic acid sequence encoding a Cas2 polypeptide, wherein the first transgene nucleic acid sequence and the second transgene nucleic acid sequence are under transcriptional control of an inducible promoter sequence, and a third transgene nucleic acid sequence including a CRISPR direct repeat (DR) sequence; wherein the CRISPR direct repeat sequence is specifically recognizable by a RT-Cas1-Cas2 complex formed by the expression products of the first transgene nucleic acid sequence and the second transgene nucleic acid sequence.

To provide a novel RNA targeting system.SOLUTION: The invention relates to CRISPR-Cas system comprising: a) one or more V type CRISPR-Cas polynucleotide sequence including a guide RNA, the guide RNA including a guide sequence coupled to a direct repeat sequence and capable of hybridizing to a target sequence, or one or more nucleotide sequence coding one or more V type CRISPR-Cas polynucleotide sequence; b) a Cpf1 effector protein or one or more nucleotide sequence coding Cpf1 effector protein, in the CRISPR-Cas system, one or more guide sequences can hybridize to the target sequence, the target sequence is positioned at 3' side of a protospacer adjacent motif (PAM), and the guide RNA forms a composite with the Cpf1 effector protein, the CRISPR-Cas system being engineered and not existing in nature.SELECTED DRAWING: None

The present invention provides for the production of improved viral and non-viral nanoplasmid vectors. Specifically, the invention provides an antibiotic-label-free covalent closed circular recombinant DNA molecule comprising: a. An antibiotic-label-free insert comprising a structured DNA sequence selected from the group consisting of a reverse repeat, a forward repeat, a homopolymerized repeat and an eukaryotic origin of replication; b. A Pol III-dependent origin of replication comprising an R6K [gamma] origin of replication having at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 18; and c. An RNA-OUT RNA selection marker, which comprises an RNA-IN modulating RNA-OUT RNA having at least 95% sequence identity with SEQ ID NO: 6, and a method for detecting the RNA-OUT RNA.

A CRISPR-Cas system and applications thereof are provided, also related to a Cas protein, a fusion protein and a guide polynucleotide. The Cas protein has at least 50% sequence identity compared to the sequence set forth in any one of SEQ ID NOs: 1-184, 426-428. The fusion protein comprises the Cas protein fused to a protein domain and / or a polypeptide tag. The guide polynucleotide comprises a direct repeat sequence having at least 70% sequence identity to any one of SEQ ID NOs: 185-199, 245-247, 287-303, 369 and 370-373, and a guide sequence engineered to hybridize to a target nucleic acid. The CRISPR-Cas system comprises a Cas protein having at least 90% sequence identity compared to the sequence set forth in any one of SEQ ID NOs: 1-184, 426-428, or a nucleic acid encoding thereof, and a guide polynucleotide or a nucleic acid encoding thereof.

A method for improving the replication of a covalently closed circular plasmid is provided. The method includes providing a covalently closed circular plasmid having a Pol I-dependent origin of replication, and an insert including a structured DNA sequence selected from inverted repeat sequences, direct repeat sequences, homopolymeric repeat sequences, eukaryotic origins of replication or eukaryotic promoter enhancer sequences, wherein the structured DNA sequence is located at a distance of less than 1000 bp from the Pol I-dependent origin of replication in the direction of replication. The method also includes modifying the covalently closed circular recombinant molecule such that the Pol I-dependent origin of replication is replaced with a Pol III-dependent origin of replication, whereby the resultant Pol III-dependent origin of replication covalently closed circular plasmid has improved replication. An antibiotic marker free covalently closed circular recombinant DNA molecule is also provided.

The present disclosure provides methods for determining whether a cancer patient with homologous recombination deficiency will benefit from treatment with PARP inhibitors or platinum agents. These methods are based on screening a cancer patient for the presence of templated insertions (TINS), including direct-repeat templated insertions (drTINS) and inverted templated insertions (iTINS).

