39 results about "Ribonucleoprotein complex" patented technology

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 application provides a double-stranded walking type DNA nanomachine and a preparation method and application thereof, and relates to the technical field of fluorescent analysis and detection.The double-stranded walking type DNA nanomachine for ctDNA fluorescent detection comprises a gold nanoparticle carrier, an anchor probe, a FAM fluorescent reporter probe, a double-stranded walking arm, a Cas12a protein and a crRNA to form a ribonucleoprotein complex.The walking arm of a conventional CRISPR / Cas12a nanomachine is a DNA single-stranded structure, which is easily cut in trans by activated Cas12a, thereby reducing the stability and sensitivity of the nanomachine.The application introduces a double-stranded DNA structure into the walking arm design to replace the conventional DNA single-stranded walking arm, constructs a double-stranded walking type three-dimensional DNA CRISPR / Cas12a nanomachine, effectively resists the reverse cutting of Cas12a on the walking arm, maintains the structural integrity and functional stability of the nanomachine, and greatly improves the detection sensitivity and specificity of lung cancer ctDNA, thereby having a good clinical application prospect.

The present application relates to the technical field of biotechnology, and particularly relates to a nanocomposite containing a cationic lipid analogue and application of the cationic lipid analogue in intracellular delivery of a gene editing ribonucleoprotein complex. The nanocomposite comprises the cationic lipid analogue and the ribonucleoprotein complex; the cationic lipid analogue has a structure as shown in formula (I). The nanocomposite has a high gene editing effect on different gene sites (AAVS1, HBB, EGFP and KRAS) in tumor tissues, which further indicates that the cationic lipid analogue delivering the ribonucleoprotein complex has a certain inhibitory effect on tumor growth; the gene editing ribonucleoprotein complex delivery carrier can achieve a high delivery efficiency in intracellular delivery and is safe and effective.

The present invention relates to a novel analytical method for quantifying the concentration of uncomplexed ("free") gRNA present in a Cas-gRNA ribonucleoprotein complex produced for genome editing.

This invention relates to lipid nanoparticles for in vivo skin gene editing in fish and their applications, belonging to the field of fish gene editing technology. The lipid nanoparticles for in vivo skin gene editing in fish comprise: a gene editing element and a lipid layer, wherein the lipid layer coats the surface of the gene editing element; the gene editing element is a CRISPR / Cas9 ribonucleoprotein complex or a nucleic acid encoding the complex; the lipid nanoparticle carrier comprises ionizable cationic lipids, auxiliary lipids, cholesterol, polyethylene glycol-modified lipids, and permanently cationic lipids, and by molar weight, the proportions are 15 parts ionizable cationic lipids, 25-30 parts auxiliary lipids, 30 parts cholesterol, 3 parts polyethylene glycol-modified lipids, and 7 parts permanently cationic lipids. The formulation of this invention is highly targeted, has high editing efficiency, is simple and safe to operate, and has broad application prospects.

The invention relates to improvements in reduction of tuber browning in potato (Solanum tuberosum) varieties utilising endonuclease-directed gene editing technologies In particular, the invention provides a Solanum tuberosum plant cell comprising a mutation which generates a phenotype of reduced or eliminated polyphenol oxidase activity when compared to a Solanum tuberosum plant cell without the mutation. The invention further provides ribonucleoprotein complexes comprising an endonuclease and a guide RNA that hybridises with a target sequence comprised within a polyphenol oxidase 2 (StPPO2) gene of a Solanum tuberosum variety, and methods of using the same.

The present disclosure provides RNA-guided CRISPR-Cas effector proteins, nucleic acids encoding same, and compositions comprising same. The present disclosure provides ribonucleoprotein complexes comprising: an RNA-guided CRISPR-Cas effector protein of the present disclosure; and a guide RNA. The present disclosure provides methods of modifying a target nucleic acid, using an RNA-guided CRISPR-Cas effector protein of the present disclosure and a guide RNA.

The invention belongs to the technical field of biology, and relates to a crRNA molecule, a CRISPR genome editing system and application thereof. The crRNA molecule is used for guiding LbCpf1 protein to perform targeted cleavage on a genome of a bivalve animal, and a nucleotide sequence of the crRNA molecule is shown as SEQ ID NO.1 or SEQ ID NO.2 in a sequence table; or a sequence having more than 90% of nucleotide homology with the sequence as shown in SEQ ID NO.1 or SEQ ID NO.2 in the sequence table. A ribonucleoprotein complex is formed by the crRNA molecule and LbCpf1 protein, the complex is applied to bivalve animal genome editing, and insertion deletion or long fragment deletion can be induced at a target site. According to the invention, a low-temperature compatible, efficient and heritable LbCpf1 genome editing platform suitable for bivalve animals is established for the first time, and a new technical path is provided for character improvement of bivalve.

