Sensor systems for target ligands and uses thereof
Disclosed herein are sensor systems, compositions comprising the sensor systems, and methods of using the same. In particular aspects, disclosed herein are sensor systems for a target intracellular ligand and uses thereof, e.g., in detection assays or in cell manipulation or therapeutic applications.
1. A sensor system comprising
a fusion protein molecule comprising at least one target ligand-binding recognition domain linked to an effector domain, wherein the target ligand-binding recognition domain is a nanobody, that specifically binds an intracellular target ligand and is configured such that
(i) in the absence of the target ligand, the target ligand-binding recognition domain is destabilized and such that the fusion protein is destabilized and the effector domain is not active, or
(ii) in the presence of the target ligand, the target ligand-binding recognition domain is stabilized upon binding of the target ligand, and the effector domain is active,
wherein the nanobody has a framework region mutated at one or more sites consisting essentially of the sites corresponding to S73, S98 and S117 relative to wild-type nanobody corresponding to SEQ ID NO: 1 to one or more of S73R, S98Y and/or S117F relative to wild-type nanobody corresponding to SEQ ID NO: 1.
2. The sensor system of claim 1 , wherein the target ligand is a polypeptide, a nucleic acid, or a combination thereof.
3. The sensor system of claim 1 , wherein the effector domain is a protein, an enzyme, a nucleic acid, a therapeutic agent, a detectable agent, or a combination thereof.
4. The sensor system of claim 1 , wherein the effector domain is a recombinase enzyme, a toxin, a DNA nuclease enzyme, or a fluorescent protein.
5. The sensor system of claim 1 , wherein the target ligand-binding recognition domain is a nanobody against a GFP protein, a nanobody against HIV C-terminal domain (CTD) or a nanobody against dihydrofolate reductase.
6. The sensor system of claim 1 , further comprising the target ligand.
7. The sensor system of claim 6 , wherein the target ligand is an endogenous, intracellular target ligand.
8. An expression vector comprising a nucleotide sequence encoding the sensor system of claim 1 , wherein nucleotide sequence encoding the nanobody is linked in frame to nucleotide sequence encoding the effector domain.
9. The expression vector of claim 8 , wherein the nucleotide sequence encoding the nanobody is linked upstream to nucleotide sequence encoding the effector domain or downstream to nucleotide sequence encoding the effector domain.
10. A method of detecting an intracellular target ligand comprising:
a. introducing to a cell the sensor system of claim 1 ;
b. detecting a detectable signal of the effector domain of the sensor system; and
c. determining the presence of an intracellular target ligand if a detectable signal of the effector domain is detected; or
determining the absence of the intracellular target ligand if a detectable signal of the effector domain is not detected.
11. The method of claim 10 , wherein the target ligand is a polypeptide, a nucleic acid, or a combination thereof.
12. A method for delivery of a therapeutic agent or pro-drug agent to a target cell in a subject comprising
administering to a subject in need thereof a composition comprising the sensor system of claim 1 , wherein the effector domain of the sensor system is a therapeutic agent or pro-drug agent; and the target ligand-binding recognition domain of the sensor system specifically binds an intracellular target ligand of a target cell to be treated,
wherein:
in a non-target cell without the target ligand, the target ligand-binding recognition domain is destabilized and such that the fusion protein is destabilized and the therapeutic agent or pro-drug agent is not active in the non-target cell, or
in a target cell with the target ligand, the target ligand-binding recognition domain is stabilized upon binding of the target ligand, and the therapeutic agent or pro-drug agent is active in the target cell.
13. A system for genome editing comprising:
a nucleic acid guide designed to be complementary to a target nucleic acid sequence to be cut; and
a fusion protein molecule comprising:
at least one target ligand-binding recognition domain linked to a nucleic acid-guided DNA endonuclease enzyme, wherein the target ligand-binding recognition domain is a nanobody, that specifically binds an intracellular target ligand and is configured such that
(i) in the absence of the target ligand, the target ligand-binding recognition domain is destabilized and such that the fusion protein is destabilized and the DNA endonuclease enzyme is not active, or
(ii) in the presence of the target ligand, the target ligand-binding recognition domain is stabilized upon binding of the target ligand, and the DNA endonuclease enzyme is active, wherein the nanobody has a framework region mutated at one or more sites consisting essentially of the sites corresponding to S73, S98 and S117 relative to wild-type nanobody corresponding to SEQ ID NO: 1 to one or more of S73R, S98Y and/or S117F relative to wild-type nanobody corresponding to SEQ ID NO: 1.
14. The system of claim 13 , wherein the nucleic acid guide is RNA.
15. The system of claim 13 , wherein the nucleic acid-guided endonuclease enzyme is CRISPR associated protein.
16. The system of claim 15 , wherein the CRISPR associated protein is Cas9.