Compositions and methods for in vivo excision of hiv-1 proviral dna

Abstract

Methods and kits for excising HIV-1 DNA in vivo are provided, which employ Clustered Regularly Interspaced Short Palindromic Repeats (CRIS-PR) and CRISPR-Associated (cas) proteins. Vectors harboring nucleic acids encoding one or more guide RNA, wherein said guide RNA hybridizes with a target

1 - 11 . (canceled) 
     
     
         12 . A method for cleaving a target human immunodeficiency virus 1 (HIV-1) DNA sequence in a eukaryotic cell comprising contacting a eukaryotic cell comprising a cellular genome and harboring the target HIV-1 DNA sequence integrated into the cellular genome with
 (a) one or more guide RNA, or nucleic acids encoding said one or more guide RNA, and   (b) a Clustered Regularly Interspaced Short Palindromic Repeats-Associated 9 (cas9) protein, or nucleic acids encoding said cas9 protein, wherein said one or more guide RNA matches said target HIV-1 DNA sequence and uniquely hybridizes to a complementary sequence of the target HIV-1 DNA sequence, thereby cleaving said target HIV-1 DNA sequence, wherein said target HIV-1 DNA sequence is selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:6.   
     
     
         13 . The method of  claim 12 , wherein the cas protein has been codon-optimized for expression in human cells. 
     
     
         14 . The method of  claim 12 , wherein the cas protein further comprises a nuclear localization sequence. 
     
     
         15 . The method of  claim 12 , wherein said target HIV-1 LTR sequence is SEQ ID NO:3, and wherein said one or more guide RNA, or nucleic acids encoding said one or more guide RNA comprise the sequence of SEQ ID NO:3, or the complement thereof 
     
     
         16 . The method of  claim 12 , wherein said target HIV-1 LTR sequence is SEQ ID NO:4, and wherein said one or more guide RNA, or nucleic acids encoding said one or more guide RNA comprise the sequence of SEQ ID NO:4, or the complement thereof 
     
     
         17 . The method of  claim 12 , wherein said target HIV-1 LTR sequence is SEQ ID NO:6, and wherein said one or more guide RNA, or nucleic acids encoding said one or more guide RNA comprise the sequence of SEQ ID NO:6, or the complement thereof. 
     
     
         18 . The method of  claim 12 , wherein said one or more guide RNA and said cas9 protein form a complex inside the eukaryotic cell, and wherein said complex cleaves the target HIV-1 DNA sequence. 
     
     
         19 . The method of  claim 12 , wherein the cas protein has been codon-optimized for expression in human cells. 
     
     
         20 . The method of  claim 13 , wherein the cas protein further comprises a nuclear localization sequence. 
     
     
         21 . The method of  claim 12 , wherein the nucleic acids encoding the one or more guide RNA, and the nucleic acids encoding the cas protein are contained in a viral vector, and wherein contacting the eukaryotic cell comprises contacting with said viral vector. 
     
     
         22 . The method of  claim 21 , wherein the contacting step is carried out in vitro. 
     
     
         23 . The method of  claim 12 , further comprising contacting the eukaryotic cell with one or more guide RNA, or nucleic acids encoding a guide RNA, wherein the guide RNA or nucleic acid encoding a guide RNA matches said target HIV-1 DNA sequence and uniquely hybridizes to a complementary sequence of the target HIV-1 DNA sequence, wherein said target HIV-1 DNA sequence is selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9. 
     
     
         24 . A kit comprising
 (a) one or more guide RNAs, or nucleic acids encoding said one or more guide RNAs, wherein said guide RNAs uniquely hybridizes with a target HIV-1 DNA sequence, wherein said target HIV-1 DNA sequence is selected from the group consisting of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:4; and   (b) a Clustered Regularly Interspaced Short Palindromic Repeats-Associated 9 (cas9) protein, or nucleic acids encoding said cas9 protein.   
     
     
         25 . The kit according to  claim 24 , wherein the cas protein is cas9. 
     
     
         26 . The kit according to  claim 24 , wherein the cas protein has been codon-optimized for expression in human cells. 
     
     
         27 . The kit according to  claim 24 , wherein the cas protein further comprises a nuclear localization sequence. 
     
     
         28 . A vector comprising nucleic acids encoding one or more guide RNAs, wherein said guide RNA uniquely hybridizes with a target HIV-1 DNA sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:6.