60 results about "Ro ribonucleoprotein" patented technology

The invention relates to a plasma biomarker for predicting the curative effect of non-small cell lung cancer immunotherapy and application of the plasma biomarker. The biomarker for predicting the curative effect of the non-small cell lung cancer immunotherapy, disclosed by the invention, comprises phosphoglycerate kinase 1 (PGK1), fibronectin 1 (FN1), chromogranin A (CHGA), elastin (ELN), heterogeneous ribonucleoprotein H3 (HNRNPH3) and an Aly / REF output factor (ALYREF). The plasma biomarkers for predicting the non-small cell lung cancer immunotherapy curative effect can be detected by adopting a mass spectrum method, and the non-small cell lung cancer immunotherapy curative effect of a patient is judged by the markers.

The invention discloses application of fluorinated ionizable lipid as a gene drug delivery carrier, and belongs to the technical field of biological medicine. The gene drug delivery carrier provided by the invention is prepared from fluorinated ionizable lipids, cationic lipids, auxiliary lipids, structural lipids and PEG lipids, wherein the molar percentage of the fluorinated ionizable lipids in the gene drug delivery carrier is 1%-10%. According to the invention, the molar percentage of the fluorinated ionizable lipid in the total lipid is optimized to 1-10%, so that the cytotoxicity is obviously reduced, and more excellent nucleic acid transfection efficiency is realized in vivo and in vitro; various nucleic acid drugs including DNA, mRNA, siRNA and CRISPR-Cas9 ribonucleoprotein can be efficiently delivered, action targets of the nucleic acid drugs are expanded to cell nucleuses and cytoplasm, and the nucleic acid drugs are more accordant with the action mechanism of modern gene therapy and editing.

A modular CRISPR-Cas system for ribonucleoprotein (RNP) delivery is provided, the CRISPR-Cas system comprising a CRISPR-Cas protein fused to a first binding peptide and a functional protein fused to a second binding peptide, where the first binding peptide and the second binding peptide are configured to bind to each other and define a binding peptide pair. Also provided are methods of assembling the CRISPR-Cas complexes for the delivery of ribonucleoprotein (RNP) and the CRISPR-Cas complexes, as well as the use of the modular CRISPR-Cas systems or CRISPR-Cas complexes for the modification of the genome of an organism.

The invention relates to lipid nanoparticles for fish in-vivo skin gene editing and application of the lipid nanoparticles, and belongs to the technical field of fish gene editing. The lipid nanoparticle for fish in-vivo skin gene editing comprises a gene editing element and a lipid layer, wherein the lipid layer wraps the surface of the gene editing element; the gene editing element is a CRISPR (clustered regularly interspaced short palindromic repeats) / Cas9 (CRISPR associated protein 9) ribonucleoprotein compound or nucleic acid for coding the compound; the lipid nanoparticle carrier comprises ionizable cationic lipid, auxiliary lipid, cholesterol, pegylated lipid and permanent cationic lipid, and the lipid nanoparticle carrier comprises the following components in parts by mole: 15 parts of ionizable cationic lipid, 25-30 parts of auxiliary lipid, 30 parts of cholesterol, 3 parts of pegylated lipid and 7 parts of permanent cationic lipid. The formula is high in pertinence, high in editing efficiency, simple, convenient and safe to operate and wide in application prospect.

Systems and methods for highly-effective CRISPR-Cas based genomic editing within human chromosome 15q11-q13 have been developed as therapeutic interventions for Angelman Syndrome (AS). Selective single guide RNA molecules (sgRNAs) that impart enhanced CRISPR-Cas editing of genomic mutations associated with Angelman Syndrome in human cells are described. The engineered sgRNAs induce activity of non-pathogenic, paternal UBE3A alleles to reduce, reverse and / or prevent the causative neurodevelopmental defects of AS in a subject in need thereof. Compositions and methods of engineered crRNAs, sgRNAs thereof and ribonucleoprotein (RNP) complexes thereof are provided for enhanced genomic engineering with increased on-off target specificity and on-target editing efficacy for treatment of AS.

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 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 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 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.

Compositions and methods are provided for genomic modification of target sequences in the genome of a cell using novel engineered Cas endonucleases. The engineered Cas polypeptides and related methods, systems, and compositions disclosed herein can be used in guide polynucleotide / endonuclease systems to modify or alter target sequences in the genome of a cell or organism. Also provided are novel effector and endonuclease systems, such as guide polynucleotide / endonuclease systems comprising endonucleases, and elements comprising such systems. Compositions and methods for guide polynucleotide / endonuclease systems comprising at least one endonuclease, optionally covalently or noncovalently linked to or assembled with at least one additional protein subunit, and compositions and methods for directly delivering an endonuclease as a ribonucleotide protein are also provided.

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.