The invention provides a CRISPR / Cas12a (clustered regularly interspaced short palindromic repeats / CRISPR associated 12a) system based on a defect PAM (polyacrylamide) lock as well as a preparation method and application of the CRISPR / Cas12a system based on the defect PAM lock. Introducing a wrong basic group at the 3'tail end of the complete complementary chain of the functionalized AFB1 aptamer to generate a mutation sequence; mixing the functionalized AFB1 aptamer with the mutation sequence, heating and slowly cooling to obtain the complete defect PAM lock recognition probe; the method comprises the following steps: mixing a complete defect PAM lock identification probe with a to-be-detected sample, and carrying out first incubation to obtain AFB1-capt; mixing the protein with pre-designed crRNA in a buffer solution, and then carrying out secondary incubation to obtain a ribonucleoprotein complex; and carrying out third incubation on the AFB1-capt and ribonucleoprotein compound to obtain a target system. According to the method, a simple identification and signal amplification system is constructed, so that the practical operation difficulty and cost are reduced, and meanwhile, background interference is reduced.

The invention relates to a ribonucleoprotein complex comprising a nuclease characterized as CRISPR-associated protein of the type II-B having a sequence identity of at least 70% to SEQ ID NO: 1, and a dual guide RNA comprising a CRISPR RNA and a transactivating crRNA, or single guide RNA having a sequence identity of at least 70% to SEQ ID NO: 5. Likewise, the present invention also relates to the use of said ribonucleoprotein to bind, modify or alter target sequences in the genome of a 0 cell or organism, or to modify the gene expression of a cell.

The invention discloses a ribonucleoprotein complex and a construction method of a hand / foot division deformity IV-type animal model, and belongs to the technical field of genetic engineering. According to the mouse model, exons 7 and 8 of the mouse genome Trp63 gene are knocked out, then a sequence is introduced, the 956th basic group is mutated from a G basic group to an A basic group, and the mouse model with the hand / foot division deformity IV is constructed, compared with an existing Trp63 gene knockout mouse model, the mouse model can better simulate the disease, and a foundation is laid for researching the pathogenic mechanism of the disease. A new method is provided for research of limb dysplasia diseases and screening of treatment drugs, and the method has a good application prospect.

Provided herein are engineered CasX systems and components thereof, including variant CasX proteins and variant guide nucleic acids (gNAs). The variant CasX proteins and variant gNAs of the present invention exhibit at least one improved characteristic when compared to a reference CasX protein or reference gNA of the invention. In some cases, the variants have one or more improved CasX ribonucleoprotein complex functions. Methods of making and using the variants are also provided.

A method for inserting a polynucleotide exogenous transgene sequence at a predetermined endogenous genetic locus in the genome of a host cell, the method comprising: (i) a donor DNA template comprising a polynucleotide insertion fragment; 5'homologous arms; and a 3'homologous arm. In some embodiments, the 5'homologous arm and the 3 'homologous arm are complementary to DNA in the target region; and (ii) a ribonucleoprotein complex (RNP) comprising (1) a Cas nuclease and at least one small guide RNA (sgRNA) complementary to at least one selected nucleic acid sequence within the predetermined genetic locus in the host cell genome.

Disclosed are Type V CRISPR nucleases. Related Type V CRISPR nuclease ribonucleoprotein complexes, guide nucleic acids, recombinant expression vectors, kits, methods, and systems are also provided.

Provided herein are lipid nanoparticles comprising a ribonucleoprotein complex (RNP) comprising a Type II Cas nuclease polypeptide (e.g., a Cas9 polypeptide) and a guide RNA (gRNA), e.g., a single guide RNA (sgRNA). Compositions and formulations comprising such lipid nanoparticles are also provided. Such lipid nanoparticles can be used for delivery of a RNP to a cell, for example, a cell that is present in a subject in vivo. Methods of producing such lipid nanoparticles, and methods of use for genome engineering, are also provided.

