Programmable genetic toggle switch systems and uses thereof

Abstract

Described herein are programmable genetic toggle switch systems that can include a first state switch and a second state switch that are mutually exclusive, such that activation of the first state switch represses expression of the second state switch and activation of the second state switch represses expression of the first state switch. Also described herein are methods of using the programmable genetic toggle switch systems.

Description

PROGRAMMABLE GENETIC TOGGLE SWITCH SYSTEMS AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U. S. Provisional Patent Application No. 63 / 754,779, filed on February 6, 2025, entitled “CRISPR TOGGLE SWITCH COMPOSITIONS, SYSTEMS, AND USES THEREOF,” the contents of which is incorporated by reference herein in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under Contract No. HR0011-25-9-0060 awarded by Defense Advanced Research Projects Agency. The government has certain rights in the invention.SEQUENCE LISTING

[0003] This application contains a sequence listing filed in electronic form as an xml file entitled “VTIP-PAT159-PCT01_ST26.xml”, created on February 6, 2026, and having a size of 73,673 bytes. The content of the sequence listing is incorporated herein in its entirety.TECHNICAL FIELD

[0004] The subject matter disclosed herein is generally directed to engineered genetic circuits.BACKGROUND

[0005] Toggle-switch genetic circuits or bistable genetic switches turn on or off gene expression in response to a brief external signal such as a chemical or light and remain in the current on- or off-state upon removal of this signal. Genetic toggle- switches have applications in biomanufacturing, biomedicine, biosensors, and biocontainment among other fields, in which control of gene expression in an on-off manner is needed with minimal inputs to the biological system. As such there exists a need for toggle-switches.

[0006] Citation or identification of any document in this application is not an admission that such a document is available as prior art to the present invention.SUMMARY

[0007] In some aspects, the techniques described herein relate to a programmable genetic toggle switch system including: a first state switch including a first control expression construct; and a second state switch including a second control expression construct wherein the first control expression construct is configured, upon activation, to positively regulate transcription of the first control expression construct and repress transcription of the second control expression construct, and wherein the second control expression construct is configured, upon activation, to positively regulate transcription of the second control expression construct and repress transcription of the first control expression construct.

[0008] In some aspects, the techniques described herein relate to a programmable genetic toggle switch system including: a first state switch including a first control expression construct; and a second state switch including a second control expression construct wherein the first control expression construct is configured, upon activation, to positively regulate transcription of the first control expression construct and repress transcription of the second control expression construct, and wherein the second control expression construct is configured, upon activation, to positively regulate transcription of the second control expression construct and repress transcription of the first control expression construct.

[0009] In some aspects, the techniques described herein relate to a system, wherein the first state switch further includes a first functional expression construct, wherein the second state switch further includes a second functional expression construct, or both.

[0010] In some aspects, the techniques described herein relate to a system, wherein the first control expression construct is configured, upon activation, to positively regulate the first functional expression construct and repress the second functional expression construct.

[0011] In some aspects, the techniques described herein relate to a system, wherein the second control expression construct is configured, upon activation, to positively regulate the second functional expression construct and repress the first functional expression construct.

[0012] In some aspects, the techniques described herein relate to a system, wherein the first control expression construct includes a first control expression construct promoter, first controlexpression construct regulatory signal target sites, and first control expression construct regulatory element region, wherein the first control expression construct regulatory element region encodes one or more first control expression construct regulatory elements, and wherein the first control expression construct promoter is operatively coupled to the first control expression construct regulatory signal target sites and the first control expression construct regulatory element region.

[0013] In some aspects, the techniques described herein relate to a system, wherein the second control expression construct includes a second control expression construct promoter, second control expression construct regulatory signal target sites, and a second control construct regulatory element region, wherein the second control expression construct regulatory element region encodes one or more second control construct regulatory elements, and wherein the second control expression construct promoter is operatively coupled to the second control expression construct regulatory signal target sites and the second control expression construct regulatory element region.

[0014] In some aspects, the techniques described herein relate to a system, wherein the first control expression construct regulatory signal target sites include a first control expression construct promoter activator signal target site and a first control expression construct promoter repressor signal target site.

[0015] In some aspects, the techniques described herein relate to a system, wherein the first control expression construct regulatory element region encodes a first control expression construct promoter activator and a second control expression construct promoter repressor.

[0016] In some aspects, the techniques described herein relate to a system, wherein the first control expression construct promoter activator is configured to bind or interact with first control expression construct promoter activator signal target site so as to activate the first control expression construct promoter.

[0017] In some aspects, the techniques described herein relate to a system, wherein the second control expression construct regulatory signal target sites include a second control expression construct promoter activator signal target site and a second control expression construct promoter repressor signal target site.

[0018] In some aspects, the techniques described herein relate to a system, wherein the second control expression construct regulatory element region encodes a second controlexpression construct promoter activator and a first control expression construct promoter repressor.

[0019] In some aspects, the techniques described herein relate to a system, wherein the second control expression construct promoter activator is configured to bind or interact with second control expression construct promoter activator signal target site so as to activate the second control expression construct promoter.

[0020] In some aspects, the techniques described herein relate to a system, wherein the first control expression construct promoter repressor is configured to bind or interact with the first control expression construct promoter repressor signal target site so as to repress activity of the first control expression construct promoter.

[0021] In some aspects, the techniques described herein relate to a system, wherein the second control expression construct promoter repressor is configured to bind or interact with the second control expression construct promoter repressor signal target site so as to repress activity of the second control expression construct promoter.

[0022] In some aspects, the techniques described herein relate to a system, wherein the first functional expression construct includes a first functional element region that encodes one or more first functional elements.

[0023] In some aspects, the techniques described herein relate to a system, wherein the first functional expression construct further includes a first functional expression construct activator signal target site.

[0024] In some aspects, the techniques described herein relate to a system, wherein the first functional expression construct includes a first functional expression construct repressor signal target site.

[0025] In some aspects, the techniques described herein relate to a system, wherein the first functional expression construct includes a first functional expression construct promoter, wherein the first functional expression construct promoter is operatively coupled to the first functional element region.

[0026] In some aspects, the techniques described herein relate to a system, wherein the second functional expression construct includes a second functional element region that encodes one or more second functional elements.

[0027] In some aspects, the techniques described herein relate to a system, wherein the second functional expression construct further includes a second functional expression construct activator signal target site.

[0028] In some aspects, the techniques described herein relate to a system, wherein the second functional expression construct includes a second functional expression construct repressor signal target site.

[0029] In some aspects, the techniques described herein relate to a system, wherein the second functional expression construct includes a second functional expression construct promoter, wherein the second functional expression construct promoter is operatively coupled to the second functional element region.

[0030] In some aspects, the techniques described herein relate to a system, wherein the first control expression construct regulatory element region encodes a first functional expression construct activator.

[0031] In some aspects, the techniques described herein relate to a system, wherein the first control expression construct regulatory element region encodes a second functional expression construct repressor.

[0032] In some aspects, the techniques described herein relate to a system, wherein the second control expression construct regulatory element region encodes a second functional expression construct activator.

[0033] In some aspects, the techniques described herein relate to a system, wherein the second control expression construct regulatory element region encodes a first functional expression construct repressor.

[0034] In some aspects, the techniques described herein relate to a system, wherein the one or more first functional elements includes one or more regulatory functional elements, effector functional elements, reporter functional elements, payload functional elements, or any combination thereof.

[0035] In some aspects, the techniques described herein relate to a system, wherein the one or more second functional elements includes one or more regulatory functional elements, effector functional elements, reporter functional elements, payload functional elements, or any combination thereof.

[0036] In some aspects, the techniques described herein relate to a system, wherein the one or more regulatory functional elements include sequence-directed regulatory elements, regulatory RNAs, regulatory proteins.

[0037] In some aspects, the techniques described herein relate to a system, wherein the sequence-directed regulatory elements are RNA sequence-directed regulatory elements, optionally guide RNAs.

[0038] In some aspects, the techniques described herein relate to a system, wherein the reporter functional element is an optically active protein.

[0039] In some aspects, the techniques described herein relate to a system, wherein at least one of the one or more first functional elements is different than at least one of the one or more second functional elements.

[0040] In some aspects, the techniques described herein relate to a system, wherein all of the one or more first functional elements is different than the one or more second functional elements.

[0041] In some aspects, the techniques described herein relate to a system, wherein the one or more first control expression construct regulatory elements are sequence-directed regulatory elements.

[0042] In some aspects, the techniques described herein relate to a system, wherein the one or more second control expression construct regulatory elements are sequence-directed regulatory elements.

[0043] In some aspects, the techniques described herein relate to a system, wherein the sequence-directed regulatory elements are RNA sequence-directed regulatory elements.

[0044] In some aspects, the techniques described herein relate to a system, wherein the RNA sequence-directed regulatory elements are guide RNAs.

[0045] In some aspects, the techniques described herein relate to a system, wherein the guide RNAs include one or more activation guide RNA configured to recruit a CRISPR-based transcriptional activator complex, an inhibition guide RNA configured to recruit a CRISPR-based transcriptional inhibitor complex, or both.

[0046] In some aspects, the techniques described herein relate to a system, wherein the one or more first control expression construct regulatory elements include (i) a first control expression construct activation guide RNA configured to specifically bind the first control expression construct promoter activator signal target site and recruit a CRISPR transcriptionalactivator complex, (ii) a second control expression construct repression guide RNA configured to specifically bind the second control expression construct promoter repressor signal target site and recruit a CRISPR transcriptional inhibitor complex, (iii) a first functional expression construct activation guide RNA configured to specifically bind the first functional expression construct activator signal target site and recruit a CRISPR transcriptional activator complex, (iv) a second functional expression construct repression guide RNA configured to specifically bind the second functional expression construct repressor signal target site and recruit a CRISPR transcriptional inhibitor complex, or any combination thereof.

[0047] In some aspects, the techniques described herein relate to a system, wherein the one or more second control expression construct regulatory elements include (i) a second control expression construct activation guide RNA configured to specifically bind the second control expression construct promoter activator signal target site and recruit a CRISPR transcriptional activator complex, (ii) a first control expression construct repression guide RNA configured to specifically bind the first control expression construct promoter repressor signal target site and recruit a CRISPR transcriptional inhibitor complex, (iii) a second functional expression construct activation guide RNA configured to specifically bind the second functional expression construct activator signal target site and recruit a CRISPR transcriptional activator complex, (iv) a first functional expression construct repression guide RNA configured to specifically bind the first functional expression construct repressor signal target site and recruit a CRISPR transcriptional inhibitor complex, or any combination thereof.

[0048] In some aspects, the techniques described herein relate to a system, wherein the first control expression construct activation guide RNA and the first functional expression construct activation guide RNA include guide RNA scaffold sequences that are distinct from guide RNA scaffold sequences of the second control expression construct repression guide RNA and the second functional expression construct repression guide RNA, such that recruitment of a CRISPR transcriptional activator complex or a CRISPR transcriptional inhibitor complex is determined by the scaffold sequence of the guide RNA.