A clustered regularly interspaced short palindromic repeat (CRISPR)-associated complex for adaptive antiviral defence (Cascade); the Cascade protein complex comprising at least CRISPR-associated protein subunits Cas7, Cas5 and Cas6 which includes at least one subunit with an additional amino acid sequence possessing nucleic acid or chromatin modifying, visualising, transcription activating or transcription repressing activity. The Cascade complex with additional activity is combined with an RNA molecule to produce a ribonucleoprotein complex. The RNA molecule is selected to have substantial complementarity to a target sequence. Targeted ribonucleoproteins can be used as genetic engineering tools for precise cutting of nucleic acids in homologous recombination, non-homologous end joining, gene modification, gene integration, mutation repair or for their visualisation, transcriptional activation or repression. A pair of ribonucleotides fused to FokI dimers may be used to generate double-strand breakages in the DNA to facilitate these applications in a sequence-specific manner.

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 present disclosure relates to compositions and methods for providing neuroprotection in a subject and for treating neurodegenerative diseases and traumatic brain or spinal cord injury. In embodiments, the present disclosure provides agents that bind to cytoplasmic heteronuclear kernel glyconucleoprotein (hnRNP) in neurons and are capable of inhibiting neuronal cell death.

The present invention relates to a method and application for realizing targeted gene editing of the offspring of Exopalaemon carinicauda or Litopenaeus vannamei. This method directly injects Cas9 RNP ( sgRNA a mixture formed by mixing with Cas9 ribonucleoprotein) into the body of Exopalaemon carinicauda or Litopenaeus vannamei with developing ovaries by intramuscular injection. The sgRNA used is a guide IAG designed based on the EcIAG or LvIAG gene of Exopalaemon carinicauda or Litopenaeus vannamei: RNA : EcIAG-gRNA or LvIAG-gRNA . After mixing different gRNA with Cas9 protein respectively, they are injected into the maternal body of Exopalaemon carinicauda or Litopenaeus vannamei by intramuscular injection. Cas9 RNP can enter the oocytes of the ovary through blood circulation to realize the editing of the target gene on its genome. After the edited oocytes develop into eggs, the offspring edited mutants are formed through fertilization.

The present disclosure provides CRISPR / Cas9 ribonucleoprotein compositions comprising chemically modified CRISPR RNA (crRNA) guide and trans-acting CRISPR RNA (tracrRNA) components. Methods of using the disclosed CRISPR / Cas9 ribonucleoprotein compositions are also provided.

The present invention relates to compositions for effective targeted delivery of a gene editing agent or transcriptional modulator to a target cell, as well as methods of use thereof for the treatment of diseases including cancer and genetic diseases. In some embodiments, the invention relates to a composition for targeted delivery of a genome editing agent or transcriptional modulator, wherein the composition comprises a delivery vehicle comprising a targeting moiety specific for binding to a cell or tissue of interest. In some embodiments, the genome editing agent comprises a ribonucleoprotein (RNP) complex comprising a CRISPR Cas protein and a guide RNA.

The invention belongs to the technical field of biological breeding, and particularly relates to a vegetable seed seedling cultivation method based on gene editing, which comprises the following steps: constructing a targeting nano delivery carrier loaded with gene editing ribonucleoprotein (RNP), the carrier is composed of surface modified polyethyleneimine and carbon nanodots of stem tip meristem homing peptide (MHP), and the carbon nanodots are uniformly distributed on the surface modified polyethyleneimine and the stem tip meristem homing peptide (MHP). RNP is delivered to stem tip meristem of a vegetable seedling through a micro-droplet in-situ permeation method, so that instantaneous editing of a target gene is realized, and homozygous editing seeds without exogenous gene residues are obtained through sexual reproduction. By means of the scheme, non-transgenic gene editing without genetic transformation and tissue culture is achieved, and the method has the advantages of being easy and convenient to operate, short in period, high in efficiency and wide in applicability and is suitable for character improvement of various vegetables such as tomatoes, spinach and cucumbers.

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 disclosure provides compositions of matter and assay methods to detect target nucleic acids of interest with enhanced fidelity. The cascade assays" or "signal boost assays comprise two different ribonucleoprotein complexes (RNPs) — RNP1 and RNP2 — and RNP2 activator molecules comprising a P AM-proximal region, which may be blocked in some embodiments and a blocked P AM-distal region. The blocked P AM-distal region molecules keep the second ribonucleoprotein complex or "RNP2" "locked" unless and until a target nucleic acid of interest activates the first ribonucleoprotein complex.

A lipid nanoparticle composition for delivery of genome editing ribonucleoprotein (RNP) includes a lipid component that encapsulates the genome editing ribronucleoprotein (RNP), wherein the lipid component includes an ionizable cationic lipid with a pK a > 6, at least one phospholipid, a structural lipid, and PEG-modified lipid.

Provided is a CRISPR RNA (crRNA) applicable in a CRISPR-based ribonucleoprotein (RNP) system for reacting towards one or more genetic materials derived from Burkholderia pseudomallei in a biological sample. The crRNA comprises a spacer region having one of a poly nucleotide sequence selected from SEQ ID No. 3. SEQ ID No. 5, and SEQ ID No. 7: a repeat region preceding the spacer region forming a secondary structure thereof; and a first extension region arranged immediately after the spacer region.

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.