402 results about "Guide RNA" patented technology

The present invention relates to targeted genome editing in eukaryotic cells or organisms. More particularly, the present invention relates to a composition for cleaving a target DNA in eukaryotic cells or organisms comprising a guide RNA specific for the target DNA and Cas protein-encoding nucleic acid or Cas protein, and use thereof.

The invention belongs to the field of genetic engineering, particularly relates to a method for human PD1 gene specific knockout through CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat) and sgRNA(single guide RNA)for specially targeting a PD1 gene, and provides a method for human PD1 gene specific knockout through CRISPR-Cas9 and sgRNA(single guide RNA) for specially targeting a PD1 gene. The sgRNA(single guide RNA)for specially targeting a human PD1 gene, prepared through adopting the method provided by the invention, can be used for accurately targeting the human PD1 gene and realizes gene knockout; the preparing method has simple steps, the targeting performance of the sgRNA is good, and the knockout efficiency of a CRISPR-Cas9 system is high.

The invention relates to a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas guide RNA (gRNA) comprising a targeting domain that is complementary to human genomic Epidermal Growth Factor Receptor (EGFR) DNA, and a vector system including one or more packaged vector(s) including: (a) a first regulatory element operably linked to a gRNA, and (b) a second regulatory element operably linked to a nucleic acid encoding a Cas protein. Also disclosed are methods of altering a nucleic acid sequence encoding EGFR in a cell including contacting the cell with a vector system, methods of treating lung cancer, and methods of selectively inducing apoptosis in a cell including administering a gRNA to the cell.

The invention belongs to the field of gene engineering and particularly relates to a method for specifically knocking out a human CCR5 (Chemokine Receptor 5) gene by CRISPR (clustered regularly interspaced short palindromic repeat-associated)-Cas 9 and SgRNA (single guide RNA) for specifically targeting the CCR5 gene as well as an intermediate carrier and application thereof. The sgRNA for specifically targeting the human CCR5 gene, prepared by the method disclosed by the invention, can be used for accurately targeting the human CCR5 gene and realizing gene knockout. The preparation method is simple in steps and good in sgRNA targeting property; the knockout efficiency of a CRISPR-Cas 9 system is high.

The invention belongs to the field of genetic engineering, particularly relates to a method for human CTLA4 gene specific knockout through CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat) and sgRNA(single guide RNA)for specially targeting a CTLA4 gene, and provides a method for human CTLA4 gene specific knockout through CRISPR-Cas9 and sgRNA(single guide RNA)for specially targeting a CTLA4 gene. The sgRNA(single guide RNA)for specially targeting a human CTLA4 gene, prepared through adopting the method provided by the invention, can be used for accurately targeting the human CTLA4 gene and realizes gene knockout; the preparing method has simple steps, the targeting performance of the sgRNA is good, and the knockout efficiency of a CRISPR-Cas9 system is high.

A method of inactivating a proviral DNA integrated into the genome of a host cell latently infected with a retrovirus by treating the host cell with a composition comprising a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease, and two or more different guide RNAs (gRNAs), wherein each of the at least two gRNAs is complementary to a different target nucleic acid sequence in a long terminal repeat (LTR) in the proviral DNA, and inactivating the proviral DNA. A composition for use in inactivating a proviral DNA integrated into the genome of a host cell latently infected with a retrovirus including isolated nucleic acid sequences comprising a CRISPR-associated endonuclease and a guide RNA, wherein the guide RNA is complementary to a target sequence in a human immunodeficiency virus.

Some aspects of this disclosure provide compositions, methods, systems, and kits for controlling the activity and/or improving the specificity of RNA-programmable endonucleases, such as Cas9. For example, provided are guide RNAs (gRNAs) that are engineered to exist in an “on” or “off” state, which control the binding and hence cleavage activity of RNA-programmable endonucleases.

The present disclosure provides compositions, kits, genetically modified cells, non-human transgenic organisms, and methods for binding and/or cleaving a single stranded target nucleic acid. A method of cleaving includes contacting a single stranded target nucleic acid with (e.g., introducing into a cell) a subject argonaute (Ago) polypeptide and a guide RNA (e.g., having a 5′-OH). In some embodiments, a subject Ago polypeptide includes an amino acid sequence having 70% or more sequence identity with amino acids 282-430 and/or 431-639 of the Marinitoga piezophila argonaute (MpAgo) protein set forth in SEQ ID NO: 1. The present disclosure provides variant Ago polypeptides; and methods of use of same.