[0049] In some aspects, the techniques described herein relate to a system, wherein the second control expression construct activation guide RNA and the second functional expression construct activation guide RNA include guide RNA scaffold sequences that are distinct from guide RNA scaffold sequences of the first control expression construct repression guide RNA and the first functional expression construct repression guide RNA, such thatrecruitment of a CRISPR transcriptional activator complex or a CRISPR transcriptional inhibitor complex is determined by the scaffold sequence of the guide RNA.

[0050] In some aspects, the techniques described herein relate to a system, further including a third state switch including a third control expression construct, wherein the third state control expression construct is configured, upon activation, to positively regulate transcription of the third control expression construct and repress transcription of the first control expression construct, the second control expression construct, or both.

[0051] In some aspects, the techniques described herein relate to a system, wherein the third state switch further includes a third functional expression construct.

[0052] In some aspects, the techniques described herein relate to a system, wherein the third control expression construct is configured, upon activation, to positively regulate the third functional expression construct and repress transcription of the first functional expression construct, second functional expression construct, or both.

[0053] In some aspects, the techniques described herein relate to a system, Wherein the third control expression construct includes a third control expression construct promoter, third control expression construct regulatory signal target sites, and a third control expression construct regulatory element region, wherein the third control expression construct regulatory element region encodes one or more third control expression construct regulatory elements, wherein the third control expression construct promoter is operatively coupled to the third control expression construct regulatory signal target sites and third first control expression construct regulatory element region.

[0054] In some aspects, the techniques described herein relate to a system, wherein the third control expression construct regulatory signal target sites include a third control expression construct promoter activator signal target site and a third control expression construct promoter repressor signal target site.

[0055] In some aspects, the techniques described herein relate to a system, wherein the third control expression construct regulatory element region encodes a third control expression construct promoter activator and first control expression construct promoter repressor, a second control expression construct promoter repressor, or both.

[0056] In some aspects, the techniques described herein relate to a system, wherein the third control expression construct promoter activator is configured to bind or interact with thirdcontrol expression construct promoter activator signal target site so as to activate the third control expression construct promoter.

[0057] In some aspects, the techniques described herein relate to a system, wherein the third control expression construct promoter repressor is configured to bind or interact with the first control expression construct promoter repressor signal target site so as to repress activity of the first control expression construct promoter and wherein the second control expression construct promoter repressor signal target site so as to repress activity of the second control expression construct promoter.

[0058] In some aspects, the techniques described herein relate to a system, wherein the third functional expression construct includes a third functional element region that encodes one or more third functional elements.

[0059] In some aspects, the techniques described herein relate to a system, wherein the third functional expression construct further includes a third functional expression construct activator signal target site.

[0060] In some aspects, the techniques described herein relate to a system, wherein the third functional expression construct includes a third functional expression construct repressor signal target site.

[0061] In some aspects, the techniques described herein relate to a system, wherein the third functional expression construct includes a third functional expression construct promoter, wherein the third functional expression construct promoter is operatively coupled to the third functional element region.

[0062] In some aspects, the techniques described herein relate to a system, wherein the third control expression construct regulatory element region encodes a third functional expression construct activator.

[0063] In some aspects, the techniques described herein relate to a system, wherein the third control expression construct regulatory element region encodes a first functional expression construct repressor, a second functional expression construct repressor, or both.

[0064] In some aspects, the techniques described herein relate to a system, wherein the one or more third functional elements includes one or more regulatory functional elements, effector functional elements, reporter functional elements, payload functional elements, or any combination thereof.

[0065] In some aspects, the techniques described herein relate to a system, wherein the one or more regulatory functional elements include sequence-directed regulatory elements, regulatory RNAs, regulatory proteins.

[0066] In some aspects, the techniques described herein relate to a system, wherein the sequence-directed regulatory elements are RNA sequence-directed regulatory elements, optionally guide RNAs.

[0067] In some aspects, the techniques described herein relate to a system, wherein the reporter functional element is an optically active protein.

[0068] In some aspects, the techniques described herein relate to a system, wherein at least one of the one or more third functional elements is different than at least one of the one or more first functional elements, at least one of the one or more second functional elements, or both.

[0069] In some aspects, the techniques described herein relate to a system, wherein all of the one or more third functional elements is different than the one or more first functional elements, the one or more second functional elements, or both.

[0070] In some aspects, the techniques described herein relate to a system, wherein the one or more third control expression construct regulatory elements are sequence-directed regulatory elements.

[0071] In some aspects, the techniques described herein relate to a system, wherein the sequence-directed regulatory elements are RNA sequence-directed regulatory elements.

[0072] In some aspects, the techniques described herein relate to a system, wherein the RNA sequence-directed regulatory elements are guide RNAs.

[0073] In some aspects, the techniques described herein relate to a system, wherein the guide RNAs include one or more activation guide RNA configured to recruit a CRISPR-based transcriptional activator complex, an inhibition guide RNA configured to recruit a CRISPR-based transcriptional inhibitor complex, or both.

[0074] In some aspects, the techniques described herein relate to a system, wherein the one or more third control expression construct regulatory elements include (i) a third control expression construct activation guide RNA configured to specifically bind the first control expression construct promoter activator signal target site and recruit a CRISPR transcriptional activator complex, (ii) a first control expression construct repression guide RNA configured to specifically bind the first control expression construct promoter repressor signal target site and recruit a CRISPR transcriptional inhibitor complex, a second control expression constructrepression guide RNA configured to specifically bind the second control expression construct promoter repressor signal target site and recruit a CRISPR transcriptional inhibitor complex, or both, (iii) a third functional expression construct activation guide RNA configured to specifically bind the third functional expression construct activator signal target site and recruit a CRISPR transcriptional activator complex, (iv) a first functional expression construct repression guide RNA configured to specifically bind the first functional expression construct repressor signal target site and recruit a CRISPR transcriptional inhibitor complex, a second functional expression construct repression guide RNA configured to specifically bind the second functional expression construct repressor signal target site and recruit a CRISPR transcriptional inhibitor complex, or both, or any combination thereof.

[0075] In some aspects, the techniques described herein relate to a system, wherein the third control expression construct activation guide RNA and the third functional expression construct activation guide RNA include guide RNA scaffold sequences that are distinct from guide RNA scaffold sequences of the first and the second control expression construct repression guide RNAs and the first and the second functional expression construct repression guide RNA, such that recruitment of a CRISPR transcriptional activator complex or a CRISPR transcriptional inhibitor complex is determined by the scaffold sequence of the guide RNA.

[0076] In some aspects, the techniques described herein relate to a system, wherein the first control expression construct is further configured, upon activation to repress transcription of the third control expression construct, repress transcription of the third functional expression construct, or both.

[0077] In some aspects, the techniques described herein relate to a system, wherein the second control expression construct is further configured, upon activation to repress transcription of the third control expression construct, repress transcription of the third functional expression construct, or both.

[0078] In some aspects, the techniques described herein relate to a system, wherein the first control expression construct regulatory element region encodes a third control expression construct promoter repressor, wherein the second control expression regulatory element region encodes a third control expression construct promoter repressor, or both.

[0079] In some aspects, the techniques described herein relate to a system, wherein the third control expression construct promoter repressor is configured to interact with the thirdcontrol expression construct promoter repressor signal target site so as to repress activity of the third control expression construct promoter.

[0080] In some aspects, the techniques described herein relate to a system, wherein the first control expression construct regulatory element region encodes a third functional expression construct promoter repressor, wherein the second control expression regulatory element region encodes a third functional expression construct promoter repressor, or both.

[0081] In some aspects, the techniques described herein relate to a system, wherein the third functional expression construct promoter repressor is configured to interact with the third functional expression construct promoter repressor signal target site so as to repress activity of the third functional expression construct promoter

[0082] In some aspects, the techniques described herein relate to a system, further including one or more expression constructs encoding one or more components of CRISPR transcriptional regulatory machinery configured to interact with the activation guide RNAs and the repression guide RNAs.

[0083] In some aspects, the techniques described herein relate to a system, wherein the CRISPR transcriptional regulatory machinery includes a CRISPR transcriptional activator complex and a CRISPR transcriptional inhibitor complex.

[0084] In some aspects, the techniques described herein relate to a system, wherein the CRISPR transcriptional activator complex and the CRISPR transcription inhibitor complex each include a dead Cas (dCas) effector.

[0085] In some aspects, the techniques described herein relate to a system, wherein the dCas effector of the CRISPR transcriptional activator complex is different than the dCas effector of the CRISPR inhibitor complex.

[0086] In some aspects, the techniques described herein relate to a system, wherein the dCas effector of the CRISPR inhibitor complex is a dCas9.

[0087] In some aspects, the techniques described herein relate to a system, wherein the dCas effector of the CRISPR activator complex is a dCas12.

[0088] In some aspects, the techniques described herein relate to a system, further including an expression construct encoding an endoribonuclease, optionally wherein the endoribonuclease is configured to process one or more RNA transcripts encoded by the system

[0089] In some aspects, the techniques described herein relate to a system, wherein the expression constructs are contained in one or more vectors.

[0090] In some aspects, the techniques described herein relate to an isolated polynucleotide or set of isolated polynucleotides encoding the system of the present disclosure.

[0091] In some aspects, the techniques described herein relate to a vector or set of vector of comprising or encoding a system of the present disclosure or component thereof.

[0092] In some aspects, the techniques described herein relate to a cell or population containing a system of the present disclosure or a vector or vectors encoding the same.

[0093] In some aspects, the techniques described herein relate to a cell or population thereof, wherein the cell or organism is a prokaryotic cell, a eukaryotic cell, or a multicellular organism.

[0094] In some aspects, the techniques described herein relate to a cell or population thereof, wherein the multicellular organism is not a human.

[0095] In some aspects, the techniques described herein relate to a cell or population thereof, wherein the cell or organism is a yeast.

[0096] In some aspects, the techniques described herein relate to a host cell line including the system of the present disclosure.

[0097] In some aspects, the techniques described herein relate to a method of switching between regulatory states in a programmable genetic toggle switch system of the present disclosure, the method including: (a) applying a first stimulus or a second stimulus to the system, wherein the first stimulus selectively activates a first state switch and the second stimulus selectively activates a second state switch; (b) upon activation of the first state switch, inducing positive regulation of transcription of a first control expression construct and repression of transcription of a second control expression construct; or (c) upon activation of the second state switch, inducing positive regulation of transcription of the second control expression construct and repression of transcription of the first control expression construct; thereby switching the system into a first regulatory state or a second regulatory state that is maintained by self-activation of the activated state switch and repression of the non-activated state switch.

[0098] In some aspects, the techniques described herein relate to a method of switching between regulatory states in a programmable genetic toggle switch system of the present disclosure, wherein the programmable genetic toggle switch system is present in a cell or population thereof or an organism, optionally a yeast.

[0099] In some aspects, the techniques described herein relate to a method of switching between regulatory states in a programmable genetic toggle switch system of the present disclosure, further including, after switching the system into the first regulatory state, applying the second stimulus to the system to deactivate the first state switch and activate the second state switch, thereby repressing transcription of the first control expression construct and inducing positive regulation of transcription of the second control expression construct so as to switch the system from the first regulatory state to the second regulatory state.

[0100] In some aspects, the techniques described herein relate to a method of switching between regulatory states in a programmable genetic toggle switch system of the present disclosure, wherein the first stimulus or the second stimulus is applied transiently, and wherein the regulatory state established by activation of the first state switch or the second state switch is maintained after removal of the transient stimulus.

