RNA-Guided Systems for In Vivo Gene Editing
First Claim
1. A method of producing an altered gene product in a eukaryotic cell comprisingproviding to the cell two or more guide RNAs and a Cas9 protein,wherein the two or more guide RNAs are complementary to two or more target genomic DNA sequences flanking a target excision sequence including one or more exons in a target gene encoding a biologically functional polypeptide,wherein the two or more guide RNAs bind to the two or more complementary target genomic DNA sequences and the Cas9 protein cleaves the two or more target genomic DNA sequences thereby removing the one or more exons from the target gene to produce an altered target gene and wherein the altered target gene recombines, and wherein the eukaryotic cell expresses the altered target gene to produce an altered biologically functional polypeptide.
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Accused Products
Abstract
Methods of editing target nucleic acids are provided using a guide RNA and a Cas9 protein to excise exons in a target gene and where the edited gene is expressed to produce a truncated polypeptide.
6 Citations
72 Claims
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1. A method of producing an altered gene product in a eukaryotic cell comprising
providing to the cell two or more guide RNAs and a Cas9 protein, wherein the two or more guide RNAs are complementary to two or more target genomic DNA sequences flanking a target excision sequence including one or more exons in a target gene encoding a biologically functional polypeptide, wherein the two or more guide RNAs bind to the two or more complementary target genomic DNA sequences and the Cas9 protein cleaves the two or more target genomic DNA sequences thereby removing the one or more exons from the target gene to produce an altered target gene and wherein the altered target gene recombines, and wherein the eukaryotic cell expresses the altered target gene to produce an altered biologically functional polypeptide.
- 36. A skeletal muscle cell including a Cas9 protein and two or more guide RNAs complementary to two or more target genomic DNA sequences flanking a target excision sequence including one or more exons in a target gene encoding dystrophin protein.
- 39. A skeletal muscle cell including a first nucleic acid encoding two or more guide RNAs complementary to two or more target genomic DNA sequences flanking a target excision sequence including one or more exons in a target gene encoding dystrophin protein and a second nucleic acid encoding a Cas9 protein.
- 46. A skeletal muscle cell including a Cas9 protein and two or more guide RNAs complementary to two or more target genomic DNA sequences flanking a target excision sequence including one or more exons in a target gene encoding dystrophin protein.
- 49. A muscle stem cell including a first nucleic acid encoding two or more guide RNAs complementary to two or more target genomic DNA sequences flanking a target excision sequence including one or more exons in a target gene encoding dystrophin protein and a second nucleic acid encoding a Cas9 protein.
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56. A genetically modified skeletal muscle cell including a first nucleic acid encoding two or more guide RNAs complementary to two or more target genomic DNA sequences flanking a target excision sequence including one or more exons in a target gene encoding dystrophin protein and a second nucleic acid encoding a Cas9 protein and wherein the target gene encoding dystrophin protein lacks one or more of exon 23, exon 52 or exon 53.
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57. A genetically modified muscle stem cell including a first nucleic acid encoding two or more guide RNAs complementary to two or more target genomic DNA sequences flanking a target excision sequence including one or more exons in a target gene encoding dystrophin protein and a second nucleic acid encoding a Cas9 protein and wherein the target gene encoding dystrophin protein lacks one or more of exon 23, exon 52 or exon 53.
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58. A method of producing an altered gene product in a eukaryotic cell within a mammal comprising
injecting two plasmids into the mammal, wherein the two plasmids include a first nucleic acid encoding two or more guide RNAs complementary to two or more target genomic DNA sequences flanking a target excision sequence including one or more exons in a target gene encoding dystrophin protein and a second nucleic acid encoding a Cas9 protein, wherein the two or more guide RNAs bind to the two or more complementary target genomic DNA sequences and the Cas9 protein cleaves the two or more target genomic DNA sequences thereby removing the one or more exons from the target gene to produce an altered target gene and wherein the altered target gene recombines, and wherein the eukaryotic cell expresses the altered target gene to produce an altered biologically functional polypeptide.
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63. A method of removing one or more mutations from a target gene encoding a dystrophin protein in a eukaryotic cell comprising
providing to the cell two or more guide RNAs and a Cas9 protein, wherein the two or more guide RNAs are complementary to two or more target genomic DNA sequences flanking a target excision sequence including one or more exons having one or more mutations in the target gene, wherein the two or more guide RNAs bind to the two or more complementary target genomic DNA sequences and the Cas9 protein cleaves the two or more target genomic DNA sequences thereby removing the one or more exons having one or more mutations from the target gene to produce an altered target gene and wherein the altered target gene recombines, and wherein the eukaryotic cell expresses the altered target gene to produce a functional truncated dystrophin protein.
Specification