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 cleavage-induced transcript stabilizer comprising:
(i) an RNA transcript comprising a ribonucleotide sequence encoding an output molecule followed, in order, by: an RNA stabilizer, a cleavage site for an RNA cleaver, and a degradation signal that leads to degradation of the RNA transcript;
wherein the RNA cleaver is an endoribonuclease, a siRNA, a miRNA, or a ribozyme; and
wherein the cleavage-induced transcript stabilizer comprises one or more recognition sites for an RNA repressor operably linked to the nucleotide sequence encoding the output molecule.
2 . The cleavage-induced transcript stabilizer of claim 1 , further comprising:
(ii) a promoter operably linked to a nucleotide sequence encoding an RNA cleaver that cleaves the RNA transcript at the cleavage site.
3 . The cleavage-induced transcript stabilizer of claim 2 , wherein the promoter of (ii) is an inducible promoter.
4 . The cleavage-induced transcript stabilizer of claim 1 , wherein the RNA transcript is degraded in the absence of the RNA cleaver.
5 . The cleavage-induced transcript stabilizer of claim 1 , wherein the RNA transcript is expressed in the presence of the RNA cleaver.
6 . The cleavage-induced transcript stabilizer of claim 5 , wherein the cleavage of the RNA transcript stabilizes the RNA transcript and results in expression of the output molecule.
7 . The cleavage-induced transcript stabilizer of claim 1 , wherein the output molecule is a detectable molecule.
8 . The cleavage-induced transcript stabilizer of claim 1 , wherein the output molecule is a therapeutic molecule.
9 . The cleavage-induced transcript stabilizer of claim 1 , wherein the output molecule is a functional molecule.
10 . The cleavage-induced transcript stabilizer of claim 9 , wherein the functional molecule is selected from the group consisting of: TetR, CNOT7, DDX6, PPR10, L7Ae, Csy4, Cas6, CasE, and Cse3.
11 . An isolated cell comprising the cleavage-induced transcript stabilizer of claim 1 .
12 . The isolated cell of claim 11 , wherein a genetic circuit that encodes the cleavage-induced transcript stabilizer is inserted into the genome of the cell.
13 . A method comprising maintaining the isolated cell of claim 11 .
14 . A method comprising delivering the cleavage-induced transcript stabilizer of claim 1 to a cell and detecting the output molecule.
15 . A method of detecting an RNA cleaver activity, comprising:
delivering the cleavage-induced transcript stabilizer of claim 1 to a cell and detecting the output molecule.
16 . A method of treating a disease or disorder comprising delivering the cleavage-induced transcript stabilizer of claim 1 to a cell, wherein the output molecule is a therapeutic molecule that is effective for treating the disease or disorder.
17 . A method of diagnosing a disease or disorder comprising delivering the cleavage-induced transcript stabilizer of claim 1 to a cell and detecting the output molecule.
18 . A method of treating a disease or disorder comprising administering an effective amount of a composition comprising the cleavage-induced transcript stabilizer of claim 1 to a subject in need thereof, wherein the output molecule is a therapeutic molecule that is effective for treating the disease or disorder.
19 . A method of diagnosing a disease or disorder comprising administering an effective amount of a composition comprising the cleavage-induced transcript stabilizer of claim 1 to a subject in need thereof and detecting the output molecule.