An RNA interference-based product and method for inhibiting the expression of the double homeobox 4 (DUX4) gene on human chromosome 4q35 are disclosed. The invention comprises the silencing of the Cas13 protein of RNA, wherein Cas13 is specifically targeted to a DUX4 region of interest using a sequence-specific guide RNA (gRNA). The recombinant adeno-associated virus of the invention is constructed with a Cas13 direct repeat sequence and delivers DNA encoding an inhibitory gRNA that knocks down DUX4 expression. The method is applicable to the treatment of muscular dystrophy, including but not limited to facial-scapulohumeral muscle dystrophy (FSHD), and other disorders, including cancer, associated with elevated DUX4 expression.

The present invention relates to a crRNA primer set, a kit and an application for detecting pine wood nematodes. The crRNA primer set includes a direct repeat sequence and an interval guide sequence; the direct repeat sequence has a Cas protein binding function; the target gene of the interval guide sequence includes an expansin gene. The crRNA primer set and the kit provided by the present invention can realize rapid and accurate screening of living pine wood nematodes by specifically detecting the expansin gene in living pine wood nematodes. The crRNA primer set and the kit of the present invention are applied to the detection of pine wood nematodes, which has the characteristics of strong specificity and high detection efficiency, and can effectively prevent the spread of living nematodes with the import, export and transportation of pine wood and other breeding materials, and have excellent prospects for promotion and use for port detection and practical applications.

The invention discloses a DNA (Deoxyribose Nucleic Acid) modification and transfer integrated escherichia coli engineering strain and application thereof. Three engineering strains of E.coli GB06-DLP12Tra-alpha beta gamma, GB05-recETTra-alpha beta gamma and DH5G-GTra-alpha beta gamma are constructed by integrating components with conjugational transfer and recombination functions, namely, Tra-red alpha beta gamma onto genomes of different Escherichia coli, and the E.coli GB06-DLP12Tra-alpha beta gamma has the best stability on repetitive sequences, so that plasmids containing 500bp direct repetitive sequences can stably exist, and the expression quantity of the E.coli GB06-DLP12Tra-alpha beta gamma is greatly increased. The possibility of unexpected intramolecular recombination of large plasmids in genetic manipulation is reduced as much as possible; when the E.coli GB05-recETTra-alpha beta gamma is used as a donor, the conjugational transfer efficiency is the highest, and is higher than that of an existing donor E.coli ET12567 expressed by a plasmid pUZ8002; the E.coli GB05-recETtra-alpha beta gamma and the DH5G-Gtra-alpha beta gamma have high electrotransfection efficiency on the plasmids, and the plasmids can be modified under the induction of rhamnose. The bifunctional engineering bacteria E.coli GB05-recETtrar-alpha beta gamma and DH5G-Gtrar-alpha beta gamma can be used for mining a natural product synthesis gene cluster, and the cloned gene cluster is directly transferred to a proper streptomyces host for heterologous expression after being modified.

A method for improving the replication of a covalently closed circular plasmid is provided. The method includes providing a covalently closed circular plasmid having a Pol I- dependent origin of replication, and an insert including a structured DNA sequence selected from inverted repeat sequence, direct repeat sequence, homopolymeric repeat sequence, eukaryotic origin of replication or eukaryotic promoter enhancer sequence, wherein the structured DNA sequence is located at a distance of less than 1000 bp from the Pol I-dependent origin of replication in the direction of replication. The method also includes modifying the covalently closed circular recombinant molecule such that the Pol I-dependent origin of replication is replaced with a Pol Ill-dependent origin of replication, whereby the resultant Pol Ill-dependent origin of replication covalently closed circular plasmid has improved replication. An antibiotic marker free covalently closed circular recombinant DNA molecule is also provided. Recombinant DNA molecules and methods related thereto which include one or more primosomal sites and / or a selectable marker oritented to transcribe away from a structured DNA sequence are also provided.

