RNA-Guided Systems for In Vivo Gene Editing

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.

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.   
     
     
         2 . The method of  claim 1  wherein the altered biologically functional polypeptide lacks a polypeptide sequence corresponding to the one or more removed exons. 
     
     
         3 . The method of  claim 1  wherein the two or more guide RNAs and the Cas9 protein are foreign to the eukaryotic cell. 
     
     
         4 . The method of  claim 1  wherein the two or more guide RNAs and the Cas9 protein are foreign to each other. 
     
     
         5 . The method of  claim 1  wherein the two or more guide RNAs and the Cas9 protein are non-naturally occurring. 
     
     
         6 . The method of  claim 1  wherein the two or more guide RNAs are provided to the cell by electroporation of the two or more guide RNAs into the cell. 
     
     
         7 . The method of  claim 1  wherein the Cas9 protein is provided to the cell by electroporation of the Cas9 protein into the cell. 
     
     
         8 . The method of  claim 1  wherein the two or more guide RNAs are provided to the cell by introducing into the cell a first foreign nucleic acid sequence encoding the two or more guide RNAs. 
     
     
         9 . The method of  claim 1  wherein the two or more guide RNAs are provided to the cell by introducing into the cell a first foreign nucleic acid sequence encoding the two or more guide RNAs present in a plasmid or vector. 
     
     
         10 . The method of  claim 1  wherein the Cas 9 protein is provided to the cell by introducing into the cell a second foreign nucleic acid sequence encoding the Cas 9 protein. 
     
     
         11 . The method of  claim 1  wherein the Cas 9 protein is provided to the cell by introducing into the cell a second foreign nucleic acid sequence encoding the Cas 9 protein present in a plasmid or vector. 
     
     
         12 . The method of  claim 1  wherein the eukaryotic cell is a yeast cell, a plant cell, a vertebrate cell, a mammalian cell or a human cell. 
     
     
         13 . The method of  claim 1  wherein the eukaryotic cell is within a mammal 
     
     
         14 . The method of  claim 1  wherein the eukaryotic cell is a skeletal muscle cell. 
     
     
         15 . The method of  claim 1  wherein the target excision sequence is greater than 45 kb. 
     
     
         16 . The method of  claim 1  wherein the target gene encodes dystrophin protein. 
     
     
         17 . The method of  claim 1  wherein the target gene encodes dystrophin protein and the one or more exons is exon 23. 
     
     
         18 . The method of  claim 1  wherein the target gene encodes dystrophin protein and the one or more exons is exon 52 and exon 53. 
     
     
         19 . The method of  claim 1  wherein the RNA includes between about 10 to about 250 nucleotides. 
     
     
         20 . The method of  claim 1  wherein the RNA includes between about 20 to about 100 nucleotides. 
     
     
         21 . The method of  claim 1  wherein the guide RNA includes a guide sequence fused to a trans-activating cr (tracr) sequence. 
     
     
         22 . The method of  claim 1  wherein the ratio of plasmid encoding the Cas9 protein to the plasmid encoding the guide RNA is between 1:5 and 2:1. 
     
     
         23 . The method of  claim 1  wherein the plasmid encoding the guide RNA is modified to increase the expression of the RNA by removing a potential premature transcription termination site. 
     
     
         24 . The method of  claim 1  wherein the one or more exons includes a mutation. 
     
     
         25 . The method of  claim 1  wherein the Cas9 protein is provided to the cell by electroporation of the Cas9 mRNA into the cell. 
     
     
         26 . The method of  claim 1  wherein the guide RNA and the Cas9 protein co-localize to the target genomic DNA sequence to form a complex. 
     
     
         27 . The method of  claim 1  wherein the target nucleic acid is chromosomal DNA. 
     
     
         28 . The method of  claim 1  wherein the Cas9 protein is wild type Cas9, Cas9 nickase or a nuclease null Cas9 including a nuclease. 
     
     
         29 . The method of  claim 1  wherein the guide RNA and the Cas9 protein are combined and then contacted with the target gene. 
     
     
         30 . The method of  claim 1  wherein the guide RNA and the Cas9 protein are combined and then contacted with the target gene within a cell. 
     
     
         31 . The method of  claim 1  comprising providing to the cell a plurality of guide RNAs with each having a portion complementary to a target genomic DNA sequence. 
     