The present invention generally relates to libraries, kits, methods, applications and screens used in functional genomics that focus on gene function in a cell and that may use vector systems and other aspects related to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas systems and components thereof. The present invention also relates to rules for making potent single guide RNAs (sgRNAs) for use in CRISPR-Cas systems. Provided are genomic libraries and genome wide libraries, kits, methods of knocking out in parallel every gene in the genome, methods of selecting individual cell knock outs that survive under a selective pressure, methods of identifying the genetic basis of one or more medical symptoms exhibited by a patient, and methods for designing a genome-scale sgRNA library.

Some aspects of this disclosure provide compositions, methods, systems, and kits for controlling the activity and/or improving the specificity of RNA-programmable endonucleases, such as Cas9. For example, provided are guide RNAs (gRNAs) that are engineered to exist in an “on” or “off” state, which control the binding and hence cleavage activity of RNA-programmable endonucleases. Some aspects of this disclosure provide gRNAs that modulate the activity of an RNA-programmable endonuclease based on the presence or absence of an extended DNA (xDNA).

The invention relates to the field of gene engineering and particularly discloses a tool for efficient site-specific transposition of genes. The tool comprises or can produce a gRNA-Cas9 protein (without incision enzyme activity)-PB transposase compound, and gRNA targets the specific site of a genome; the Cas9 protein without incision enzyme activity is double-mutant Cas9 protein with 10th-site amino acid residue D mutating into A and 840th-site amino acid residue H mutating into A. Through design of gRNA (guide RNA) targeting the animal genome, under the coexpression condition of gRNA and site-specific transposase, PB transposase is targeted to a specific position of the genome along with a CRISPR/cas9 and gRNA compound, the PB transposase realizes a transposition function at the specific site, accordingly, donor plasmids (carriers containing exogenous target genes) are simultaneously co-transfected, the exogenous target carriers can be transposed to the specific site in the genome through transposition, and site-specific transposition of the genes is realized.

The invention belongs to the technical field of molecular biology and biomedicine and in particular relates to application of a gRNA (guide Ribonucleic Acid) sequence based on a CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9) system on knockout of a human Lin28A gene and application to tumor treatment. According to a design principle of CRISPR/Cas9, 6 guide RNAs (gRNA) are designed and a sequence table is shown as SEQ ID NO.1 to SEQ ID NO.6; the guide RNAs are constructed on a PX458 carrier and two efficient target gRNAs are obtained through activity detection and screening. The CRISPR/Cas9 system guided by the two gRNAs is utilized in human hepatocellular carcinoma cell lines (HepG2) and human melanoma cell lines (A375), and the human Lin28A gene can be effectively knocked out; the system is easy to operate and the human Lin28A gene knockout efficiency is high, so that the system is applicable to various tumor cell models. The gRNA provided by the invention is hopefully applied to a new drug for treating tumors.

The invention discloses a CRISPR-Cas9 system used for assembling DNA and a DNA assembly method. The CRISPR-Cas9 system includes the following parts: a plasmid used for expressing Cas9 gene, a first guide RNA, and/or a plasmid used for expressing the first guide RNA, wherein the first guide RNA has a CRISPR site. The CRISPR site is combined with a first reporter gene carried by a semisynthetic chromosome used for assembling according to base complementation pairing rule. The CRISPR-Cas9 system has advantages of high replacement success rate of homologous recombination, less species of guide RNA which needs to design and use, less harmful effect subjected by the genomic sequence, and low off-target rate.

The invention relates to the field of microorganisms, in particular to a site-directed mutation method for genomes of saccharomyces cerevisiae. To-be-repaired single-base mutation sites on the genomes of the saccharomyces cerevisiae can be repaired by the aid of CRISPR/Cas9 technologies. Effects can be realized near the to-be-repaired sites by Cas9 proteins under the guidance actions of guide RNA (ribonucleic acid) if the mutation sites are positioned in PAM sequences (NGG) or front 11bp of the PAM sequences, and the genomes of the saccharomyces cerevisiae can be subjected to double-strand break at cut positions, so that donor DNA (deoxyribonucleic acid) can be efficiently recombined. Compared with existing results, the site-directed mutation method has the advantages that the sites of the genomes of the saccharomyces cerevisiae can be efficiently and quickly mutated, and screening markers do not need to be integrated for the genomes of the saccharomyces cerevisiae.

