RNA-Guided Human Genome Engineering
A method of altering a eukaryotic cell is provided including transfecting the eukaryotic cell with a nucleic acid encoding RNA complementary to genomic DNA of the eukaryotic cell, transfecting the eukaryotic cell with a nucleic acid encoding an enzyme that interacts with the RNA and cleaves the genomic DNA in a site specific manner, wherein the cell expresses the RNA and the enzyme, the RNA binds to complementary genomic DNA and the enzyme cleaves the genomic DNA in a site specific manner.
1 . A method of altering a eukaryotic cell comprising
providing to the eukaryotic cell a guide RNA sequence complementary to a target nucleic acid sequence,
providing to the eukaryotic cell a Cas9 enzyme that interacts with the guide RNA sequence and cleaves the target nucleic acid sequence in a site specific manner,
wherein the guide RNA sequence binds to the complementary target nucleic acid sequence and the Cas9 enzyme cleaves the target nucleic acid sequence in a site specific manner;
wherein the guide RNA sequence includes a guide sequence of GN 19 complementary to the target nucleic acid sequence and a scaffold sequence and wherein the guide RNA sequence is between about 100 to about 250 nucleotides.
2 . The method of claim 1 wherein the guide RNA is about 250 nucleotides.
3 . The method of claim 1 wherein the guide RNA is about 100 nucleotides.
4 . The method of claim 1 wherein the eukaryotic cell is a yeast cell, a plant cell or a mammalian cell.
5 . The method or claim 1 wherein the eukaryotic cell is a human cell.
6 . The method of claim 1 wherein a plurality of guide RNAs are provided to the eukaryotic cell that are complementary to different target nucleic acid sequences and the Cas9 enzyme cleaves the different target nucleic acid sequences in a site specific manner
7 . The method of claim 1
wherein the guide RNA is provided to the eukaryotic cell by introducing to the eukaryotic cell a nucleic acid encoding the guide RNA,
wherein the Cas9 enzyme is provided to the eukaryotic cell by introducing to the eukaryotic cell a nucleic acid encoding the Cas9 enzyme,
wherein the eukaryotic cell expresses the guide RNA and the Cas9 enzyme, the guide RNA binds to complementary target nucleic acid and the Cas9 enzyme cleaves the target nucleic acid in a site specific manner.
8 . A eukaryotic cell including
a nucleic acid encoding a guide RNA sequence complementary to a target nucleic acid sequence,
a nucleic acid encoding a Cas9 enzyme that interacts with the guide RNA sequence and cleaves the target nucleic acid sequence in a site specific manner,
wherein the eukaryotic cell expresses the guide RNA and the Cas9 enzyme, the guide RNA binds to the complementary target nucleic acid and the Cas9 enzyme cleaves the target nucleic acid in a site specific manner,
wherein the guide RNA sequence includes a guide sequence of GN 19 complementary to the target nucleic acid sequence and a scaffold sequence and wherein the guide RNA sequence is between about 100 to about 250 nucleotides.
9 . The eukaryotic cell of claim 8 wherein the guide RNA is about 250 nucleotides.
10 . The eukaryotic cell of claim 8 wherein the guide RNA is about 100 nucleotides.
11 . The eukaryotic cell of claim 8 wherein the eukaryotic cell is a yeast cell, a plant cell or a mammalian cell.
12 . The eukaryotic cell of claim 8 wherein the eukaryotic cell is a human cell.
13 . The eukaryotic cell of claim 8 further including a plurality of nucleic acids encoding a plurality of guide RNA sequences complementary to different target nucleic acid sequences.
14 . A method of altering a eukaryotic cell comprising
providing to the eukaryotic cell a guide RNA complementary to a target nucleic acid sequence,
providing to the eukaryotic cell a Cas9 enzyme that interacts with the guide RNA and cleaves the target nucleic acid sequence in a site specific manner,
wherein the guide RNA binds to the complementary target nucleic acid sequence and the Cas9 enzyme cleaves the target nucleic acid sequence in a site specific manner;
wherein the guide RNA includes a guide sequence complementary to the target nucleic acid sequence and a scaffold sequence connected to the guide sequence and having the following nucleic acid sequence and structure:
15 . The method of claim 14
wherein the guide RNA is provided to the eukaryotic cell by introducing to the eukaryotic cell a nucleic acid encoding the guide RNA,
wherein the Cas9 enzyme is provided to the eukaryotic cell by introducing to the eukaryotic cell a nucleic acid encoding the Cas9 enzyme,
wherein the eukaryotic cell expresses the guide RNA and the Cas9 enzyme, the guide RNA binds to complementary target nucleic acid and the Cas9 enzyme cleaves the target nucleic acid in a site specific manner.
16 . The method of claim 14 wherein the eukaryotic cell is a yeast cell, a plant cell or a mammalian cell.
17 . The method or claim 14 wherein the eukaryotic cell is a human cell.
18 . The method of claim 14 wherein a plurality of guide RNAs are provided to the eukaryotic cell that are complementary to different target nucleic acid sequences and the Cas9 enzyme cleaves the different target nucleic acid sequences in a site specific manner.
19 . A method of altering expression of a target nucleic acid sequence in a eukaryotic cell comprising
providing to the eukaryotic cell a guide RNA sequence complementary to the target nucleic acid sequence,
providing to the eukaryotic cell a Cas9 enzyme that interacts with the guide RNA sequence and cleaves the target nucleic acid sequence in a site specific manner,
wherein the guide RNA sequence binds to the complementary target nucleic acid sequence and the Cas9 enzyme cleaves the target nucleic acid sequence in a site specific manner whereby expression of the target nucleic acid sequence is altered;
wherein the guide RNA sequence includes a guide sequence of GN 19 complementary to the target nucleic acid sequence and a scaffold sequence and wherein the guide RNA sequence is between about 100 to about 250 nucleotides.
20 . The method of claim 19 wherein the guide RNA is about 250 nucleotides.
21 . The method of claim 19 wherein the guide RNA is about 100 nucleotides.
22 . The method of claim 19 wherein the eukaryotic cell is a yeast cell, a plant cell or a mammalian cell.
23 . The method or claim 19 wherein the eukaryotic cell is a human cell.
24 . The method of claim 19 wherein a plurality of guide RNAs are provided to the eukaryotic cell that are complementary to different target nucleic acid sequences and the Cas9 enzyme cleaves the different target nucleic acid sequences in a site specific manner.
25 . The method of claim 19
wherein the guide RNA is provided to the eukaryotic cell by introducing to the eukaryotic cell a nucleic acid encoding the guide RNA,
wherein the Cas9 enzyme is provided to the eukaryotic cell by introducing to the eukaryotic cell a nucleic acid encoding the Cas9 enzyme,
wherein the eukaryotic cell expresses the guide RNA and the Cas9 enzyme, the guide RNA binds to complementary target nucleic acid and the Cas9 enzyme cleaves the target nucleic acid in a site specific manner.