RNA cleavage-induced transcript stabilizer and uses thereof
Provided herein are genetic circuits and encoded RNA transcripts that produce an output molecule in response to an RNA cleavage event that removes a degradation signal. In some embodiments, the genetic circuits described herein may be used for detecting RNA cleaver activities (e.g., in a cell). Methods of using the genetic circuits described herein in diagnostic or therapeutic applications are also provided.
1. A composition comprising:
(i) a first genetic circuit encoding an RNA transcript, the genetic circuit comprising
a first promoter operably linked to a nucleotide sequence encoding an output molecule followed, from 5′ to 3′, by an RNA stabilizer, a cleavage site for an RNA cleaver, and a degradation signal, wherein the RNA transcript comprises one or more recognition sites for an RNA repressor operably linked to the nucleotide sequence encoding the output molecule; and
(ii) a second genetic circuit comprising a second promoter operably linked to a nucleotide sequence encoding the RNA repressor, and one or more cleavage sites for the RNA cleaver.
2. The composition of claim 1 , further comprising:
(iii) a third genetic circuit comprising a third promoter operably linked to a nucleotide sequence encoding the RNA cleaver.
3. The composition of claim 1 , wherein the RNA cleaver is selected from the group consisting of: endoribonucleases, RNAi molecules, and ribozymes.
4. The composition of claim 1 , wherein the RNA repressor is an RNA binding protein.
5. The composition of claim 4 , wherein the RNA binding protein is fused to a modifying domain.
6. The composition of claim 1 , wherein the RNA stabilizer is selected from the group consisting of: MALAT1 triplex, MENβ triplex, KSHV PAN triplex, histone stem loop, and a polyA signal.
7. A cell comprising the first and second genetic circuits of claim 1 .
8. The cell of claim 7 , wherein the first and second genetic circuits are inserted into the genome of the cell.
9. A method comprising maintaining the cell of claim 7 .
10. The method of claim 2 , further comprising detecting the output molecule.
11. The method of claim 10 , further comprising classifying the cell.
12. A method comprising delivering the RNA transcript encoded by the first genetic circuit of claim 1 to a cell and detecting the output molecule.
13. A method of treating a disease or disorder comprising administering an effective amount of a composition comprising the composition of claim 2 to a subject in need thereof, wherein the output molecule is a therapeutic molecule that is effective for treating the disease or disorder.
14. An RNA transcript encoded by the first genetic circuit of claim 1 .