[0101] In some aspects, the techniques described herein relate to a method of switching between regulatory states in a programmable genetic toggle switch system of the present disclosure, wherein the first stimulus or the second stimulus is applied for a sustained duration, and wherein the regulatory state is maintained during the sustained application of the stimulus.

[0102] In some aspects, the techniques described herein relate to a method of switching between regulatory states in a programmable genetic toggle switch system of the present disclosure, wherein the first stimulus and / or the second stimulus includes one or more of an optical stimulus, a thermal stimulus, a chemical stimulus, a pH stimulus, an osmolarity stimulus, an electrical stimulus, or a mechanical stimulus.

[0103] In some aspects, the techniques described herein relate to a method of switching between regulatory states in a programmable genetic toggle switch system of the present disclosure, wherein the first stimulus and the second stimulus are orthogonal stimuli, such that application of the first stimulus selectively activates the first state switch without activating the second state switch, and application of the second stimulus selectively activates the second state switch without activating the first state switch.

[0104] In some aspects, the techniques described herein relate to a method of switching between regulatory states in a programmable genetic toggle switch system of the present disclosure, wherein the first stimulus and the second stimulus are applied in combination or sequentially.

[0105] In some aspects, the techniques described herein relate to a method of switching between regulatory states in a programmable genetic toggle switch system of the present disclosure, wherein switching the system into the first regulatory state or the second regulatory state results in a corresponding change in expression of one or more functional elements encoded by a functional expression construct associated with the activated state switch and repression of expression of one or more functional elements encoded by a functional expression construct associated with the non-activated state switch.

[0106] In some aspects, the techniques described herein relate to a method of switching between regulatory states in a programmable genetic toggle switch system of the present disclosure, wherein the first regulatory state or the second regulatory state is maintained through one or more cell divisions following activation of the first state switch or the second state switch.

[0107] In some aspects, the techniques described herein relate to a method of switching between regulatory states in a programmable genetic toggle switch system of the present disclosure, wherein the first regulatory state or the second regulatory state is maintained upon exposure of the system to one or more environmental changes selected from changes in temperature, nutrient availability, pH, osmolarity, oxygen availability, growth conditions, or any combination thereof.

[0108] These and other aspects, objects, features, and advantages of the example embodiments will become apparent to those having ordinary skill in the art upon consideration of the following detailed description of example embodiments.BRIEF DESCRIPTION OF THE DRAWINGS

[0109] An understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention may be utilized, and the accompanying drawings of which:

[0110] FIG. 1A-1B shows construct schematics of an exemplary toggle-switch design that allows bistable switching between two different metabolic states (FIG. 1A) or two reporters (FIG. IB) In FIG. 1A, sgRNA array 1 is activated and solid green lines represent activating interactions whereas solid orange lines represent repressive interactions. Dotted lines represent interactions that would be active only when sgRNA array 2 is active. In this case sgRNA array1 was activated by chemical or light activation driving initial production of the Metl, Met2, and Met3 sgRNAs as well as the self-activating Selfl and repressive Otherl sgRNAs. In FIG. IB, The sgRNAs have similar self-activation and other-inhibition, similar to that of FIG. 1 A, but instead of activation of metabolic genes, different fluorescent proteins (GFP or RFP) are activated. Exemplary chemicals such as IPTG or aTc can be employed for chemical activation. Activating sgRNAs interact with the endonuclease deactivated Cas protein dCpfl fused to a transcriptional activation domain, whereas repressive sgRNAs interact with an endonuclease deactivated dCas9 fused to a transcriptional inhibitor / repressor domain. The guide RNA handle sequences are unique between Cpfl and Cas9 allowing this handle sequence to confer activation or repression regulatory capacity, while the target sequence confers gene specificity. While 2 arrays are shown in FIG. 1A-1B for simplicity, this concept is extensible to larger numbers of arrays and larger arrays only limited by current assembly and expression technology.

[0111] FIG. 2 shows a schematic representation of expression constructs for combinatorial libraries of Green and Red state toggle switch guide RNA arrays and reporters, as well as CRISPRai machinery variants.

[0112] FIG. 3 shows general CRISPRai logic. Synthetic guide RNAs targeting genes for repression or activation are expressed from Pol II promoters containing Csy4 RNA endonuclease sites. Csy4 cleaves individual sgRNAs from the array expressed as an mRNA. Deactivated, nuclease-dead dCas9 and dCas12a proteins fused to an activation domain and repression domain, as well as Csy4 are expressed separately. Guide RNA handles specific to dCas9 or dCas12a encode whether the target gene will be activated or repressed. 20 bp target sites allows activation or repression of nearly any gene in the genome only limited by PAM sites in promoters and interactions with endogenous regulators.

[0113] FIG. 4 shows a schematic of CRISPRai machinery construct with exemplary repressors and activators noted beneath the respective construct element.

[0114] FIG. 5 shows EL222 activation a pC120::mCherry reporter in the presence of blue light. As proof of concept Applicant is using a repressive guide RNA to inhibit EL222 activation in the presence of light and an activating guide RNA to drive mCherry expression in the absence of light.

[0115] FIG. 6 shows a schematic demonstrating that sgRNA target sites vary between activators and repressors.

[0116] FIG. 7 shows a schematic of constructs for Golden-Gate drop in for high-throughput promoter-sgRNA combinatorial screening

[0117] FIG. 8 shows a schematic of a construct for two-state toggle switch strains was based on two plasmids one containing the toggle switch arrays and reporters and another vector containing the CRISPRai machinery as well as optoswitch machinery. Expression cassettes are driven by medium strength (med, approximately 10-fold increase above background) constitutive promoters. All combinations of GFP / RFP switch-only arrays and empty spacer cassettes have been constructed in single vectors with pC120 and pGal reporters and transformed into YB392. Low basal fluorescence of these strains is expected without CRISPRai regulatory machinery.

[0118] FIG. 9A-9B shows screening clones of initial toggle switch parental strains. mCherry and YFP reporter fluorescence levels (as fold change over YB392 intrinsic fluorescence) of Y. lipolytica YB392 transformants with various toggle switch and control vectors. Four individual transformants of each construct were cultured individually overnight in 500 pL YPAD medium in a 96-well deepwell plate at 30°C with shaking at 300 RPM. The following day the fluorescence of these cultures was measured by flow cytometry. Each dot represents the mean of fluorescence >20,000 cells sampled from a single transformant’s culture. Some dots are overlapping. Green dots were chosen for initial individual transformations with CRISPRai machinery vectors. All positive transformants (>5-fold mCherry and >1.5-fold YFP) were also mixed for combinatorial transformations.

[0119] FIG. 10A-10B shows a schematic of constructs for constitutive expression of the guide RNAs responsible for involved in the toggle switches will test if the self-activating and other-repressing guide RNAs and CRISPRai machinery is functional (FIG. 10A) and reporter-only controls exclude the self-activation and other switch repression guide RNAs (FIG. 10B).Reporter-only arrays activate the respective reporter, but the reporter response should decay more rapidly than complete and functional toggle-switch arrays.

[0120] FIG. 11 shows a schematic of an optoswitch Drop-in testing vector, which was also previously used by Applicant to confirm EL222-pC120-blue light activation).

[0121] FIG. 12 shows a schematic of constructs built based upon the initial designs in FIG.8 but with the green state construct built reflecting a different design in which the reporter activation guide for the red-state (ak) was included in the green-state switch (top construct) andshowing that it would have both activation and repression on the Red-state switch reporter construct.

[0122] FIG. 13A-13B shows results from YB-392 wildtype and YB-392 with EL222 and a pC 120:mCherry reporter integration that were cultured overnight prior to exposure to varying light treatments for 10 hours from two experiments (Experiment 1, FIG. 13 A and Experiment 2, FIG. 13B). Following 10 hours of light treatment (indicated by the grey vertical bars) in all cultures in Experiment 1 were returned to dark conditions. In Experiment 2, experimental cultures were split after 10 hours and one replicate was switched to the opposite treatment, e.g. Dark-to-Light (D-to-L) or Light-to-Dark (L-to-D). mCherry fluorescence was measured by flow cytometry. Each point represents the median of 5000 events. Some measurements were excluded due to bubble errors during flow cytometry.

[0123] FIG. 14 shows mCherry fluorescence representing both pC120:mCherry and pGakmCherry reporters in the switch-only control strains. Switch-only control parental strains (NA, right-most column) were co-transformed with the Optoswitch vector and CRISPRai vectors (left three columns). 2-4 transformants of each strain (represented as separate lines and points) were assayed as described in e.g., Example 4.

[0124] FIG. 15 shows an overview schematic of a toggle switch system of the present disclosure. Dotted lines represent repression and solid lines represent activation.

[0125] FIG. 16 shows a schematic of activation and repression control of two orthogonal switches of a toggle switch system of the present disclosure. Dotted lines represent repression and solid lines represent activation, atsce refers to the activator target site of the control expression construct, rtsce refers to the repressor target site of the control expression construct, atsfe refers to the activator target site of the functional expression construct, rtsfe refers to the repressor target site of the functional expression construct.

[0126] FIG. 17A and 17B shows exemplary configurations of the regulatory element regions of two orthogonal switches of a toggle switch system of the present disclosure. The gray diamonds in FIG. 17B represent endoribonuclease cleavage sites. aferefers to an activator of the functional expression construct. acerefers to an activator of the control expression construct. rcerefers to a repressor of the control expression construct. rferefers to a repressor of the functional expression construct.

[0127] The figures herein are for illustrative purposes only and are not necessarily drawn to scale.DETAILED DESCRIPTION OF THE EXAMPLE EMBODIMENTS

[0128] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0129] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0130] All publications and patents cited in this specification are cited to disclose and describe the methods and / or materials in connection with which the publications are cited. All such publications and patents are herein incorporated by references as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. Such incorporation by reference is expressly limited to the methods and / or materials described in the cited publications and patents and does not extend to any lexicographical definitions from the cited publications and patents. Any lexicographical definition in the publications and patents cited that is not also expressly repeated in the instant application should not be treated as such and should not be read as defining any terms appearing in the accompanying claims. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

[0131] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0132] Where a range is expressed, a further aspect includes from the one particular value and / or to the other particular value. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g., the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g. ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of Tess than x’, less than y’, and Tess than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’. In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.

[0133] It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0134] It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-rangeis explicitly recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the subranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.General Definitions

[0135] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Definitions of common terms and techniques in molecular biology may be found in Molecular Cloning: A Laboratory Manual, 2ndedition (1989) (Sambrook, Fritsch, and Maniatis); Molecular Cloning: A Laboratory Manual, 4thedition (2012) (Green and Sambrook); Current Protocols in Molecular Biology (1987) (F. M. Ausubel et al. eds.); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (1995) (M. J. MacPherson, B. D. Hames, and G. R. Taylor eds.): Antibodies, A Laboratory Manual (1988) (Harlow and Lane, eds.): Antibodies A Laboratory Manual, 2ndedition 2013 (E. A. Greenfield ed.); Animal Cell Culture (1987) (R. I. Freshney, ed.); Benjamin Lewin, Genes IX, published by Jones and Bartlett, 2008 (ISBN 0763752223); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0632021829); Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 9780471185710); Singleton etal., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N. Y. 1994), March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, N. Y. 1992); and Marten H. Hofker and Jan van Deursen, Transgenic Mouse Methods and Protocols, 2ndedition (2011).