Described herein is a DNA template plasmid for generating genome modified immune cells including a plasmid backbone, a first insert, and a second insert. The first insert includes a transgene, wherein the transgene is flanked by left and right homology arms that are complementary to sequences on both sides of a cleavage site in a target expressed gene in an unmodified immune cell. The second insert includes a cleavage target including a protospacer sequence defining the cleavage site in the target expressed gene, and a protoadjacent motif sequence (PAM) for recognition by a Cas9 ribonucleoprotein complex (Cas9-RNP). The Cas9-RNP includes a Cas9 polypeptide and a single guide RNA (sgRNA) comprising a sequence complementary to the protospacer sequence. The Cas9-RNP binds the second insert and linearizes the DNA template plasmid by Cas9-RNP-directed cleavage at the cleavage site. Also included are RNP complexes, methods of genome modifying immune cells, and treatment methods.

The invention relates to a vector for improving gene knock-in efficiency and a method for verifying accurate gene knock-in efficiency. The vector comprises a gene knock-out vector, an auxiliary knock-in vector and a donor template vector, the gene knockout vector comprises sgRNA and a knockout vector skeleton, the sgRNA comprises a sequence complementary to a target gene, and the skeleton comprises a sequence for coding a Cas protein and has cleavage activity; the auxiliary knock-in vector comprises crRNA of a 5'and / or 3 'homologous arm of a targeted donor template and an auxiliary knock-in vector skeleton, the crRNA comprises a sequence complementary to the homologous arm, the skeleton comprises a sequence for coding nickase inactivation type Cas protein, and the crRNA and the sequence form a ribonucleoprotein complex to realize an auxiliary knock-in function; the donor template carrier comprises a donor template and a donor template carrier skeleton. The method realizes breakthrough improvement of the gene accurate knock-in efficiency, can quantitatively evaluate the gene accurate knock-in efficiency, and shows important potential in the field of gene therapy.

The present disclosure is directed to methods for performing size exclusion chromatographic (SEC) separations. Embodiments of the present disclosure feature methods for improved separations of biomolecule analytes, such as CRISPR-related proteins, nucleotides, and ribonucleoprotein complexes, in SEC, for example, by using hydroxy-terminated polyethylene glycol surface modified stationary phase materials and / or C2 / PEG surface modified column hardware.

The invention relates to the technical field of gene editing, in particular to an ultra-long fragment gene precise fixed-point insertion method and application thereof.According to the method, virus-like particles VICEP are adopted to deliver a ribonucleoprotein complex (RNP), and an integrated defective lentivirus IDLV is adopted to deliver a donor template containing a 5 ' / 3' homologous arm; the two genes have a synergistic effect in the same time window to trigger homologous recombination repair, so that high-efficiency and low-off-target integration of a large-fragment gene at a target site is realized. By adding an inhibitor HDR promoter during infection, non-homologous end ligation (NHEJ) can be further inhibited and insertion efficiency can be improved. The method shows stable knock-in efficiency greater than or equal to 15% in various difficult-to-transfect cells such as iPSC, HUDEP2, K562 and the like, breaks through the traditional AAV capacity upper limit and electrotransfection toxicity bottleneck, and can be widely applied to gene therapy, cell engineering and synthetic biology.

This application relates to the technical field of virus detection, specifically to a composition, kit, and method for simultaneously detecting seven G genotypes of group A rotavirus. The composition comprises: components A-G for detecting upstream and downstream primers and probes for group A rotavirus G1, G2, G3, G4, G8, G9, and G12 genotypes; and component H for detecting the upstream and downstream primers and probes for human ribonucleoprotein complex. Detection using this composition or a kit containing it offers advantages such as high sensitivity and specificity, ease of operation, short detection time, small sample volume required, and low contamination. It can directly detect nucleic acids extracted from the sample to be tested, and has high application value in rapid virus detection.

The application belongs to the technical field of gene editing, and particularly relates to a CRISPR-assisted genome site-directed insertion marker system and a cell editing method. The system comprises a first guide RNA targeting a genome site, a second guide RNA targeting a donor vector, a high-fidelity Cas9 protein, and a universal donor vector, wherein the universal donor vector contains a P2A-fluorescent protein-T2A-HSV-tk expression frame. The ribonucleoprotein complex formed by electrotransferring the above components realizes efficient and accurate insertion of an exogenous fragment. Compared with the prior art, the application significantly improves the site-directed knock-in efficiency (the positive rate of organoids under resistance screening reaches 48.28%) and fidelity in cells and organoids, realizes the double functions of fluorescent tracing and inducible clearance of target cells and visualization, simplifies the design of the donor, reduces the experimental period and cost, and is particularly suitable for complex systems such as cells and organoids with limited homologous recombination activity.

