IP Library › Patent Application 17004338
Patent Application
App. No. 17/004,338

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

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Quick Facts
Patent No.
US None
App. No.
17/004,338
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 (18)

1 - 57 . (canceled)

58 . 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 complex, wherein the CRISPR/Cas complex 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,

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

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

a) a Cas9 polypeptide comprising a nuclear localization signal, 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,

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

60 . The method of claim 58 or 59 , wherein the nuclear localization signal is located at the C terminus of the Cas9 polypeptide.

61 . The method of claim 58 or 59 , wherein the eukaryotic cell is a mammalian cell.

62 . The method of claim 61 , wherein the mammalian cell is a human cell.

63 . The method of claim 58 or 59 , wherein the target nucleic acid sequence is a genomic sequence located at its endogenous site in the genome of the eukaryotic cell.

64 . The method of claim 58 , wherein the nucleic acid encoding the Cas9 polypeptide is a vector.

65 . The method of claim 58 or 59 , wherein the Cas9 polypeptide is a Streptococcus Cas9 polypeptide.

66 . The method of claim 65 , wherein the Cas9 polypeptide is a Streptococcus pyogenes Cas9 polypeptide.

67 . The method of claim 58 or 59 , wherein the nucleic acid encoding the Cas9 polypeptide is introduced into the eukaryotic cell before introducing the guide RNA into the eukaryotic cell.

68 . The method of claim 58 or 59 , wherein the target DNA sequence comprises a first strand having a region complementary to the crRNA portion of the chimeric guide RNA and a second strand having a trinucleotide protospacer adjacent motif (PAM), wherein the PAM consists of the trinucleotide 5′-NGG-3′.