[0136] Definitions of common terms and techniques in chemistry and organic chemistry can be found in Smith. Organic Synthesis, published by Academic Press. 2016; Tinoco et al. Physical Chemistry, 5thedition (2013) published by Pearson; Brown et al., Chemistry, The Central Science 14thed. (2017), published by Pearson, Clayden et al., Organic Chemistry, 2nded. 2012, published by Oxford University Press; Carey and Sunberg, Advanced Organic Chemistry, Part A: Structure and Mechanisms, 5thed. 2008, published by Springer; Carey and Sunberg, Advanced Organic Chemistry, Part B: Reactions and Synthesis, 5thed. 2010,published by Springer, and Vollhardt and Schore, Organic Chemistry, Structure and Function; 8thed. (2018) published by W. H. Freeman.

[0137] Definitions of common terms, analysis, and techniques in genetics can be found in e.g., Hartl and Clark. Principles of Population Genetics. 4thEd. 2006, published by Oxford University Press. Published by Booker. Genetics: Analysis and Principles, 7thEd. 2021, published by McGraw Hill; Isik et la., Genetic Data Analysis for Plant and Animal Breeding. First ed. 2017. published by Springer International Publishing AG; Green, E. L. Genetics and Probability in Animal Breeding Experiments. 2014, published by Palgrave; Bourdon, R. M. Understanding Animal Breeding. 2000 2ndEd. published by Prentice Hall; Pal and Chakravarty. Genetics and Breeding for Disease Resistance of Livestock. First Ed. 2019, published by Academic Press; Fasso, D. Classification of Genetic Variance in Animals. First Ed. 2015, published by Callisto Reference; Megahed, M. Handbook of Animal Breeding and Genetics, 2013, published by Omniscriptum Gmbh & Co. Kg., LAP Lambert Academic Publishing; Reece. Analysis of Genes and Genomes. 2004, published by John Wiley & Sons. Inc; Deonier et al., Computational Genome Analysis. 5thEd. 2005, published by Springer-Verlag, New York; Meneely, P. Genetic Analysis: Genes, Genomes, and Networks in Eukaryotes. 3rdEd. 2020, published by Oxford University Press.

[0138] As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise.

[0139] As used herein, "about," "approximately," “substantially,” and the like, when used in connection with a measurable variable such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value including those within experimental error (which can be determined by e.g. given data set, art accepted standard, and / or with e.g., a given confidence interval (e.g. 90%, 95%, or more confidence interval from the mean), such as variations of + / - 10% or less, + / -5% or less, + / -1% or less, and + / -0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” can mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off,1measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0140] The term “optional” or “optionally” means that the subsequent described event, circumstance or substituent may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0141] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

[0142] As used herein, a “biological sample” refers to a sample obtained from, made by, secreted by, excreted by, or otherwise containing part of or from a biologic entity. A biologic sample can contain whole cells and / or live cells and / or cell debris, and / or cell products, and / or virus particles. The biological sample can contain (or be derived from) a “bodily fluid”. The biological sample can be obtained from an environment (e.g., water source, soil, air, and the like). Such samples are also referred to herein as environmental samples. As used herein “bodily fluid” refers to any non-solid excretion, secretion, or other fluid present in an organism and includes, without limitation unless otherwise specified or is apparent from the description herein, amniotic fluid, aqueous humor, vitreous humor, bile, blood or component thereof (e.g. plasma, serum, etc.), breast milk, cerebrospinal fluid, cerumen (earwax), chyle, chyme, endolymph, perilymph, exudates, feces, female ejaculate, gastric acid, gastric juice, lymph, mucus (including nasal drainage and phlegm), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum (skin oil), semen, sputum, synovial fluid, sweat, tears, urine, vaginal secretion, vomit and mixtures of one or more thereof. Biological samples include cell cultures, bodily fluids, cell cultures from bodily fluids. Bodily fluids may be obtained from an organism, for example by puncture, or other collecting or sampling procedures.

[0143] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues,cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.

[0144] As used herein with reference to the relationship between DNA, cDNA, cRNA, RNA, protein / peptides, and the like “corresponding to” or “encoding” (used interchangeably herein) refers to the underlying biological relationship between these different molecules. As such, one of skill in the art would understand that operatively “corresponding to” can direct them to determine the possible underlying and / or resulting sequences of other molecules given the sequence of any other molecule which has a similar biological relationship with these molecules. For example, from a DNA sequence an RNA sequence can be determined and from an RNA sequence a cDNA sequence can be determined.

[0145] As used herein, the term “encode” refers to principle that DNA can be transcribed into RNA, which can then be translated into amino acid sequences that can form proteins. Thus, if a polynucleotide is said to encode another polynucleotide (e.g., RNA) or a protein it means that its sequence is such that when transcribed and / or translated it forms the noted polynucleotide or protein.

[0146] As used herein, “culturing” can refer to maintaining cells under conditions in which they can proliferate and avoid senescence as a group of cells. “Culturing” can also include conditions in which the cells also or alternatively differentiate.

[0147] As used herein, “expression” refers to the process by which polynucleotides are transcribed into RNA transcripts. In the context of mRNA and other translated RNA species, “expression” also refers to the process or processes by which the transcribed RNA is subsequently translated into peptides, polypeptides, or proteins. In some instances, “expression” can also be a reflection of the stability of a given RNA. For example, when one measures RNA, depending on the method of detection and / or quantification of the RNA as well as other techniques used in conjunction with RNA detection and / or quantification, it can be that increased / decreased RNA transcript levels are the result of increased / decreased transcription and / or increased / decreased stability and / or degradation of the RNA transcript. One of ordinary skill in the art will appreciate these techniques and the relation “expression” in these various contexts to the underlying biological mechanisms.

[0148] As used herein, the terms “guide polynucleotide,” “guide sequence,” or “guide RNA” as can refer to any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence. The degree of complementarity between a guide polynucleotide and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g. the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). A guide polynucleotide (also referred to herein as a guide sequence and includes single guide sequences (sgRNA)) can be about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, 90, 100, 110, 112, 115, 120, 130, 140, or more nucleotides in length. The guide polynucleotide can include a nucleotide sequence that is complementary to a target DNA sequence. This portion of the guide sequence can be referred to as the complementary region of the guide RNA. In some contexts, the two are distinguished from one another by calling one the complementary region or target region and the rest of the polynucleotide the guide sequence or tracrRNA. The guide sequence can also include one or more miRNA target sequences coupled to the 3 ’ end of the guide sequence. The guide sequence can include one or more MS2 RNA aptamers incorporated within the portion of the guide strand that is not the complementary portion. As used herein the term guide sequence can include any specially modified guide sequences, including but not limited to those configured for use in synergistic activation mediator (SAM) implemented CRISPR (Nature 517, 583-588 (29 January 2015) or suppression (Cell Volume 154, Issue 2, 18 July 2013, Pages 442-451). A guide polynucleotide can be less than about 150, 125, 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length. The ability of a guide polynucleotide to direct sequence-specific binding of a CRISPR complex to a target sequence may be assessed by any suitable assay. For example, the components of a CRISPR system sufficient to form a CRISPR complex, including the guide polynucleotide to be tested, may be provided to a host cell having the corresponding target sequence, such as by transfection with vectors encoding the components of the CRISPR sequence, followed by an assessment of preferential cleavage within the target sequence. Similarly, cleavage of a target polynucleotide sequence may be evaluated in a test tube by providing the target sequence, components of a CRISPR complex, including the guidepolynucleotide to be tested and a control guide polynucleotide different from the test guide polynucleotide, and comparing binding or rate of cleavage at the target sequence between the test and control guide polynucleotide reactions. Other assays are possible, and will occur to those skilled in the art.

[0149] A complementary region of the gRNA can be configured to target any DNA region of interest. The complementary region of the gRNA and the gRNA can be designed using a suitable gRNA design tool. Suitable tools are known in the art and are available to the skilled artisan. As such, the constructs described herein are enabled for any desired target DNA so long as it is CRISPR compatible according to the known requirements for CRISPR activation or inhibiton.

[0150] A guide polynucleotide can be selected to reduce the degree of secondary structure within the guide polynucleotide. Secondary structure may be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculating the minimal Gibbs free energy. An example of one such algorithm is mFold, as described by Zuker & Stiegler ((1981) Nucleic Acids Res. 9, 133-148). Another example folding algorithm is the online webserver RNAfold, developed at Institute for Theoretical Chemistry at the University of Vienna, using the centroid structure prediction algorithm (see e.g. Gruber et al., (2008) Cell 106: 23-24; and Carr & Church (2009) Nature Biotechnol. 27: 1151-1162).

[0151] As used herein, “deoxyribonucleic acid (DNA)” and “ribonucleic acid (RNA)” can generally refer to any polyribonucleotide or polydeoxyribonucleotide (collectively polynucleotides), which may be unmodified RNA or DNA or modified RNA or DNA. RNA can be in the form of non-coding RNA such as tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), anti-sense RNA, RNAi (RNA interference construct), siRNA (short interfering RNA), microRNA (miRNA), long non-coding RNA (IncRNA) ribozymes, aptamers, guide RNA (gRNA), coding mRNA (messenger RNA), cell-free DNA (cfDNA), circulating cfDNA, and / or the like.

[0152] As used herein, “isolated” means separated from constituents, cellular and otherwise, in which the polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, are normally associated with in nature. A non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, do not require “isolation” to distinguish it from its naturally occurring counterpart.

[0153] As used herein, “nucleic acid,” “nucleotide sequence,” and “polynucleotide” can be used interchangeably herein and can generally refer to a string of at least two base-sugar-phosphate combinations and refers to, among others, single-and double-stranded DNA, DNA that is a mixture of single-and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, polynucleotide as used herein can refer to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions can be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple-helical region often is an oligonucleotide. “Polynucleotide” and “nucleic acids” also encompasses such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells, inter alia. For instance, the term polynucleotide as used herein can include DNAs or RNAs as described herein that contain one or more modified bases. Thus, DNAs or RNAs including unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. “Polynucleotide”, “nucleotide sequences” and “nucleic acids” also includes PNAs (peptide nucleic acids), phosphorothioates, and other variants of the phosphate backbone of native nucleic acids. Natural nucleic acids have a phosphate backbone, artificial nucleic acids can contain other types of backbones, but contain the same bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are “nucleic acids” or "polynucleotides" as that term is intended herein. As used herein, “nucleic acid sequence” and “oligonucleotide” also encompasses a nucleic acid and polynucleotide as defined elsewhere herein.

[0154] As used herein, a “population" of cells (or “cell population”) is any number of cells greater than 1. In some embodiments, the cell population contains at least 1x102, at least 1x103 cells, at least 1x104 cells, at least at least 1x105 cells, at least 1x106 cells, at least 1x107 cells, at least 1x108 cells, at least 1x109 cells, at least 1x1010, at least 1x1020, at least 1x1030, at least 1x1040, or at least 1x1050 cells.

