Compositions for the inactivation of virus replication and methods of making and using the same

Abstract

Provided herein are recombinant constructs, vectors and expression cassettes including a first promoter which is suitably a tRNA promoter operably connected to a first polynucleotide encoding a first single guide RNA and a second promoter operably connected to a second polynucleotide encoding a Cas9 polypeptide. The first single guide RNA includes a first portion complementary to a strand of a target sequence of a DNA virus and a second portion capable of interacting with the Cas9 polypeptide. Also provided are codon optimized Staphylococcus aureus derived Cas9 polynucleotides and polypeptides with nuclear localization signals and optionally an epitope tag. Also provided are constructs for production of sgRNAs including a tRNA. Methods of inhibiting viral replication, inhibiting expression of a target sequence from a virus or treating a viral infection or viral induced cancer using the compositions are also provided.

1 .- 57 . (canceled) 
     
     
         58 . A recombinant construct for expression of a single guide RNA comprising a first polynucleotide encoding a mammalian or viral tRNA operably connected to a second polynucleotide encoding at least a second portion of a single guide RNA capable of interacting with a Cas9 polypeptide. 
     
     
         59 . The construct of  claim 58 , wherein the first polynucleotide is selected from SEQ ID NO: 41-50. 
     
     
         60 . The construct of  claim 58 , wherein the second polynucleotide further encodes a first portion of the single guide RNA complementary to a target sequence 5′ to the second portion capable of interacting with the Cas9 polypeptide. 
     
     
         61 . The construct of  claim 58 , wherein the second polynucleotide further encodes a first portion of the single guide RNA having one or more recognition site for a restriction endonuclease. 
     
     
         62 . The construct of  claim 58 , further comprising a promoter operably connected to a third polynucleotide encoding a Cas9 polypeptide. 
     
     
         63 .- 65 . (canceled) 
     
     
         66 . The recombinant construct of  claim 58 , wherein the first polynucleotide comprises a tRNA selected from the group consisting of a Gln tRNA, Pro tRNA, Gly tRNA, Asn tRNA, Cys tRNA, Glu tRNA, and a mouse gamma herpesvirus-68 (MHV68) tRNA. 
     
     
         67 . The recombinant construct of  claim 58 , further comprising a first inverted terminal repeat and a second inverted terminal repeat, wherein the inverted terminal repeats flank the construct for packaging in an adeno-associated virus (AAV) vector. 
     
     
         68 . The recombinant construct of  claim 62 , wherein the Cas9 polypeptide is selected from the Cas9 polypeptides from  S. pyogenes, S. aureus,  or nickase Cas9 derivatives thereof. 
     
     
         69 . The recombinant construct of  claim 68 , wherein the  S. aureus  Cas9 (Sau Cas9) polypeptide having SEQ ID NO: 57 is selected. 
     
     
         70 . The recombinant construct of  claim 68 , wherein the Sau Cas9 polypeptide is encoded by a polynucleotide comprising SEQ ID NO: 55. 
     
     
         71 . The recombinant construct of  claim 62 , further comprising a nuclear localization signal operably linked to the Cas9 polypeptide. 
     
     
         72 . The recombinant construct of  claim 58 , further comprising an intron. 
     
     
         73 . The recombinant construct of  claim 62 , further comprising a poly(A) addition site linked to the third polynucleotide encoding the Cas9 polypeptide. 
     
     
         74 . The recombinant construct of  claim 58 , further comprising a polynucleotide encoding a second mammalian or viral tRNA operably connected to a second single guide RNA comprising a first portion complementary to a strand of a second target sequence and a second portion of a single guide RNA capable of interacting with a Cas9 polypeptide. 
     
     
         75 . The recombinant construct of  claim 74 , wherein the first target sequence and the second target sequence are located within 30 base pairs of each other on opposite strands of the DNA in a target. 
     
     
         76 . A recombinant vector comprising the construct of  claim 58 . 
     
     
         77 . The recombinant vector of  claim 76 , wherein the vector is a viral vector. 
     
     
         78 . The recombinant vector of  claim 77 , wherein the viral vector is selected from the group consisting of a retrovirus, a lentivirus, an adenovirus or an adeno-associated virus. 
     
     
         79 . The recombinant vector of  claim 78 , wherein the vector is an adeno-associated virus (AAV).