IP Library › Patent Application 18313946
Patent Application
App. No. 18/313,946

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.
18/313,946
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 (16)

1 - 57 . (canceled)

58 . A composition for inducing targeted disruption of endogenous genes in a eukaryotic cell, the composition comprising a Cas9/RNA complex comprising:

a Cas9 protein linked to one or more tagging sequences, wherein the tagging sequence comprises a nuclear localization signal (NLS); and

a guide RNA having a CRISPR RNA (crRNA) and a transactivating crRNA (tracrRNA), wherein the crRNA comprises: i) a portion to be hybridized with a portion of the tracrRNA, and ii) a portion complementary to a target DNA of the endogenous genes,

wherein the ribonucleoprotein functions as an endonuclease that induces targeted disruption of the target DNA.

59 . The composition of claim 58 , wherein the Cas9 protein is complexed with the guide RNA to form a ribonucleoprotein before being introduced into the eukaryotic cell.

60 . The composition of claim 58 , wherein the guide RNA is one of (a) a dual RNA and (b) a single-chain guide RNA (sgRNA) comprising the crRNA portion fused to the tracrRNA portion.

61 . The composition of claim 58 , wherein the Cas9 protein has the NLS in proximity to its N-terminal, its C-terminal, or both its N-terminal and C-terminal

62 . The composition of claim 58 , wherein the Cas9 protein is derived from S. pyogene.

63 . The composition of claim 58 , wherein the tagging sequence further comprises a peptide tag.

64 . The composition of claim 63 , wherein the peptide tag is a HA-tag.

65 . The composition of claim 63 , wherein the peptide tag is a His-tag.

66 . The composition of claim 58 , wherein the Cas9 protein is expressed in and purified from a bacterial system for a transgene-free use to avoid the host genome integration.

67 . The composition of claim 58 , wherein the guide RNA is transcribed in vitro.

68 . The composition of claim 58 , wherein the guide RNA is chemically synthesized.

69 . The composition of claim 58 , wherein the target DNA comprises a first strand having a region complementary to the crRNA and a second strand having a trinucleotide protospacer adjacent motif (PAM), wherein the PAM consists of the trinucleotide 5′-NGG-3′.