[0155] As used herein, “polypeptides” or “proteins” refers to amino acid residue sequences. Those sequences are written left to right in the direction from the amino to the carboxyterminus. In accordance with standard nomenclature, amino acid residue sequences are denominated by either a three letter or a single letter code as indicated as follows: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C), Glutamine (Gin, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine (His, H), Isoleucine (He, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (Vai, V). “Protein” and “Polypeptide” can refer to a molecule composed of one or more chains of amino acids in a specific order. The term protein is used interchangeable with “polypeptide.” The order is determined by the base sequence of nucleotides in the gene coding for the protein. Proteins can be required for the structure, function, and regulation of the body ’ s cells, tissues, and organs.

[0156] As used herein, the term “recombinant” or “engineered” can generally refer to a non-naturally occurring nucleic acid, nucleic acid construct, or polypeptide. Such non-naturally occurring nucleic acids may include natural nucleic acids that have been modified, for example that have deletions, substitutions, inversions, insertions, etc., and / or combinations of nucleic acid sequences of different origin that are joined using molecular biology technologies (e.g., a nucleic acid sequences encoding a fusion protein (e.g., a protein or polypeptide formed from the combination of two different proteins or protein fragments), the combination of a nucleic acid encoding a polypeptide to a promoter sequence, where the coding sequence and promoter sequence are from different sources or otherwise do not typically occur together naturally (e.g., a nucleic acid and a constitutive promoter), etc. Recombinant or engineered can also refer to the polypeptide encoded by the recombinant nucleic acid. Non-naturally occurring nucleic acids or polypeptides include nucleic acids and polypeptides modified by man.

[0157] As used herein, the term “specific binding” refers to non-covalent physical association of a first and a second moiety wherein the association between the first and second moi eties is at least 2 times as strong, at least 5 times as strong as, at least 10 times as strong as, at least 50 times as strong as, at least 100 times as strong as, or stronger than the association of either moiety with most or all other moieties present in the environment in which binding occurs. Binding of two or more entities may be considered specific if the equilibrium dissociation constant, Kd, is 10-3M or less, 10-4M or less, 10-5M or less, 10-6M or less, 10-7M or less, 10-8M or less, 10-9M or less, 10-10M or less, 10-11M or less, or 10-12M or less under the conditions employed, e.g., under physiological conditions such as those inside a cellor consistent with cell survival. In some embodiments, specific binding can be accomplished by a plurality of weaker interactions (e.g., a plurality of individual interactions, wherein each individual interaction is characterized by a Kd of greater than 10-3M). In some embodiments, specific binding, which can be referred to as “molecular recognition,” is a saturable binding interaction between two entities that is dependent on complementary orientation of functional groups on each entity. Examples of specific binding interactions include primer-polynucleotide interaction, aptamer-aptamer target interactions, antibody-antigen interactions, avidin-biotin interactions, ligand-receptor interactions, metal-chelate interactions, hybridization between complementary nucleic acids, etc.

[0158] As used herein, “therapeutic” refers to treating, healing, and / or ameliorating a disease, disorder, condition, or side effect, or to decreasing in the rate of advancement of a disease, disorder, condition, or side effect. A “therapeutically protein” or “therapeutic molecule” can therefore refer to protein or molecule that is configured to, designed to, or is effective to treat a disease or a symptom thereof.

[0159] Various embodiments are described hereinafter. It should be noted that the specific embodiments are not intended as an exhaustive description or as a limitation to the broader aspects discussed herein. One aspect described in conjunction with a particular embodiment is not necessarily limited to that embodiment and can be practiced with any other embodiment s). Reference throughout this specification to “one embodiment”, “an embodiment,” “an example embodiment,” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment,” “in an embodiment,” or “an example embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention. For example, in the appended claims, any of the claimed embodiments can be used in any combination.

[0160] All publications, published patent documents, and patent applications cited herein are hereby incorporated by reference to the same extent as though each individual publication,published patent document, or patent application was specifically and individually indicated as being incorporated by reference.OVERVIEW

[0161] Existing genetic switch systems often rely on tightly balanced expression levels, constitutive repressors, or externally maintained inputs to preserve bistability, which can limit robustness and scalability. Such systems frequently exhibit state leakage, sensitivity to cellular context, loss of memory across cell divisions, or unintended cross-activation when expanded beyond two states. In addition, many conventional switch architectures are difficult to reprogram, require extensive redesign to accommodate new targets or inputs, or fail to support orthogonal control of multiple regulatory states within the same cell. These limitations constrain the reliability, flexibility, and extensibility of current genetic switch technologies, particularly in complex or dynamic biological environments. As such there exists a need for alternative systems.

[0162] With that said, embodiments disclosed herein describe a programmable genetic toggle switch system that includes at least a first state switch and a second state switch configured to be mutually exclusive, such that activation of one state switch results in repression of the other. As set forth in the claims, each state switch includes a control expression construct that supports self-activation and cross-repression and can regulate one or more functional expression constructs through CRISPR-based transcriptional activation and inhibition. This architecture allows a selected regulatory state to be established, maintained, and reversed in response to defined stimuli, including orthogonal stimuli, and supports extension beyond two states. By decoupling state maintenance from continuous external input and by using programmable regulatory elements rather than fixed repressors, the claimed systems reduce state leakage, improve robustness across cell divisions, and support scalable, reprogrammable control of functional output. The systems further allow coordinated regulation of reporters, effectors, and payloads in cells, populations of cells, or organisms, addressing limitations of prior genetic switch designs that lack stability, extensibility, or orthogonal control. Other compositions, compounds, methods, features, and advantages of the present disclosure will be or become apparent to one having ordinary skill in the art upon examination of the following drawings, detailed description, and examples. It is intended that all such additional compositions, compounds, methods, features, and advantages be included within this description, and be within the scope of the present disclosure.PROGRAMMABLE GENETIC TOGGLE SWITCH SYSTEMS

[0163] Described herein are programmable genetic toggle switch systems that include at least a first state switch and a second state switch configured to operate in a mutually exclusive manner, such that activation of one state switch represses expression associated with the other state switch. The systems enable controlled switching between discrete regulatory states and maintenance of a selected regulatory state through self-reinforcing regulatory interactions. It will be appreciated that using the logic demonstrated herein, that any number of switches can be configured and included in the system to provide mutually exclusive or tailored switch control and operation.

[0164] As used herein, a “state switch” refers to a regulatory unit that includes one or more expression constructs configured to establish, maintain, and enforce a regulatory state. Each state switch includes at least a control expression construct and can further include one or more functional expression constructs. The state switches are configured such that activation of a first state switch induces positive regulation of transcription associated with the first state switch and repression of transcription associated with the second state switch, while activation of the second state switch induces positive regulation of transcription associated with the second state switch and repression of transcription associated with the first state switch.

[0165] In the disclosed systems, the first state switch and the second state switch are mutually exclusive, meaning that regulatory activity associated with one state switch suppresses establishment or maintenance of the alternative regulatory state. Mutual exclusivity is enforced through coordinated activation and repression mediated by the control expression constructs of the respective state switches.

[0166] Upon activation, a state switch is configured to self-activate, thereby reinforcing its own transcriptional activity, while simultaneously repressing transcription associated with the opposing state switch. This regulatory architecture allows the system to function as a toggle, wherein the system occupies either a first regulatory state associated with the first state switch or a second regulatory state associated with the second state switch. Once a regulatory state is established, that state can be maintained in the absence of continued stimulation.

[0167] The systems described herein implement a separation between control-layer regulation and functional output, which provides modularity, scalability, and programmability. The control layer is implemented through control expression constructs, which enforce state selection, self-activation, and repression of the opposing state. The functional layer isimplemented through functional expression constructs, which encode one or more functional elements whose expression is regulated by the control layer.

[0168] This separation allows regulatory logic to be encoded independently of functional output. As a result, a single toggle switch architecture can be coupled to different functional expression constructs to achieve diverse outputs, including reporting, regulation of downstream genes, or execution of biological functions, without altering the underlying control logic.

[0169] Each state switch includes a control expression construct that contains a promoter, one or more regulatory signal target sites, and a regulatory element region encoding one or more control expression construct regulatory elements. The promoter is operatively coupled to the regulatory signal target sites and the regulatory element region such that transcription of the regulatory elements is regulated by interactions at the regulatory signal target sites.

[0170] The regulatory elements encoded by the regulatory element region are configured to interact with regulatory signal target sites associated with the same control expression construct and with regulatory signal target sites associated with the control expression construct of the opposing state switch. Through these interactions, the control expression constructs mediate both self-activation and cross-repression, thereby enforcing mutual exclusivity between the first and second state switches.

[0171] Each state switch can further include one or more functional expression constructs. Functional expression constructs include a promoter and a functional element region encoding one or more functional elements. Expression of the functional expression constructs is regulated by the control expression constructs such that activation of a state switch induces expression of functional elements associated with that state switch while repressing expression of functional elements associated with the opposing state switch.

[0172] The functional elements encoded by functional expression constructs provide outputs associated with the regulatory state of the system. Functional elements can function as reporters, effectors, regulatory molecules, or payloads depending on the intended application. Because functional expression constructs are regulated by the control layer, functional output is directly coupled to the regulatory state established by the toggle switch system.

[0173] FIGS. 15-17B illustrate non-limiting schematic representations of programmable genetic toggle switch architectures consistent with the systems described herein. In the illustrated architectures, each state switch includes a control expression construct and a functional expression construct, with regulatory interactions configured to enable self-activation and cross-repression between the state switches. It will be appreciated that additional switches can be included and configured based upon the logic presented in the context of two switches. The illustrated figures provide conceptual representations of regulatory relationships and system organization and do not limit the scope of the disclosed systems, which can be implemented using alternative configurations, numbers of expression constructs, or regulatory interactions consistent with the claims.Control Expression Constructs

[0174] Control expression constructs form the regulatory core of each state switch within the programmable genetic toggle switch systems described herein. A control expression construct functions to establish, maintain, and enforce a regulatory state by directing selfactivation of the associated state switch and repression of the opposing state switch.

[0175] Each state switch includes a control expression construct that operates as part of a coordinated regulatory architecture. Through interactions mediated by regulatory signal target sites and regulatory elements encoded by the control expression construct, the system can transition between regulatory states and maintain a selected regulatory state over time.General Architecture of Control Expression Constructs

[0176] A control expression construct includes a promoter, one or more regulatory signal target sites associated with the promoter, and a regulatory element region encoding one or more control expression construct regulatory elements. The promoter is operatively coupled to the regulatory signal target sites and the regulatory element region such that transcription of the regulatory elements responds to regulatory signals interacting with the regulatory signal target sites.

[0177] The regulatory elements encoded by the regulatory element region are configured to interact with regulatory signal target sites associated with the same control expression construct and with regulatory signal target sites associated with the control expression construct of the opposing state switch. Through these interactions, the control expression constructs participate in feedback regulation that reinforces the active regulatory state and suppresses the inactive regulatory state.Control Expression Construct Promoters

[0178] The promoter of a control expression construct directs transcription of the regulatory element region and participates in regulatory feedback through interactions with regulatory signal target sites. The promoter can be selected to provide appropriatetranscriptional strength, responsiveness, and stability for self-activation and cross-repression within a state switch.