The present disclosure provides a Cas12i protein comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity with the amino acid sequence represented by any one of SEQ ID NO: 1-10 (preferably, SEQ ID NO: 1-3 and 6, more preferably, SEQ ID NO: 1). The present disclosure also provides an engineered, non-naturally occurring CRISPR-Cas system comprising: (1) the Cas12i protein or a polynucleotide encoding the Cas12i protein, and (2) a Cas12i protein or a polynucleotide encoding the Cas12i protein; and (2) a CRISPR RNA (crRNA) or a polynucleotide encoding the crRNA, the crRNA comprising: (i) a spacer sequence capable of hybridizing to a target sequence of a target DNA, and (ii) a direct repeat (DR) sequence linked to the spacer sequence capable of directing the binding of the Cas12i protein to the crRNA to form a CRISPR-Cas complex that targets the target sequence.

To provide DUX4 RNA silencing using RNA-targeting CRISPR-CAS13B.SOLUTION: RNA interference-based products and methods for inhibiting the expression of the double homeobox 4 (DUX4) gene on human chromosome 4q35 are disclosed. The disclosure includes the Cas13 protein silencing of an RNA, where Cas13 is specifically targeted to a DUX4 region of interest using a sequence-specific guide RNA (gRNA). Recombinant adeno-associated viruses of the disclosure deliver DNAs encoding inhibitory gRNAs that are constructed with Cas13 direct repeats to knock down the expression of DUX4. The methods have application to the treatment of muscular dystrophies including, but not limited to, facioscapulohumeral muscular dystrophy (FSHD), and other disorders associated with elevated DUX4 expression, including cancer.SELECTED DRAWING: None

Disclosed is a Cast 2a protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 3 (CrmCas12a) and SEQ ID NO: 4 (SrmCas12a) or a Cas12a protein of the same clade; where the Cas12a protein comprises a maltose-binding protein (MBP) tag, and a CRISPR RNA (crRNA) for detecting Brucella. Also disclosed is a detection kit for Brucella comprising the crRNA as disclosed herein and a method of detecting a nucleic acid of Brucella in a sample using the detection kit. Further disclosed is a CRISPR-Cas12a composition comprising the Cast 2a protein and a crRNA comprising a direct repeat (DR) and a guide domain (protospacer), wherein the DR and the guide domain are connected in the 5' to 3' direction of the crRNA, and the crRNA is configured to target a nucleic acid of a predetermined target sequence and form a complex with the Cast 2a protein as disclosed herein.

The invention relates to a bacterial cell comprising a Cas1 RT fusion protein, a Cas2 protein and a CRISPR direct repeat (DR) sequence, wherein an RNA polymerase promoter in addition to the leader sequence is associated with the DR sequence. The invention further relates to a composition comprising two bacterial cell populations, each comprising a Cas1 RT fusion protein and Cas2 protein. The two cell types contain different versions of a CRISPR direct repeat (DR) sequence. The invention further relates to methods for analysis of transcription recording events of bacteria having passed through a subject's intestine, to assign a probability to the subject having a condition, such as malnutrition or inflammation of the intestine.

Provided in the present application are a CRISPR-Cas system and the use thereof. The present application further relates to a Cas protein, a fusion protein, and a guide polynucleotide. The Cas protein has at least 50% sequence identity with any one among SEQ ID NOs: 1-184 and 426-428. The fusion protein comprises the Cas protein fused to a protein domain and / or a polypeptide tag. The guide polynucleotide comprises a direct repeat sequence and a guide sequence engineered to hybridize with a target nucleic acid, where the direct repeat sequence has at least 70% sequence identity with any one among SEQ ID NOs: 185-199, 245-247, 287-303, 369, and 370-373. The CRISPR-Cas system comprises a Cas protein having at least 90% sequence identity with any one sequence among SEQ ID NOs: 1-184 and 426-428 or an encoding nucleic acid thereof, as well as a guide polynucleotide or an encoding nucleic acid thereof.