Composition for cleaving a target DNA comprising a guide RNA specific for the target DNA and cas protein-encoding nucleic acid or cas protein, and use thereof

a technology of target dna and guide rna, which is applied in the direction of hydrolases, enzymes, biochemical apparatus and processes, etc., can solve the problems of inability to develop a genome editing method using the rna-guided endonuclease system based on crispr/cas, the rflp limit is limited, and the enzymes tend to underestimate the mutation frequency

Inactive Publication Date: 2015-10-08
TOOLGEN INC
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Benefits of technology

[0039]The present composition for cleaving a target DNA or inducing a targeted mutagenesis in eukaryotic cells or organisms, comprising a guide RNA specific for the target DNA and Cas protein-encoding nucleic acid or Cas protein, the kit comprising the composition, and the method for inducing targeted mutagenesis provide a new convenient genome ed...

Problems solved by technology

However, until now, a genome editing method using the RNA-guided endonuclease (RGEN) based on CRISPR/Cas system has not been developed.
Meanwhile, Restriction fragment length polymorphism (RFLP) is one of the oldest, most convenient, and least expensive methods of genotyping that is still used widely in molecular biology and genetics but is often limited by the lack of appropriate sites recogni...

Method used

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  • Composition for cleaving a target DNA comprising a guide RNA specific for the target DNA and cas protein-encoding nucleic acid or cas protein, and use thereof
  • Composition for cleaving a target DNA comprising a guide RNA specific for the target DNA and cas protein-encoding nucleic acid or cas protein, and use thereof
  • Composition for cleaving a target DNA comprising a guide RNA specific for the target DNA and cas protein-encoding nucleic acid or cas protein, and use thereof

Examples

Experimental program
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Effect test

example 1

Genome Editing Assay

[0160]1-1. DNA Cleavage Activity of Cas9 Protein

[0161]Firstly, the DNA cleavage activity of Cas9 derived from Streptococcus pyogenes in the presence or absence of a chimeric guide RNA in vitro was tested.

[0162]To this end, recombinant Cas9 protein that was expressed in and purified fromE. coli was used to cleave a predigested or circular plasmid DNA that contained the 23-base pair (bp) human CCR5 target sequence. A Cas9 target sequence consists of a 20-bp DNA sequence complementary to crRNA or a chimeric guide RNA and the trinucleotide (5′-NGG-3′) protospacer adjacent motif (PAM) recognized by Cas9 itself (FIG. 1A).

[0163]Specifically, the Cas9-coding sequence (4,104 bp), derived from Streptococcus pyogenes strain M1 GAS (NC—002737.1), was reconstituted using the human codon usage table and synthesized using oligonucleotides. First, 1-kb DNA segments were assembled using overlapping ˜35-mer oligonucleotides and Phusion polymerase (New England Biolabs) and cloned i...

example 2

Proteinaceous RGEN-Mediated Genome Editing

[0185]RGENs can be delivered into cells in many different forms. RGENs consist of Cas9 protein, crRNA, and tracrRNA. The two RNAs can be fused to form a single-chain guide RNA (sgRNA). A plasmid that encodes Cas9 under a promoter such as CMV or CAG can be transfected into cells. crRNA, tracrRNA, or sgRNA can also be expressed in cells using plasmids that encode these RNAs. Use of plasmids, however, often results in integration of the whole or part of the plasmids in the host genome. The bacterial sequences incorporated in plasmid DNA can cause unwanted immune response in vivo. Cells transfected with plasmid for cell therapy or animals and plants derived from DNA-transfected cells must go through a costly and lengthy regulation procedure before market approval in most developed countries. Furthermore, plasmid DNA can persist in cells for several days post-transfection, aggravating off-target effects of RGENs.

[0186]Here, we used recombinant Ca...

example 3

RNA-Guided Genome Editing in Mice

[0189]To examine the gene-targeting potential of RGENs in pronuclear (PN)-stage mouse embryos, the forkhead box N1 (Foxn1) gene, which is important for thymus development and keratinocyte differentiation (Nehls et al., 1996), and the protein kinase, DNA activated, catalytic polypeptide (Prkdc) gene, which encodes an enzyme critical for DNA DSB repair and recombination (Taccioli et al., 1998) were used.

[0190]To evaluate the genome-editing activity of the Foxn1-RGEN, we injected Cas9 mRNA (10-ng / μl solution) with various doses of the sgRNA (FIG. 5a) into the cytoplasm of PN-stage mouse embryos, and conducted T7 endonuclease I (T7E1) assays (Kim et al. 2009) using genomic DNAs obtained from in vitro cultivated embryos (FIG. 6a).

[0191]Alternatively, we directly injected the RGEN in the form of recombinant Cas9 protein (0.3 to 30 ng / μl) complexed with the two-fold molar excess of Foxn1-specific sgRNA (0.14 to 14 ng / μl) into the cytoplasm or pronucleus of ...

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Abstract

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.

Description

TECHNICAL FIELD[0001]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.BACKGROUND ART[0002]CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) are loci containing multiple short direct repeats that are found in the genomes of approximately 40% of sequenced bacteria and 90% of sequenced archaea. CRISPR functions as a prokaryotic immune system, in that it confers resistance to exogenous genetic elements such as plasmids and phages. The CRISPR system provides a form of acquired immunity. Short segments of foreign DNA, called spacers, are incorporated into the genome between CRISPR repeats, and serve as a memory of past exposures. CRISPR spacers are then used to recognize and silence exoge...

Claims

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Application Information

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IPC IPC(8): C12N15/52C12N9/16
CPCC12N9/16C12N15/52C12N2310/531C12N15/8216C12Y301/21C12N9/22C12N15/102C12N15/63C12N15/111C12N2310/20C12N15/113C12N15/8509C12Q1/683C12N15/85C12N15/907C12N2310/10
Inventor KIM, JIN-SOOCHO, SEUNG WOOKIM, SOJUNGKIM, JONG MINKIM, SEOKJOONG
Owner TOOLGEN INC
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