[0179] Control expression construct promoters can include constitutive promoters, inducible promoters, repressible promoters, optogenetically regulated promoters, synthetic promoters, or hybrid promoters. Promoters can be selected to support sustained transcription following activation or to support dynamic switching behavior in response to regulatory signals.

[0180] Exemplary constitutive promoters that can be used as promoters in the control expression construct include, but are not limited to, bacterial promoters such as J23100, J23101, J23105, J23110, J23115, lac-derived constitutive promoters, and other synthetic constitutive promoters; yeast promoters such as TEF1, ADH1, PGK1, TDH3, RPL18B, RPL41A, and CYC1; and mammalian promoters such as CMV, EF1α, PGK, SV40, CAG, and ubiquitin promoters.

[0181] Exemplary inducible or regulatable promoters that can be used as promoters in the control expression construct include, but are not limited to, yeast galactose-responsive promoters such as GALI, GAL10, GAL7, GAL2, GAL3, and bidirectional GAL1–GAL10 promoters; bacterial promoters such as lac, tet, araBAD, rhaBAD, and xyl promoters; and mammalian promoters responsive to small molecules or signaling pathways, including tetracycline-responsive promoters, steroid hormone–responsive promoters, metal-responsive promoters, and stress-responsive promoters.

[0182] Exemplary optogenetically regulated promoters that can be used as promoters in the control expression construct include, but are not limited to, promoters regulated by blue light, red light, or other wavelengths, including promoters responsive to light-regulated transcription factors, light-activated dimerization systems, or light-controlled DNA-binding systems. Such promoters include synthetic promoters derived from light-responsive transcription factor binding sites, promoters regulated by EL222-based systems, promoters regulated by red-light-responsive systems such as phytochrome-based or bacteriophytochrome-based systems, and promoters incorporating light-responsive regulatory modules operably linked to core promoter elements.

[0183] Exemplary repressible promoters that can be used as promoters in the control expression construct include promoters subject to repression by transcriptional repressors, CRISPR transcriptional inhibitor complexes, or environmental conditions such as glucoserepression, nutrient repression, or stress-mediated repression. Such promoters include glucose-repressible GAL promoters, methionine-repressible promoters, copper-repressible promoters, and synthetic repressible promoters.

[0184] Synthetic or engineered promoters can also be used, including promoters designed with defined regulatory signal target sites, altered basal transcriptional activity, tunable responsiveness, or modular architectures. Hybrid promoters incorporating features of natural promoters and synthetic regulatory elements can be used to support desired regulatory behavior within the control expression construct.

[0185] The selection of a promoter for a control expression construct can be coordinated with the number, identity, and arrangement of regulatory signal target sites and with the regulatory elements encoded by the regulatory element region, such that the promoter supports self-activation of the associated state switch and repression of the opposing state switch. Promoter Responsiveness Across State Switches

[0186] The control expression construct promoters associated with different state switches can be selected such that the promoters respond to different regulatory stimuli. In this manner, activation of one state switch can be driven by a stimulus that does not activate the control expression construct promoters of other state switches. This arrangement supports selective activation of a desired regulatory state and reduces unintended cross-activation.

[0187] While the programmable genetic toggle switch systems described herein are illustrated using a first state switch and a second state switch as a base example, the same regulatory logic can be applied to systems that include more than two state switches. In such systems, each state switch can be associated with a distinct control expression construct promoter responsive to a different stimulus, allowing the system to switch among multiple regulatory states in a mutually exclusive or selectively exclusive manner.

[0188] Promoters associated with different state switches can be responsive to distinct classes of stimuli, including different chemical signals, environmental conditions, signaling pathways, or physical inputs. For example, a first control expression construct promoter can respond to a first stimulus, a second control expression construct promoter can respond to a second stimulus, and a third control expression construct promoter can respond to a third stimulus, where each stimulus is orthogonal to the others. Orthogonality in promoter responsiveness allows independent and selective control of multiple state switches within the same system.

[0189] In configurations in which control expression construct promoters are regulated by optical stimuli, promoters associated with different state switches can be responsive to different wavelengths of light. For example, a first control expression construct promoter can respond to light within a first wavelength range, a second control expression construct promoter can respond to light within a second wavelength range distinct from the first, and additional control expression construct promoters can respond to additional wavelength ranges. Selection of a particular wavelength can selectively activate a corresponding state switch without activating other state switches.

[0190] Optical regulation can include promoters responsive to blue light, red light, green light, far-red light, or other regions of the electromagnetic spectrum. Control expression construct promoters responsive to different wavelengths can be used in combination within the same system to provide multiplexed and orthogonal optical control of regulatory states.

[0191] The use of distinct promoters responsive to different stimuli, including different wavelengths of light, supports precise control of state switching, facilitates reversible and repeatable switching among multiple regulatory states, and allows integration of multiple independent control inputs within a single programmable genetic toggle switch system.

[0192] Additional suitable promoters are described elsewhere herein such as in the section titled Vectors.Control Expression Construct Regulatory Signal Target Sites

[0193] Control expression construct regulatory signal target sites are nucleotide sequences operatively coupled with the control expression construct promoter that interact with regulatory signals to modulate promoter activity. Control expression construct regulatory signal target sites can function as activator signal target sites, repressor signal target sites, or combinations thereof.

[0194] Control expression construct regulatory signal target sites can be positioned upstream of, downstream of, or within the control expression construct promoter region. Multiple control expression construct regulatory signal target sites can be arranged in series, in tandem, or in other configurations to support cooperative, competitive, additive, or hierarchical regulatory interactions or responses. The number, sequence, and arrangement of control expression regulatory signal target sites can be selected to support desired self-activation and cross-repression behaviors.

[0195] Control expression construct regulatory signal target sites can be present in varying numbers within the control expression constructs. In some embodiments, the control expression construct regulatory signal target sites are operatively coupled to the promoter of the control expression construct. In some embodiments, the control expression construct regulatory signal target sites can be within the promoter of the control expression construct. In some embodiments, the control expression construct contains at least two regulatory signal target sites, including one or more activator signal target sites and one or more repressor signal target sites. In some embodiments, the control expression construct contains 2-50 or more regulatory signal target sites. In some embodiments, the control expression construct contains two regulatory signal target sites, three regulatory signal target sites, four regulatory signal target sites, or more than four regulatory signal target sites. In some configurations, the control expression construct contains includes between two and ten regulatory signal target sites, between two and eight regulatory signal target sites, or between two and five regulatory signal target sites.

[0196] Regulatory signal target sites included in control expression constructs can include one or more activator signal target sites and one or more repressor signal target sites. In some configurations, a control expression construct promoter includes one or more activator signal target sites and one or more repressor signal target sites. Exemplary configurations include control expression construct promoters including one activator signal target site and one repressor signal target site, two activator signal target sites and one repressor signal target site, one activator signal target site and two repressor signal target sites, or multiple activator and multiple repressor signal target sites.

[0197] In some configurations, the number of activator signal target sites associated with a control expression construct can range from one to fifty or more. Exemplary configurations include between one and twenty activator signal target sites, between one and ten activator signal target sites, between two and eight activator signal target sites, or between two and five activator signal target sites. Similarly, the number of repressor signal target sites associated with a control expression construct can range from one to fifty or more, including between one and twenty repressor signal target sites, between one and ten repressor signal target sites, between two and eight repressor signal target sites, or between two and five repressor signal target sites.

[0198] Control expression construct regulatory signal target sites can support interactions with a variety of regulatory element types, including RNA-based regulatory elements, proteinbased regulatory elements, and regulatory elements that function as part of ribonucleoprotein complexes. Interactions between regulatory elements and regulatory signal target sites can occur through sequence-specific recognition, structural or conformational recognition, protein–DNA interactions, cooperative binding, or combinations thereof.

[0199] Control expression construct regulatory signal target sites can include binding sites for transcription factors, response elements for inducible regulatory proteins, operator sequences, enhancer elements, or synthetic regulatory sequences. Such target sites can interact with regulatory elements based on binding affinity, structural complementarity, or cooperative interactions with additional regulatory components.

[0200] In some configurations, regulatory signal target sites function as activator signal target sites that promote transcription when bound by corresponding regulatory elements. In other configurations, regulatory signal target sites function as repressor signal target sites that suppress transcription when bound by corresponding regulatory elements. The control expression construct includes both activator and repressor signal target sites, allowing combinatorial regulation of transcription of the control expression construct.

[0201] In some embodiments, control expression construct regulatory signal target sites include guide RNA target sites configured for interaction with guide RNAs encoded by control expression construct regulatory element regions. These guide RNA target sites direct recruitment of CRISPR transcriptional regulatory machinery to defined locations relative to a promoter.

[0202] Guide RNA target sites associated with control expression constructs can be selected to support recruitment of CRISPR transcriptional activator complexes or CRISPR transcriptional inhibitor complexes by control expression construct regulatory elements. The regulatory outcome associated with a given guide RNA target site is determined by the control expression construct regulatory element interacting with the site, including the identity of the guide RNA and its associated regulatory machinery.

[0203] Guide RNA target sites can be positioned upstream of a promoter, overlapping a promoter, downstream of a promoter, or within regions that influence transcriptional initiation or elongation. Placement of guide RNA target sites relative to the transcription start site can beselected to support transcriptional activation, transcriptional repression, or modulation of promoter accessibility mediated by control expression construct regulatory elements.

[0204] Guide RNA target sites used by control expression construct regulatory elements can be selected based on sequence characteristics, promoter context, and desired regulatory outcome. Design considerations can include proximity to the transcription start site, orientation relative to the promoter, spacing between multiple guide RNA target sites, and compatibility with the CRISPR transcriptional regulatory machinery interacting with the control expression construct regulatory elements.

[0205] Multiple guide RNA target sites can be included within a single promoter region to support cooperative regulation, graded regulatory responses, redundancy, or robustness. Guide RNA target sites can be identical or distinct and can be arranged to support binding of multiple control expression construct regulatory elements simultaneously or sequentially.

[0206] In configurations in which control expression construct regulatory elements mediate transcriptional activation, guide RNA target sites can be selected to position recruited transcriptional activator complexes in regions that promote transcriptional initiation or enhancer-like activity. In configurations in which control expression construct regulatory elements mediate transcriptional repression, guide RNA target sites can be selected to position recruited transcriptional inhibitor complexes in regions that interfere with transcriptional initiation, transcriptional elongation, or promoter accessibility.

[0207] Control expression construct regulatory signal target sites can be selected to support multiplexed regulation by multiple control expression construct regulatory elements. In such configurations, distinct guide RNA target sites are designed to interact with distinct guide RNAs encoded by control expression construct regulatory element regions without crossreactivity.

[0208] In systems that include multiple state switches, different sets of control expression construct regulatory signal target sites can be associated with different state switches to support selective activation and repression across regulatory states. Orthogonal sets of guide RNA target sites can be used to support independent regulation of multiple promoters by different control expression construct regulatory elements within the same system.

[0209] Through selection and arrangement of regulatory signal target sites responsive to control expression construct regulatory elements, the system supports coordinated self-activation, cross-repression, and regulation of functional expression constructs while preserving specificity and scalability across multiple regulatory states.Regulatory Element Regions

[0210] The regulatory element region of a control expression construct encodes one or more regulatory elements that participate in transcriptional regulation. The regulatory element region is transcribed under control of the promoter and produces regulatory elements that interact with regulatory signal target sites.