The application provides a CRISPR / Cas12a system based on a defective PAM lock, a preparation method and application, and the application introduces a PAM sequence at the 5' end of an AFB1 aptamer sequence to obtain a functionalized AFB1 aptamer; an error base is introduced on the functionalized AFB1 aptamer to generate a mutant sequence; the functionalized AFB1 aptamer and the mutant sequence are mixed to obtain a complete defective PAM lock recognition probe; the complete defective PAM lock recognition probe is mixed with a sample to be detected and is subjected to first incubation to obtain AFB1-capt; a protein is mixed with a pre-designed crRNA and is subjected to second incubation to obtain a ribonucleoprotein complex; AFB1-capt and the ribonucleoprotein complex are subjected to third incubation to obtain a target system. The application constructs a simple recognition and signal amplification system, reduces the operation difficulty and cost, and reduces background interference.

The application discloses a CRISPR / Cas9-drug co-assembly biomimetic nano system and preparation and application thereof, and belongs to the technical field of biological medicines. The biomimetic nano system is composed of a natural product active small molecule drug as a nano inner core, a gene editing system and an auxiliary component co-assembled into a nano drug particle, and then a biomimetic membrane is further wrapped on the surface to form the biomimetic nano system; wherein the natural active small molecule drug is ursolic acid, the CRISPR / Cas9 gene editing system is a ribonucleoprotein complex containing a nuclear localization sequence, the auxiliary component is a cell-penetrating peptide, and the biomimetic membrane is a tumor cell membrane. The biomimetic nano system is constructed on the basis of revealing the combined immune regulation characteristics and molecular mechanisms of immune checkpoint PD-L1 gene therapy and natural product ursolic acid anti-tumor, has a significant inhibitory effect on the occurrence of liver cancer, and has a wide application prospect in the preparation of anti-liver cancer drugs.

The present disclosure provides RNA-guided CRISPR-Cas effector proteins, nucleic acids encoding the effector proteins, and compositions comprising the effector proteins. The present disclosure provides ribonucleoprotein complexes comprising: an RNA-guided CRISPR-Cas effector protein of the present disclosure; and a guide RNA. The present disclosure provides methods of modifying a target nucleic acid using an RNA-guided CRISPR-Cas effector protein and a guide RNA of the present disclosure. The present disclosure provides methods of modulating transcription of a target nucleic acid.

Provided is a programmable nuclease based ultra-sensitive method for enriching and detecting a target nucleic acid. Based on specific cleavage of a programmable nuclease, by designing and optimizing an sgRNA, a guide DNA or a guide RNA, the enrichment method uses the sgRNA, the guide DNA, or the guide RNA, and a Cas protein or an Ago protein to form a ribonucleoprotein complex. Thus, non-target nucleic acids can be removed continuously and specifically while amplifying at a constant temperature. The number of target nucleic acids in a sample continuously increases exponentially, and the target nucleic acids such as low-abundance mutant genes and methylated DNAs are enriched or detected.

TITLE: PRODUCTION OF TOXIC VECTORS The present invention relates generally to the field of medicine. More particularly, it relates to a eukaryotic cell expressing a ribonucleoprotein complex and its use for the production of toxic vectors. The method for producing these toxic vectors also belongs to the invention. (Figure 7)

Provided are engineered virus-like particles comprising a ribonucleoprotein complex of a base editor and a guide RNA that targets gene(s) associated with glaucoma, and applications thereof.

This invention relates to a novel analytical method for quantifying the concentration of non-complexed ("free") gRNA present in Cas-gRNA ribonucleoprotein complexes manufactured for genome editing.

Provided herein are lipid nanoparticles comprising a ribonucleoprotein complex (RNP) comprising a Type II Cas nuclease polypeptide (e.g., a Cas9 polypeptide) and a guide RNA (gRNA), e.g., a single guide RNA (sgRNA). Compositions and formulations comprising such lipid nanoparticles are also provided. Such lipid nanoparticles can be used for delivery of a RNP to a cell, for example, a cell that is present in a subject in vivo. Methods of producing such lipid nanoparticles, and methods of use for genome engineering, are also provided.