Methods for screening and using CRISPR/Cas9 guidance RNA sequence from HIV provirus genome
View Patent ↗The invention pertains to compositions and methods for identifying gRNAs that are effective in treating a latent HIV infection. An embodiment of the invention provides a cell having incorporated into its genome: a gene that expresses a CRISPR-Cas protein and an HIV pseudovirus genome, wherein the HIV pseudovirus genome has a first marker gene encoding a first marker protein under the control of HIV-1 LTR promoter and a second marker gene inserted into the nef gene of the HIV pseudovirus and encoding a second marker protein under the control of a constitutive promoter. Screening methods for identifying gRNAs that can treat a latent HIV infection are also provided. Further, compositions comprising a CRISPR-Cas protein and gRNAs that can treat a latent HIV infection are provided. Furthermore, a method for treating a latent HIV infection in a subject by administering the compositions of CRISPR-Cas protein and gRNAs are described.
1. A composition comprising a CRISPR-Cas protein, one or more gRNAs that are effective for removing or disrupting an HIV provirus genome integrated into the genome of a cell, magnetic nanoparticles, and liposomes.
2. The composition of claim 1 , wherein the one or more gRNAs are selected from gLTR having the sequence of SEQ ID NO: 13; gnef having the sequence of SEQ ID NO: 14; gtat having the sequence of SEQ ID NO: 15; and gpol having the sequence of SEQ ID NO: 16.
3. The composition of claim 2 , wherein the one or more gRNAs comprise a combination of gnef and gtat or a combination of gnef and gpol.
4. The composition of claim 2 , wherein the liposomes carry on their surface one or more binding agents that bind specifically to molecules present on the surface of a target cell.
5. The composition of claim 1 , wherein the CRISPR-Cas protein is Cas9.
6. A composition comprising a CRISPR-Cas plasmid encoding a Cas9 protein, one or more gRNAs that are effective for removing or disrupting an HIV provirus genome integrated into the genome of a cell, magnetic nanoparticles, and liposomes.
7. The composition of claim 6 , wherein the one or more gRNAs are selected from gLTR having the sequence of SEQ ID NO: 13; gnef having the sequence of SEQ ID NO: 14; gtat having the sequence of SEQ ID NO: 15; and gpol having the sequence of SEQ ID NO: 16.
8. The composition of claim 7 , wherein the one or more gRNAs comprise a combination of gnef and gtat or a combination of gnef and gpol.
9. The composition of claim 6 , wherein the liposomes carry on their surface one or more binding agents that bind specifically to molecules present on the surface of a target cell.
10. A kit comprising:
a polynucleotide molecule comprising a HIV pseudovirus genome comprising a first marker gene operably linked to a HIV-1 LTR promoter and encoding a first marker protein and a second marker gene operably linked to a constitutive promoter and encoding a second marker protein, wherein the second promoter and marker gene are located in the nef gene of the HIV pseudovirus genome;
a CRISPR-Cas protein;
at least one polynucleotide molecule comprising one or more gRNAs;
magnetic nanoparticles;
liposomes;
reagents for detecting the expression of the marker proteins; and optionally, a cell.
11. The kit of claim 10 , wherein the CRISPR-Cas protein is a Cas9 protein.
12. The kit of claim 10 , wherein the at least one polynucleotide molecule comprises one or more gRNAs selected from gLTR having the sequence of SEQ ID NO: 13; gnef having the sequence of SEQ ID NO: 14; gtat having the sequence of SEQ ID NO: 15; and gpol having the sequence of SEQ ID NO: 16, or a combination.
13. The kit of claim 12 , wherein the at least one polynucleotide molecule comprises a combination of gRNA gnef and gRNA gtat or a combination of gRNA gnef and gRNA gpol.
14. The kit of claim 10 , wherein the second promoter and marker gene are located in the pol gene of the HIV pseudovirus genome.
15. The kit of claim 10 , wherein the second promoter and marker gene are located in the tat gene of the HIV pseudovirus genome.