[0211] The regulatory element region can encode a single regulatory element or multiple regulatory elements. In some configurations, the regulatory element region encodes regulatory elements that function in self-activation by interacting with regulatory signal target sites associated with the same control expression construct. In other configurations, the regulatory element region encodes regulatory elements that function in repression of the opposing state switch by interacting with regulatory signal target sites associated with the control expression construct of the opposing state switch.

[0212] Control expression construct regulatory element regions can be organized in a variety of architectures to support expression, processing, and function of one or more control expression construct regulatory elements. The organization of the regulatory element region influences the number, identity, timing, and relative activity of regulatory elements produced by a control expression construct.

[0213] In some configurations, a regulatory element region encodes a single control expression construct regulatory element transcribed as an individual regulatory molecule. In other configurations, a regulatory element region encodes multiple control expression construct regulatory elements arranged as an array or cluster within a single transcript. Such arrays can include regulatory elements arranged in series, separated by spacer sequences, processing signals, or structural motifs.

[0214] Where multiple control expression construct regulatory elements are encoded within a single transcript, the regulatory element region can include sequence features that support processing of the transcript into individual regulatory elements. Processing can occur through the action of RNA processing enzymes, ribozymes, or other processing mechanisms present in the system. In some configurations, processing yields individual regulatory elements with defined termini and functional activity.

[0215] The regulatory element region can be organized to support coordinated or differential expression of regulatory elements. For example, regulatory elements encoded within a single regulatory element region can be produced at similar stoichiometries, while regulatory elements encoded in separate regulatory element regions or under different promoters can be produced at different relative levels. Such organization allows tuning of selfactivation strength, cross-repression strength, and regulation of functional expression constructs.

[0216] In configurations using guide RNAs as control expression construct regulatory elements, regulatory element regions can encode guide RNAs arranged in arrays suitable for processing into individual guide RNAs. Guide RNAs within an array can target regulatory signal target sites associated with control expression constructs, functional expression constructs, or both. The order, spacing, and composition of guide RNAs within an array can be selected to support desired regulatory logic, timing, or hierarchy.

[0217] Regulatory element regions can also encode combinations of different types of control expression construct regulatory elements. For example, a regulatory element region can encode multiple guide RNAs, a guide RNA and a protein-based regulatory element, or regulatory elements that interact with different transcriptional regulatory mechanisms. Such mixed regulatory element regions support integration of multiple regulatory modalities within a single control expression construct.

[0218] Through selection and organization of regulatory element regions, the programmable genetic toggle switch system supports flexible implementation of selfactivation, cross-repression, multiplexed regulation, and coordination of control-layer regulation with functional expression constructs across simple and complex regulatory architectures.Control Expression Construct Regulatory Elements

[0219] Control expression construct regulatory elements refer to molecules encoded by the regulatory element region that participate in transcriptional regulation within the system. These regulatory elements can interact with regulatory signal target sites that are operatively coupled with promoters of control expression constructs and functional expression constructs through a variety of mechanisms, including sequence-specific interactions, structural or conformational recognition, protein–DNA interactions, protein-protein interactions, or combinations thereof.

[0220] In some configurations, regulatory elements are sequence-directed regulatory elements that interact with regulatory signal target sites through base-pairing or sequencespecific binding. In other configurations, regulatory elements include transcription factors or other regulatory proteins that interact with regulatory signal target sites based on structural recognition, binding affinity, or cooperative interactions with additional regulatory components. Through these mechanisms, regulatory elements can modulate transcriptional activity of control expression constructs and functional expression constructs. Additional suitable regulatory elements are described elsewhere herein such as in the section titled Vectors.

[0221] Control expression construct regulatory elements can include DNA or RNA-based regulatory elements, protein-based regulatory elements, or regulatory elements that form ribonucleoprotein complexes. The regulatory elements can act directly or indirectly to promote transcriptional activation or transcriptional repression at the associated regulatory signal target sites.

[0222] Control expression construct regulatory elements can function independently or in cooperation with other regulatory elements or regulatory complexes present in the system.

[0223] In some embodiments, control expression construct regulatory elements include guide RNAs configured to interact with regulatory signal target sites and to recruit transcriptional regulatory machinery. Activation guide RNAs can interact with promoter activator signal target sites to promote transcription of the associated regulatory element region. Repression guide RNAs can interact with promoter repressor signal target sites to suppress transcription of the associated regulatory element region.

[0224] Guide RNAs encoded by a control expression construct can include distinct classes associated with activation or repression. In some configurations, activation guide RNAs and repression guide RNAs include distinct guide RNA scaffold sequences such that recruitment of transcriptional activator complexes or transcriptional inhibitor complexes is determined by guide RNA scaffold identity. This arrangement allows regulatory logic to be encoded within the guide RNAs themselves.

[0225] Control expression construct regulatory elements can regulate transcription of functional expression constructs in addition to regulating transcription of control expression constructs. Functional expression constructs include promoters associated with regulatory signal target sites that respond to control expression construct regulatory elements.

[0226] In some configurations, control expression construct regulatory elements interact with regulatory signal target sites associated with functional expression construct promoters to activate transcription of functional elements associated with an active state switch. In other configurations, control expression construct regulatory elements interact with regulatory signal target sites associated with functional expression construct promoters to repress transcription of functional elements associated with an inactive or opposing state switch.

[0227] A single control expression construct regulatory element can interact with regulatory signal target sites present in promoters of both control expression constructs and functional expression constructs. Alternatively, different control expression construct regulatory elements encoded by the same regulatory element region can interact with distinct regulatory signal target sites associated with control expression constructs and functional expression constructs. Through these interactions, control expression construct regulatory elements couple regulatory state selection to functional output.

[0228] Control expression construct regulatory element regions can encode multiple control expression construct regulatory elements configured to operate in a multiplexed and orthogonal manner. In such configurations, different regulatory elements encoded by the same regulatory element region or by different regulatory element regions are configured to regulate different promoters or different regulatory signal target sites without unintended crossinterference.

[0229] Multiplexing of control expression construct regulatory elements allows a single state switch to encode multiple regulatory functions. For example, a regulatory element region can encode a first control expression construct regulatory element that participates in selfactivation of the associated state switch and a second control expression construct regulatory element that participates in repression of one or more other state switches. In other configurations, a regulatory element region can encode control expression construct regulatory elements that regulate both control expression constructs and functional expression constructs.

[0230] Orthogonality among control expression construct regulatory elements can be achieved by selection of regulatory elements that differ in sequence specificity, binding characteristics, scaffold structure, associated regulatory machinery, or mode of interaction with regulatory signal target sites. Orthogonal regulatory elements can interact with distinct sets of regulatory signal target sites such that each regulatory element selectively regulates a defined promoter or set of promoters within the system.

[0231] In configurations using guide RNAs as control expression construct regulatory elements, orthogonality can be achieved by selection of guide RNAs with distinct target sequences, distinct guide RNA scaffold architectures, or distinct associated transcriptional regulatory complexes. Guide RNAs encoded by a control expression construct regulatory element region can be designed to act independently or cooperatively, allowing coordinated regulation of multiple promoters by a single state switch.

[0232] Control expression construct regulatory elements can be arranged to act simultaneously, sequentially, or conditionally. For example, multiple regulatory elements encoded by a regulatory element region can act in parallel to reinforce a regulatory state, or different regulatory elements can act at different stages of state switching to support transition dynamics, stabilization, or repression of alternative states.

[0233] Through multiplexing and orthogonality at the level of control expression construct regulatory elements, the programmable genetic toggle switch system supports scalable expansion from two-state configurations to multi-state configurations, coordinated regulation of control and functional expression constructs, and precise control over regulatory logic across complex genetic circuits.

[0234] In some configurations, the number of control expression construct activator regulatory elements encoded by a regulatory element region can range from one to fifty or more. Exemplary configurations include regulatory element regions encoding between one and twenty activator regulatory elements, between one and ten activator regulatory elements, between two and eight activator regulatory elements, or between two and five activator regulatory elements. Similarly, the number of control expression construct repressor regulatory elements encoded by a regulatory element region can range from one to fifty or more, including configurations encoding between one and twenty repressor regulatory elements, between one and ten repressor regulatory elements, between two and eight repressor regulatory elements, or between two and five repressor regulatory elements.

[0235] With respect to regulation of functional expression constructs, the number of control expression construct activator regulatory elements configured to activate functional expression construct promoters can range from one to fifty or more, including configurations encoding between one and twenty such activator regulatory elements or between one and ten such activator regulatory elements. The number of control expression construct repressor regulatory elements configured to repress functional expression construct promoters cansimilarly range from one to fifty or more, including configurations encoding between one and twenty or between one and ten such repressor regulatory elements. Activator and repressor regulatory elements acting on functional expression constructs can be present in equal or unequal numbers relative to those acting on control expression constructs, allowing independent tuning of state maintenance and functional output.Self-Activation and Cross-Repression

[0236] Control expression constructs are configured to support both self-activation and cross-repression. Self-activation occurs when regulatory elements encoded by a control expression construct interact with regulatory signal target sites associated with the same control expression construct to promote continued transcription of the regulatory element region. Cross-repression occurs when regulatory elements encoded by a control expression construct interact with regulatory signal target sites associated with the control expression construct of the opposing state switch to suppress transcription.

[0237] Control expression constructs associated with state switches are configured to support regulatory architectures based on self-activation and cross-repression. In the base two-state configuration, a control expression construct associated with an activated state switch reinforces its own transcriptional activity while repressing transcription associated with the opposing state switch. The same regulatory logic can be extended to systems that include more than two state switches.

[0238] In systems including three or more state switches, each state switch can include a control expression construct configured to self-activate upon activation and to repress one or more other state switches. Cross-repression can occur in a pairwise manner, such that each state switch represses all other state switches, or in a selective manner, such that each state switch represses a defined subset of other state switches. These configurations allow the system to operate as a mutually exclusive multi-state switch or as a selectively exclusive state-selection system.

[0239] In some configurations, the control expression constructs of multiple state switches are arranged in a winner-take-all architecture, in which activation of a particular state switch results in dominant self-activation and coordinated repression of all other state switches. In other configurations, hierarchical or directional repression architectures can be used, in which activation of one state switch represses some state switches while remaining permissive orneutral with respect to others. Such architectures support ordered state transitions or predefined state progression pathways.

[0240] Self-activation of a state switch can be mediated by regulatory elements encoded by the control expression construct interacting with regulatory signal target sites associated with the same control expression construct. Cross-repression can be mediated by regulatory elements encoded by a control expression construct interacting with regulatory signal target sites associated with control expression constructs of other state switches. The number, identity, and arrangement of regulatory signal target sites and regulatory elements can be selected to support desired regulatory relationships among multiple state switches.

[0241] Through coordinated self-activation and cross-repression across multiple state switches, the system can transition among multiple regulatory states and maintain a selected regulatory state over time. This architecture supports scalable expansion of the programmable genetic toggle switch system from two-state configurations to multi-state configurations while preserving selective activation, state stability, and controlled switching behavior.

