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
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.
1 - 57 . (canceled)
58 . A composition for cleaving a target nucleic acid sequence in a mammalian cell, the composition comprising a Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas system in the mammalian cell, wherein the Type II CRISPR/Cas system comprises:
a) a nucleic acid encoding a Cas9 polypeptide, wherein the Cas9 polypeptide comprises a nuclear localization signal, and
b) a chimeric guide RNA comprising a CRISPR RNA (crRNA) portion fused to a trans activating crRNA (tracrRNA) portion,
wherein the Cas9 polypeptide and the chimeric guide RNA form a Cas9/RNA complex in the mammalian cell and wherein the crRNA portion has sufficient sequence complementarity to the target nucleic acid sequence in the mammalian cell to allow the Cas9 polypeptide to mediate double stranded cleavage at the target nucleic acid sequence.
59 . The composition of claim 58 , wherein the nuclear localization signal is located at the C terminus of the Cas9 polypeptide.
60 . The composition of claim 58 , wherein the mammalian cell is a human cell.
61 . The composition of claim 58 , wherein the nucleic acid encoding the Cas 9 polypeptide is codon-optimized for expression in mammalian cells.
62 . The composition of claim 58 , wherein the chimeric guide RNA is in vitro transcribed RNA.
63 . The composition of claim 58 , wherein the target nucleic acid sequence is a genomic sequence located at its endogenous site in the genome of the mammalian cell.
64 . The composition of claim 58 , wherein the Cas9 polypeptide is a Streptococcus pyogenes Cas9 polypeptide.
65 . The composition of claim 58 , wherein the target nucleic acid sequence consists of 20 nucleotides complementary to the crRNA portion of the chimeric guide RNA and a trinucleotide protospacer adjacent motif (PAM), and wherein the PAM consists of the trinucleotide 5′-NGG-3′.
66 . A method of introducing a site-specific, double-stranded break at a target nucleic acid sequence in a mammalian cell, the method comprising introducing into the mammalian 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, wherein the Cas9 polypeptide comprises a nuclear localization signal, and
b) a chimeric guide RNA comprising a CRISPR RNA (crRNA) portion fused to a trans activating crRNA (tracrRNA) portion,
wherein the Cas9 polypeptide and the chimeric guide RNA form a Cas9/RNA complex in the mammalian cell and wherein the crRNA portion has sufficient sequence complementarity to the target nucleic acid sequence in the mammalian cell to allow the Cas9 polypeptide to mediate double stranded cleavage at the target sequence.
67 . The method of claim 66 , wherein the nuclear localization signal is located at the C terminus of the Cas9 polypeptide.
68 . The method of claim 66 , wherein the mammalian cell is a human cell.
69 . The method of claim 66 , wherein the nucleic acid encoding the Cas 9 polypeptide is codon-optimized for expression in mammalian cells.
70 . The method of claim 66 , wherein the target nucleic acid sequence is a genomic sequence located at its endogenous site in the genome of the mammalian cell.
71 . The method of claim 66 , wherein the chimeric guide RNA is transcribed in vitro before introduction into the mammalian cell.
72 . The method of claim 66 , wherein the Cas9 polypeptide is a Streptococcus pyogenes Cas9 polypeptide.
73 . The method of claim 66 , wherein the target nucleic acid sequence consists of 20 nucleotides complementary to the crRNA portion of the chimeric guide RNA and a trinucleotide protospacer adjacent motif (PAM), wherein the PAM consists of the trinucleotide 5′-NGG-3′.
74 . The method of claim 66 , wherein the nucleic acid encoding the Cas9 protein is introduced into the mammalian cell before introducing the chimeric guide RNA into the mammalian cell.
75 . A Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas system for site-specific, double stranded cleavage of a target nucleic acid sequence in a mammalian cell, the CRISPR/Cas system comprising:
a) a Cas9 polypeptide, wherein the Cas9 polypeptide comprises a nuclear localization signal, and
b) a chimeric guide RNA comprising a CRISPR RNA (crRNA) portion and a trans-activating crRNA (tracrRNA) portion,
wherein the Cas9 polypeptide and the chimeric guide RNA form a Cas9/RNA complex in the mammalian cell and wherein the crRNA portion has sufficient sequence complementarity to the target nucleic acid sequence in the mammalian cell to allow the Cas9 polypeptide to mediate double stranded cleavage at the target sequence.
76 . The CRISPR/Cas system of claim 75 , wherein the nuclear localization signal is located at the C terminus of the Cas9 polypeptide.
77 . The CRISPR/Cas system of claim 75 , wherein the mammalian cell is a human cell.
78 . The CRISPR/Cas system of claim 75 , wherein the nucleic acid encoding the Cas 9 polypeptide is codon-optimized for expression in mammalian cells.
79 . The CRISPR/Cas system of claim 75 , wherein the chimeric guide RNA is in vitro transcribed RNA.
80 . The CRISPR/Cas system of claim 75 , wherein the target nucleic acid sequence is a genomic sequence located at its endogenous site in the genome of the mammalian cell.
81 . The CRISPR/Cas system of claim 75 , wherein the Cas9 polypeptide is a Streptococcus pyogenes Cas9 polypeptide.