The invention relates to the technical field of genetic engineering, and particularly relates to a guide RNA (gRNA) sequence based on a CRISPR/Cas9 system and combinations thereof, as well as gRNA for a specific targeted knockout hepatitis B virus cccDNA P gene. In the invention, 30 gRNAs are designed according to design principles of CRISPR/Cas9, have a sequence table as shown in SEQ ID NO.1-30, and are constructed on a PX458 vector, to screen out four more efficient gRNAs. The CRISPR/Cas9 system guided by the four gRNAs and combinations thereof is utilized in a human hepatocellular carcinoma cell line (HepG2.2.15) to effectively knock out the human hepatitis B virus cccDNA P gene. The gRNA of the specific targeted hepatitis B virus cccDNA prepared in the invention can accurately target the hepatitis B virus cccDNA and realize gene knockout. The preparation method is simple in operation; the gRNA has a good targeting property; the CRISPR/Cas9 system is high in knockout efficiency.

The invention provides a genome editing method based on a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) system and belongs to the technical field of gene editing. The genome editing method provided by the invention utilizes introns to express guide RNA molecules in the CRISPR genome editing system and adopts a principle that one or more than one tRNA-gRNA or crRNA series unitsare arranged in the introns of an encoded gene, and after the introns form a fusion gene with Cas9 or Cpf1, a promoter can be used for driving. The genome editing method provided by the invention hasthe benefits that a cellular endogenous mRNA splicing system and a cellular endogenous tRNA processing system are skillfully utilized, and other elements are not required to be added, so that not onlyis the safety higher, but also more simplicity can be realized; the synchronous expression of a plurality of guide RNAs with the Cas9 or the Cpf1 is realized by utilizing the one promoter, so that anexisting CRISPR editing system is simplified, moreover, the efficiency and the ability of simultaneously editing multiple target points of the CRISPR editing system are improved.

The present disclosure provides CasX proteins, nucleic acids encoding the CasX proteins, and modified host cells comprising the CasX proteins and/or nucleic acids encoding same. CasX proteins are useful in a variety of applications, which are provided. The present disclosure provides CasX guide RNAs that bind to and provide sequence specificity to the CasX proteins, nucleic acids encoding the CasX guide RNAs, and modified host cells comprising the CasX guide RNAs and/or nucleic acids encoding same. CasX guide RNAs are useful in a variety of applications, which are provided. The present disclosure provides archaeal Cas9 polypeptides and nucleic acids encoding same, as well as their associated archaeal Cas9 guide RNAs and nucleic acids encoding same.

The invention provides specific synthetic chimeric xenonucleic acid guide RNA; s(XNA-gRNA) for enhancing crispr mediated genome editing efficiency. The invention also provides methods and compositions for inducing CRISPR/Cas-based gene editing/regulation (e.g., genome editing or gene expression) of a target nucleic acid (e.g., target DNA or target RNA) in a cell. The methods include using single guide RNAs (sgRNAs) that have been chemically modified with xeno nucleic acids which enhance gene regulation of the target nucleic acid in a primary cell for use in ex vivo therapy or in a cell in a subject for use in in vivo therapy. Additionally, provided herein are methods for preventing or treating a genetic disease in a subject by administering a sufficient amount of a sgRNA that has been chemically modified with xeno nucleic acids to correct a mutation in a target gene associated with the genetic disease.

This invention relates to reagents and methods for increasing specificity and efficiency of genome editing by CRISPR associated (Cas) protein systems, more particularly by Cas:guide RNA complexes, by blocking off-target nucleic acids from cleavage by Cas:guide RNA complexes.

The invention relates to a corn transcription factor ZmbHLH167 and an application of the corn transcription factor ZmbHLH167. A base sequence of the gene is shown as SEQ ID NO (sequence identifier number): 1. A corn immature embryo is transformed by taking a gene segment SEQ ID NO: 2 of the sequential coding protein ZmbHLH167 as guide RNA (ribonucleic acid) by use of a CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 technology, and a plant of a gene deletion mutant is obtained. Compared with a wild seed, a corn seed of the genetically modified mutant is smaller significantly, but a germination rate is not influenced. Biochemical analysis shows that a starch content of the corn seed of the genetically modified mutant is decreased obviously, a content of protein and total oil and fat is significantly increased, and a genetic resource is provided for creating high quality corn.

The invention provides a CRISPR-Cas9 system and an application thereof in treating breast cancer diseases, wherein by providing a plurality of sgRNAs (single guide RNAs), the efficient knockout of an RecQL4 gene can be achieved. In comparison with ZFNs and TALENs complex expression structures, siRNA is low in using complexity and efficiency; a Cas9 expression structure in the CRISPR-Cas9 system is fixed; for different genes, system building can be completed by interpolating recognition sequences into the sgRNA expression structure, so that operations are simplified and cost is reduced; and the system is applicable to mammal gene targeting in a large scale.