Molecular state machines using hdr template insertion

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 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.   
     
     
         2 . The method of  claim 1 , wherein the first tracking molecule is a transcription factor that activates an inducible promoter operatively linked to at least one of (i) synthesis of the first HDR template or (ii) synthesis of a nuclease that creates a double strand break (DSB) in the double-stranded polynucleotide. 
     
     
         3 . The method of  claim 1 , wherein the concentration of the first tracking molecule decays below the first functional level within a threshold time. 
     
     
         4 . The method of  claim 3 , 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. 
     
     
         5 . The method of  claim 1 , wherein the second signal is indicative of a condition in an environment surrounding the cell or a condition in an internal environment of the cell. 
     
     
         6 . The method of  claim 1 , wherein the first HDR template is inserted into a sequence that was added to the double-stranded polynucleotide by earlier insertion of a different HDR template. 
     
     
         7 . The method of  claim 1 , further comprising causing generation of the first signal by manipulation of the cell or an environment surrounding the cell. 
     
     
         8 . The method of  claim 1 , 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, wherein a middle portion of the first HDR template includes an instance of a cut site into which further copies of the first HDR template are inserted. 
     
     
         9 . The method of  claim 1 , 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.   
     
     
         10 . The method of  claim 8 , wherein reducing the concentration of the first tracking molecule comprises increasing expression of a protein that degrades the first tracking molecule. 
     
     
         11 . A molecular state machine implemented in a cell and comprising:
 means for detecting a first signal at a first engineered signaling pathway indicating a first state;   means for generating a tracking molecule in response to the first signal;   means for detecting a second signal; and   means for inserting a HDR template into a double-stranded polynucleotide in the cell in response to the second signal when a concentration of the tracking molecule is at or above a functional level.   
     
     
         12 . A molecular state machine implemented in a cell and comprising:
 an RNA product in the cell;   a first engineered signaling pathway in the cell that is configured to generate a first tracking molecule in response to a first signal indicating a first state;   a double-stranded polynucleotide in the cell having a target site;   a nuclease in the cell configured to create a double strand break (DSB) at a cut site in the target site; and   a first homology directed repair (HDR) template in the cell with homology to the target site, wherein the RNA product is configured to increase (i) an amount of the first HDR template available in the cell or an (ii) activity of the nuclease configured to create the DSB at the cut site in the target site responsive to presence of a second signal when a concentration of the first tracking molecule is at or above a functional level.   
     
     
         13 . The molecular state machine of  claim 12 , wherein the second signal is indicative of a condition in an environment surrounding the cell or a condition in an internal environment of the cell. 
     
     
         14 . The molecular state machine of  claim 12 , wherein the target site is present in the double-stranded polynucleotide when the cell is in the first state and not present when the cell is not in the first state. 
     
     
         15 . The molecular state machine of  claim 12 , wherein one or both of the first tracking molecule and the second tracking molecule are transcription factors. 
     
     
         16 . The molecular state machine of  claim 12 , wherein the RNA product is:
 mRNA that encodes the nuclease;   a gRNA and the nuclease is a CRISPR-associated protein;   the first HDR template; or   a template configured for generating the first HDR template through reverse transcription.   
     
     
         17 . The molecular state machine of  claim 12 , further comprising a bi-stable repressor, wherein the bi-stable repressor places the cell in the first state when the concentration of the first tracking molecule is at or above the functional level. 
     
     
         18 . The molecular state machine of  claim 12 , further comprising an inducible promoter that is activated by the first tracking molecule. 
     
     
         19 . The molecular state machine of  claim 12 , further comprising an inhibition system that includes a repressor which blocks activity of the RNA product. 
     
     
         20 . The molecular state machine of  claim 12 , further comprising:
 a second RNA product in the cell;   a third engineered signaling pathway in the cell that is configured to generate a third tracking molecule in response to a third signal indicating a third state; and   a second HDR template in the cell with homology to the target site, wherein the second RNA product is configured to increase (i) an amount of the second HDR template available in the cell or an (ii) activity of the nuclease configured to create the DSB at the cut site in the target site responsive to presence of the third tracking molecule when a concentration of the first tracking molecule is at or above a functional level.