Methods for cleaving a target DNA using a guide RNA specific for the target DNA and Cas protein-encoding nucleic acid or Cas protein

• US 10,851,380 B2
• Filed: 04/13/2015
• Issued: 12/01/2020
• Est. Priority Date: 10/23/2012
• Status: Active Grant

• Current Assignee: Toolgen Incorporated
• Original Assignee: Toolgen Incorporated
• Inventors: Kim, Jin-Soo, Cho, Seung Woo, Kim, Sojung
• Primary Examiner(s): Hibbert, Catherine S
• Application Number:
  US14/685,568
• Publication Number:
  US 20150322457A1
• Time in Patent Office: 2,059 Days
• Field of Search: None
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 15/8216 Methods for controlling, regulating or enhancing expression of transgenes in plant cells
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2310/10 Type of nucleic acid
• CPC Class Codes: C12N 2310/20 involving clustered regularly interspaced short palindromic repeats [CRISPRs]
• CPC Class Codes: C12N 2310/531 Stem-loop; Hairpin
• CPC Class Codes: C12N 9/16 acting on ester bonds (3.1)
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Y 301/21 Endodeoxyribonucleases producing 5'-phosphomonoesters (3.1.21)

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.

Claims

1. A method of introducing a site-specific, double-stranded break at a target nucleic acid sequence in a eukaryotic cell, the method comprising introducing into the eukaryotic cell a Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas system, wherein the CRISPR/Cas system comprises:

a) a nucleic acid encoding a Cas9 polypeptide comprising a nuclear localization signal, wherein the nucleic acid is codon-optimized for expression in eukaryotic cells, and

b) a guide RNA that hybridizes to the target nucleic acid, wherein the guide RNA is a chimeric guide RNA comprising a CRISPR RNA (crRNA) portion fused to a trans activating crRNA (tracrRNA) portion, wherein the guide RNA comprises two guanines at its 5′

end, and there are no additional nucleic acid residues between the two guanines at the 5′

end and the crRNA portion of the guide RNA;

whereby a site-specific, double stranded break at the target nucleic acid sequence is introduced.

2. The method of claim 1, wherein the nuclear localization signal is located at the C terminus of the Cas9 polypeptide.

3. The method of claim 1, wherein the eukaryotic cell is a mammalian cell.

4. The method of claim 3, wherein the mammalian cell is a human cell.

5. The method of claim 1, wherein the nucleic acid encoding the Cas 9 polypeptide is codon-optimized for expression in mammalian cells.

6. The method of claim 1, wherein the target nucleic acid sequence is a genomic sequence located at its endogenous site in the genome of the eukaryotic cell.

7. The method of claim 1, wherein the nucleic acid encoding the Cas9 polypeptide is a vector.

8. The method of claim 1, wherein the Cas9 polypeptide is a Streptococcus pyogenes Cas9 polypeptide.

9. The method of claim 1, wherein the nucleic acid encoding the Cas9 polypeptide is introduced into the eukaryotic cell before introducing the guide RNA into the eukaryotic cell.

10. The method of claim 1, wherein the Cas9 polypeptide is a Streptococcus Cas9 polypeptide.