     
         32 . The method of  claim 1  wherein the cell is a transplantable cell. 
     
     
         33 . The method of  claim 1  wherein the cell is a progenitor cell. 
     
     
         34 . The method of  claim 1  wherein the cell is a stem cell. 
     
     
         35 . The method of  claim 1  wherein the cell is a muscle stem cell. 
     
     
         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. 
     
     
         37 . The skeletal muscle cell of  claim 36  wherein the one or more exons are in the exon 45-55 region. 
     
     
         38 . The skeletal muscle cell of  claim 36  wherein the one or more exons include exon 23, exon 52 or exon 53. 
     
     
         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. 
     
     
         40 . The skeletal muscle cell of  claim 39  wherein the one or more exons are in the exon 45-55 region. 
     
     
         41 . The skeletal muscle cell of  claim 39  wherein the one or more exons include exon 23, exon 52 or exon 53. 
     
     
         42 . The skeletal muscle cell of  claim 39  wherein the first nucleic acid is within a plasmid or vector. 
     
     
         43 . The skeletal muscle cell of  claim 39  wherein the second nucleic acid is within a plasmid or vector. 
     
     
         44 . The skeletal muscle cell of  claim 39  wherein the second nucleic acid is within a viral vector. 
     
     
         45 . The skeletal muscle cell of  claim 39  wherein the second nucleic acid is within a viral vector selected from the group consisting of lentivirus, adenovirus, adeno-associated virus, retrovirus, herpes simplex virus, or sendai virus. 
     
     
         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. 
     
     
         47 . The skeletal muscle cell of  claim 46  wherein the one or more exons are in the exon 45-55 region. 
     
     
         48 . The skeletal muscle cell of  claim 46  wherein the one or more exons include exon 23, exon 52 or exon 53. 
     
     
         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. 
     
     
         50 . The muscle stem cell of  claim 49  wherein the one or more exons are in the exon 45-55 region. 
     
     
         51 . The muscle stem cell of  claim 49  wherein the one or more exons include exon 23, exon 52 or exon 53. 
     
     
         52 . The muscle stem cell of  claim 49  wherein the first nucleic acid is within a plasmid or vector. 
     
     
         53 . The muscle stem cell of  claim 49  wherein the second nucleic acid is within a plasmid or vector. 
     
     
         54 . The muscle stem cell of  claim 49  wherein the second nucleic acid is within a viral vector. 
     
     
         55 . The muscle stem cell of  claim 49  wherein the second nucleic acid is within a viral vector selected from the group consisting of lentivirus, adenovirus, adeno-associated virus, retrovirus, herpes simplex virus, or sendai virus. 
     
     
         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. 
     
     
         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. 
     
     
         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.   
     
     
         59 . The method of  claim 58  wherein the eukaryotic cell is a skeletal muscle cell. 
     
     
         60 . The method of  claim 58  wherein the eukaryotic cell is a muscle stem cell. 
     
     
         61 . The method of  claim 58  wherein the eukaryotic cell is a member of the group consisting of a skeletal muscle cell, a muscle stem cell, a progenitor cell and a stem cell. 
     
     
         62 . The method of  claim 58  wherein the one or more exons is exon 23, exon 52 or exon 53. 
     
     
         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.   
     
     
         64 . The method of  claim 63  wherein the eukaryotic cell is a skeletal muscle cell. 
     
     
         65 . The method of  claim 63  wherein the eukaryotic cell is a muscle stem cell. 
     
     
         66 . The method of  claim 63  wherein the eukaryotic cell is a member of the group consisting of a skeletal muscle cell, a muscle stem cell, a progenitor cell and a stem cell. 
     
     
         67 . The method of  claim 63  wherein the one or more exons is exon 23, exon 52 or exon 53. 
     
     
         68 . The method of  claim 63  wherein the eukaryotic cell is within a mammal 
     
     
         69 . The method of  claim 63  wherein the one or more exons are in the exon 45-55 region. 
     
     
         70 . The method of  claim 1  wherein the one or more exons are in the exon 45-55 region. 
     
     
         71 . The method of  claim 11  wherein the second nucleic acid is within a viral vector. 
     
     
         72 . The method of  claim 11  wherein the second nucleic acid is within a viral vector selected from the group consisting of lentivirus, adenovirus, adeno-associated virus, retrovirus, herpes simplex virus, or sendai virus.