Molecular State Machines

Abstract

A molecular state machine is implemented in a cell by designing the cell to use specific homology directed repair (“HDR”) templates for repairing double strand breaks in polynucleotides based on a current “state” of the cell. The state may be established by the presence of a molecule in the cell or by the availability of specific cut sites in the polynucleotides of the cell. Different HDR templates or different nucleases may be available for performing HDR based on the state. When the state is changed, the same signal or event will result in a different HDR template being incorporated into the existing polynucleotides of the cell. Signals that are internal or external to the cell may be used to change the state of the cell. The cell may create a log of molecular events, store binary data, or perform other synthetic biology/molecular computing functions based on state.

1 . A molecular state machine implemented in a cell comprising:
 an engineered signaling pathway that generates a tracking molecule in response to a first signal associated with a first state;   an operon that changes a rate of transcription of an RNA product based on the tracking molecule;   a double-stranded polynucleotide having a target site;   a homology directed repair (HDR) template with homology to the target site, wherein an amount of the HDR template available in the cell or an activity of a nuclease configured to create a double strand break (DSB) at a cut site in the target site is increased by the RNA product; and   an inhibition system that reduces the amount of HDR template available in the cell or the activity of the nuclease.   
     
     
         2 . The molecular state machine of  claim 1 , wherein the tracking molecule is a transcription factor. 
     
     
         3 . The molecular state machine of  claim 1 , wherein the RNA product is mRNA that encodes the nuclease. 
     
     
         4 . The molecular state machine of  claim 1 , wherein the RNA product is a gRNA and the nuclease is a CRISPR associated protein. 
     
     
         5 . The molecular state machine of  claim 1 , wherein the RNA product is the HDR template or is a template for generating the HDR template through reverse transcription. 
     
     
         6 . The molecular state machine of  claim 1 , wherein the inhibition system responds to a second signal associated with a second state. 
     
     
         7 . The molecular state machine of  claim 6 , wherein the inhibition system comprises CRISPRi that inactivates the operon. 
     
     
         8 . A method of encoding binary data in a polynucleotide within a cell, the method comprising:
 receiving a first external signal that indicates a first binary digit;   inserting a first HDR template into a double stranded polynucleotide by HDR, the first HDR template representative of the first binary digit according to a context-dependent code;   generating a first molecular signal based on the first binary digit, presence of the first molecular signal placing the cell into a first state;   receiving a second external signal that indicates a second binary digit; and   inserting a second HDR template into the double-stranded polynucleotide by HDR, the second HDR template representative of the second binary digit according to the context-dependent code and based on the cell being in the first state.   
     
     
         9 . The method of  claim 8 , wherein the first molecular signal comprises a transcription factor that activates a promoter associated with the first state of the cell. 
     
     
         10 . The method of  claim 8 , wherein the second HDR template comprises a 3′-end sequence and a 5′-end sequence that are homologous to corresponding portions of a target site on the double-stranded polynucleotide, and a middle region that comprises an identifier region that represents the second binary digit and an additional target site having a sequence based on the first state. 
     
     
         11 . The method of  claim 8 , wherein the context-dependent code prevents adjacent insertions of a same polynucleotide sequence into the double-stranded polynucleotide. 
     
     
         12 . The method of  claim 8 , further comprising:
 generating a second molecular signal based on the second binary digit, presence of the second molecular signal placing the cell into a second state different than the first state;   receiving a third external signal that indicates a third binary digit; and   inserting a third HDR template into the double-stranded polynucleotide by HDR, the third HDR template representative of the third binary digit according to the context-dependent code and based on the cell being in the second state.   
     
     
         13 . The method of  claim 12 , wherein the first state is a first stable state of a bi-stable molecular switch and the second state is a second state of the bi-stable molecular switch. 
     
     
         14 . A method for recording a signal in a cell based on state, the method comprising:
 detecting a first signal at a first engineered signaling pathway indicating a first state;   generating a first tracking molecule in response to the first signal;   detecting a second signal; and   responsive to the second signal and when a concentration of the first tracking molecule is at or above a first functional level, inserting a first HDR template into a double-stranded polynucleotide in the cell.   
     
     
         15 . The method of  claim 14 , wherein the first tracking molecule is a transcription factor that activates an inducible promoter operatively linked to at least one of synthesis of the HDR template or synthesis of a nuclease that creates a double strand break (DSB) in the double-stranded polynucleotide. 
     
     
         16 . The method of  claim 14 , wherein the concentration of the first tracking molecule decays below the first functional level within a threshold time. 
     
     
         17 . The method of  claim 16 , further comprising responsive to the second signal and when the concentration of the first tracking molecule is below the first functional level, inserting a second HDR template into the double-stranded polynucleotide. 
     
     
         18 . The method of  claim 14 , further comprising iteratively inserting the first HDR template while the second signal is present and the concentration of the first tracking molecule is at or above the first functional level. 
     
     
         19 . The method of  claim 14 , further comprising:
 detecting a third signal at a second engineered signaling pathway indicating a second state different from the first state;   generating a second tracking molecule in response to the third signal;   reducing the concentration of the first tracking molecule in response to the third signal;   detecting the second signal; and   responsive to the second signal and when a concentration of the second tracking molecule is at or above a second functional level, inserting a third HDR template into the double-stranded polynucleotide.   
     
     
         20 . The method of  claim 19 , wherein the reducing the concentration of the first tracking molecule causes increasing expression of a protein that degrades the first tracking molecule.