57 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 invention discloses an ultra-sensitive target nucleic acid enrichment detection method based on programmable nuclease, and belongs to the technical field of biology. According to the enrichment method disclosed by the invention, on the basis of specific cutting of programmable nuclease, wild type nucleic acid can be continuously removed while isothermal amplification is carried out, so that the number of mutant alleles in a sample is continuously and exponentially increased, and low-abundance mutant genes or methylated DNA are enriched or detected. According to the present invention, sgRNA, guide DNA or guide RNA are designed and optimized, the sgRNA, the guide DNA or the guide RNA and a Cas protein or an Ago protein are used to form a ribonucleoprotein complex, the isothermal amplification is performed while the specific cutting is performed on the target gene, the mutation-containing gene is not cut, and the low-abundance mutation gene is enriched;

The present application relates to a polynucleotide construct for gene editing suitable to hybridize with a CRISPR-Cas9 ribonucleoprotein complex. The disclosed polynucleotide construct is suitable for the spatially controlled release of CRISPR-Cas9 ribonucleoprotein for gene editing. The gene editing potential of the construct in a composition was demonstrated in vitro at single cell level. The safety and gene editing of the construct was also demonstrated. This construct offers a new strategy for the delivery of the CRISPR system with spatial control.

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 invention discloses an ultra-sensitive target nucleic acid enrichment detection method based on programmable nuclease, and belongs to the technical field of biology. According to the enrichment method disclosed by the invention, on the basis of specific cutting of programmable nuclease, wild type nucleic acid can be continuously removed while isothermal amplification is carried out, so that the number of mutant alleles in a sample is continuously and exponentially increased, and low-abundance mutant genes or methylated DNA are enriched or detected. According to the present invention, sgRNA, guide DNA or guide RNA are designed and optimized, the sgRNA, the guide DNA or the guide RNA and a Cas protein or an Ago protein are used to form a ribonucleoprotein complex, the isothermal amplification is performed while the specific cutting is performed on the target gene, the mutation-containing gene is not cut, and the low-abundance mutation gene is enriched;

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 belongs to the technical field of plant breeding, and discloses a breeding method for improving the mutagenesis efficiency of stem tips of woody plants. The CRISPR-Cas9 ribonucleoprotein complex and plant hormones 6-benzyladenine, gibberellin and jasmonic acid are co-encapsulated and then accurately delivered to stem tip meristem of woody ornamental plants such as acer truncatum, pteroceltis tatarinowii, crape myrtle and tassel, and the gene mutation obtaining rate of the stem tip meristem is effectively increased; the intelligent environment control system is used for dynamically optimizing illumination, temperature, humidity and oxidative stress, activating a plant endogenous DNA repair pathway and further enhancing homologous recombination efficiency, meanwhile, through collaborative optimization of induction hormone signals and environmental stress, the stress resistance is improved, the leaf color, the plant type and the growth vigor can be accurately regulated and controlled, and the yield of the plant is increased. According to the method, efficient, accurate and stress-resistant linked woody plant stem tip mutation breeding is realized.

The present application relates to a polynucleotide construct for gene editing suitable to hybridize with a CRISPR-Cas9 ribonucleoprotein complex. The disclosed polynucleotide construct is suitable for the spatially controlled release of CRISPR-Cas9 ribonucleoprotein for gene editing. The gene editing potential of the construct in a composition was demonstrated in vitro at single cell level. The safety and gene editing of the construct was also demonstrated. This construct offers a new strategy for the delivery of the CRISPR system with spatial control.

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 invention relates to the field of medicine, and more particularly to bioengineering, genetic engineering, gene editing, molecular medicine, nanotechnology, biotechnology, nanoengineering and protein engineering. The invention can be used for packaging Cas proteins and guide RNA (guide RNA, single-guide RNA, crRNA, tracrRNA), separately or together in the form of ribonucleoprotein complexes, and for packaging a Cas protein and a guide RNA of any class, type, form, origin and modification, as well as for packaging any proteins and RNA of any structure and sequence.

The invention discloses a method for delivering a gene editing product into a fertilized egg of an animal, and belongs to the technical field of biology. The method comprises the following steps: delivering a gene editing substance into a fertilized egg of an animal by using an electroporation method, wherein the gene editing substance is a ribonucleic acid protein complex formed by a leading editing protein and RNA; and the RNA comprises pegRNA, gRNA or / or sgRNA (small guide RNA). According to the system, a RNP (ribonucleic acid protein) compound is formed by purified pilot editing protein and pegRNA transcribed in vitro, and is directly delivered to a sheep embryo by using an electroporation method. Compared with a traditional microinjection technology, the method is simple, convenient, safe and efficient. By optimizing the concentration ratio of pegRNA to PE protein in RNP, efficient editing of sheep embryo genes is realized, and the highest editing rate reaches 29.94%.

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.

The present invention relates in general to the field of medicine. More particularly, it relates to an eukaryotic cell expressing a ribonucleoprotein complex and to the use thereof for producing toxic vectors. The invention also relates to the method for producing these toxic vectors.

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.

The invention discloses an ultra-sensitive target nucleic acid enrichment detection method based on programmable nuclease, and belongs to the technical field of biology. According to the enrichment method disclosed by the invention, on the basis of specific cutting of programmable nuclease, wild type nucleic acid can be continuously removed while isothermal amplification is carried out, so that the number of mutant alleles in a sample is continuously and exponentially increased, and low-abundance mutant genes or methylated DNA are enriched or detected. According to the present invention, sgRNA, guide DNA or guide RNA are designed and optimized, the sgRNA, the guide DNA or the guide RNA and a Cas protein or an Ago protein are used to form a ribonucleoprotein complex, the isothermal amplification is performed while the specific cutting is performed on the target gene, the mutation-containing gene is not cut, and the low-abundance mutation gene is enriched;

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 discloses a base editing system and application thereof. The base editing system comprises a guide RNA (Ribonucleic Acid) and a base editor; the guide RNA is targeted to an exon 4 of the KCNQ4 gene; the guide RNA and the base editor form a nuclear glycoprotein compound and perform base editing on the exon 4 of the KCNQ4 gene, so that the 229 amino acid of the protein expressed by the KCNQ4 after base editing is glycine. The base editing system disclosed by the invention can correct pathogenic mutation sites of cochlear hair cells, obviously improve auditory functions of Kcnq4 + / G229D mice and Kcnq4G229D / G229D mice, increase the survival of the hair cells, improve the electrophysiological functions of the hair cells, and have a good application prospect.

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.