[0242] Through coordinated self-activation and cross-repression, control expression constructs enforce mutual exclusivity between the first and second state switches and support stable maintenance of the selected regulatory state.Functional Expression Constructs

[0243] Functional expression constructs provide the output layer of the programmable genetic toggle switch systems described herein. Functional expression constructs are regulated by control expression constructs and produce functional elements whose expression state corresponds to the regulatory state selected by the system. Through regulation of functional expression constructs, changes in regulatory state are translated into changes in functional output.

[0244] Functional expression constructs are associated with one or more state switches. In some configurations, a functional expression construct is associated with a single state switch and is activated when that state switch is active. In other configurations, functional expression constructs associated with one state switch are repressed when a different state switch is active. Through this arrangement, functional output reflects the mutually exclusive or selectively exclusive regulatory state of the system.

[0245] A functional expression construct includes a promoter and a functional element region encoding one or more functional elements. The promoter is associated with one or moreregulatory signal target sites that interact with control expression construct regulatory elements to regulate transcription of the functional element region.

[0246] Functional expression constructs can be configured such that transcription of functional elements is activated by control expression construct regulatory elements associated with an active state switch and repressed by control expression construct regulatory elements associated with an inactive or opposing state switch. In this manner, functional expression constructs operate downstream of the control layer and respond to the regulatory logic implemented by the control expression constructs.Functional Expression Construct Promoters

[0247] Functional expression construct promoters direct transcription of functional element regions and respond to regulatory signals mediated by control expression construct regulatory elements. Functional expression construct promoters can be selected to provide appropriate transcriptional strength, dynamic range, and responsiveness to control-layer regulation.

[0248] Functional expression construct promoters can include constitutive promoters, inducible promoters, repressible promoters, optogenetically regulated promoters, synthetic promoters, or hybrid promoters. In some configurations, functional expression construct promoters are similar to or the same as control expression construct promoters. In other configurations, functional expression construct promoters differ from control expression construct promoters to allow independent tuning of functional output relative to control-layer regulation.

[0249] Functional expression construct promoters can include regulatory signal target sites positioned to support activation, repression, or combinatorial regulation by control expression construct regulatory elements. The number, identity, and arrangement of regulatory signal target sites can be selected to support desired functional output behavior.

[0250] Exemplary constitutive promoters that can be used as promoters in functional expression constructs include, but are not limited to, bacterial promoters such as J23100, J23101, J23105, J23110, and other synthetic constitutive promoters; yeast promoters such as TEF1, ADH1, PGK1, TDH3, RPL18B, RPL41A, and CYC1; and mammalian promoters such as CMV, EF1α, PGK, SV40, CAG, ubiquitin promoters, and tissue-preferred constitutive promoters. Such promoters are commonly used to drive expression of functional elements including fluorescent proteins, luminescent reporters, or other detectable output molecules.

[0251] Exemplary inducible or regulatable promoters that can be used as promoters in functional expression constructs include, but are not limited to, yeast galactose-responsive promoters such as GALI, GAL10, GAL7, GAL2, GAL3, and bidirectional GAL1–GAL10 promoters; bacterial promoters such as lac, tet, araBAD, rhaBAD, and xyl promoters; and mammalian promoters responsive to small molecules, hormones, or signaling pathways, including tetracycline-responsive promoters, steroid hormone–responsive promoters, metal-responsive promoters, heat-shock promoters, and stress-responsive promoters. Such promoters can be used to conditionally express functional elements including reporter proteins in response to defined stimuli.

[0252] Exemplary optogenetically regulated promoters that can be used as promoters in functional expression constructs include, but are not limited to, promoters responsive to blue light, red light, green light, far-red light, or other regions of the electromagnetic spectrum. Such promoters include synthetic promoters regulated by light-responsive transcription factors, promoters incorporating binding sites for optogenetic regulatory proteins, promoters regulated by EL222-based systems, promoters regulated by phytochrome-based or bacteriophytochromebased systems, and promoters incorporating modular light-responsive regulatory domains operatively linked to core promoter elements. Optogenetically regulated promoters can be used to drive expression of fluorescent proteins or other reporters that provide real-time readout of regulatory state switching.

[0253] Exemplary repressible promoters that can be used as promoters in functional expression constructs include promoters subject to repression by transcriptional repressors, CRISPR transcriptional inhibitor complexes, or environmental conditions. Such promoters include glucose-repressible GAL promoters, methionine-repressible promoters, copper-repressible promoters, tetracycline-repressible promoters, and synthetic repressible promoters incorporating repressor binding sites. Repressible promoters can be used to suppress expression of functional elements associated with inactive or opposing regulatory states.

[0254] Synthetic or engineered promoters can also be used as functional expression construct promoters. Such promoters can be designed with defined regulatory signal target sites, altered basal transcriptional activity, tunable responsiveness, or modular architectures. Hybrid promoters combining features of natural promoters and synthetic regulatory elements can be used to support desired functional output behavior, including strong expression offluorescent proteins, low background expression in inactive states, or rapid transitions between expression states.

[0255] The selection of a functional expression construct promoter can be coordinated with the number, identity, and arrangement of regulatory signal target sites and with the control expression construct regulatory elements that interact with those target sites, such that expression of functional elements, including reporter proteins, corresponds to the regulatory state of the programmable genetic toggle switch system.

[0256] In some embodiments, the functional expression construct promoter is a low, medium, or high expression promoter. As used herein, a “low expression promoter” (abbreviated pLow) is a promoter that drives expression that is above background expression but less than 10-fold above background expression level. As used herein “a medium expression promoter” (abbreviated pMed) refers to a promoter that results in about 10-fold increase in expression above background. As used herein, a “high expression promoter” refers to a promoter that results in a much greater than 10-fold increase in expression above background. In some embodiments, the pMed promoter is P34 or P97. In some embodiments, the pLow promoter is P92 or P94. Additional suitable promoters are described elsewhere herein such as in the section titled Vectors.Functional Expression Construct Regulatory Binding Sites

[0257] Each functional expression construct includes at least two functional expression construct regulatory element signal target sites including at least one functional expression construct activator signal target site and at least one functional expression construct repressor signal target site. The at least two functional expression construct regulatory element signal target sites can be operatively coupled with a functional expression construct promoter. Functional expression construct regulatory element signal target sites can interact with control expression construct regulatory elements to regulate transcription of a functional element region.

[0258] Interaction of control expression construct regulatory elements with a functional expression construct activator signal target site promotes transcription of the associated functional expression construct, while interaction of control expression construct regulatory elements with a functional expression construct repressor signal target site suppresses transcription of the associated functional expression construct. Through the presence of both activator and repressor signal target sites, functional expression constructs can responddifferentially to regulatory elements associated with different regulatory states as described in greater detail elsewhere herein.

[0259] Functional expression construct regulatory element signal target sites function as loci through which regulatory state information established by control expression constructs is translated into functional output. Changes in regulatory state result in corresponding changes in expression of functional elements through coordinated activation and repression mediated by these signal target sites.

[0260] Functional expression construct regulatory element signal target sites can be positioned upstream of, downstream of, or within the functional expression construct promoter region. Multiple functional expression construct regulatory signal target sites can be arranged in series, in tandem, or in other configurations to support cooperative, competitive, additive, or hierarchical regulatory interactions or responses. The number, sequence, and arrangement of control expression regulatory signal target sites can be selected to support desired selfactivation and cross-repression behaviors.

[0261] Functional expression construct regulatory signal target sites can be present in varying numbers within the control expression constructs. In some embodiments, the functional expression construct regulatory signal target sites are operatively coupled to the promoter of the functional expression construct. In some embodiments, the functional expression construct regulatory signal target sites can be within the promoter of the functional expression construct. In some embodiments, the functional expression construct contains at least two regulatory signal target sites, including one or more activator signal target sites and one or more repressor signal target sites. In some embodiments, the functional expression construct contains 2-50 or more regulatory signal target sites. In some embodiments, the functional expression construct contains two regulatory signal target sites, three regulatory signal target sites, four regulatory signal target sites, or more than four regulatory signal target sites. In some configurations, the functional expression construct contains includes between two and ten regulatory signal target sites, between two and eight regulatory signal target sites, or between two and five regulatory signal target sites.

[0262] Regulatory signal target sites associated with functional expression constructs can include, without limitation, activator signal target sites and repressor signal target sites. In some configurations, a functional expression construct includes one or more activator signal target sites and one or more repressor signal target sites. Exemplary configurations include oneactivator signal target site and one repressor signal target site, two activator signal target sites and one repressor signal target site, one activator signal target site and two repressor signal target sites, or multiple activator and multiple repressor signal target sites.

[0263] In some configurations, the number of activator signal target sites in a functional expression construct can range from one to fifty or more. Exemplary configurations include between one and twenty activator signal target sites, between one and ten activator signal target sites, between two and eight activator signal target sites, or between two and five activator signal target sites. Similarly, the number of repressor signal target sites in a functional expression construct can range from one to fifty or more, including between one and twenty repressor signal target sites, between one and ten repressor signal target sites, between two and eight repressor signal target sites, or between two and five repressor signal target sites.

[0264] Functional expression constructs can include regulatory signal target sites in the same or different numbers relative to control expression constructs. In some configurations, a functional expression construct includes between one and fifty or more activator signal target sites or between one and fifty or more repressor signal target sites. The number and arrangement of regulatory signal target sites associated with functional expression constructs can be selected independently from those associated with control expression constructs, allowing independent tuning of functional output relative to regulatory state maintenance.

[0265] Functional expression construct regulatory signal target sites can support interactions with a variety of regulatory element types, including RNA-based regulatory elements, protein-based regulatory elements, and regulatory elements that function as part of ribonucleoprotein complexes. Interactions between regulatory elements and regulatory signal target sites can occur through sequence-specific recognition, structural or conformational recognition, protein–DNA interactions, cooperative binding, or combinations thereof.

[0266] Functional expression construct regulatory signal target sites can include binding sites for transcription factors, response elements for inducible regulatory proteins, operator sequences, enhancer elements, or synthetic regulatory sequences. Such target sites can interact with regulatory elements based on binding affinity, structural complementarity, or cooperative interactions with additional regulatory components.

[0267] In some configurations, regulatory signal target sites function as activator signal target sites that promote transcription when bound by corresponding regulatory elements. In other configurations, regulatory signal target sites function as repressor signal target sites thatsuppress transcription when bound by corresponding regulatory elements. The functional expression construct includes both activator and repressor signal target sites, allowing combinatorial regulation of transcription of the functional expression construct.

[0268] In some embodiments, functional expression construct regulatory signal target sites include guide RNA target sites configured for interaction with guide RNAs encoded by control expression construct regulatory element regions. These guide RNA target sites direct recruitment of CRISPR transcriptional regulatory machinery to defined locations relative to a promoter.

[0269] Guide RNA target sites associated with functional expression constructs can be selected to support recruitment of CRISPR transcriptional activator complexes or CRISPR transcriptional inhibitor complexes by control expression construct regulatory elements. The regulatory outcome associated with a given guide RNA target site is determined by the control expression construct regulatory element interacting with the site, including the identity of the guide RNA and its associated regulatory machinery.

[0270] Guide RNA target sites can be positioned upstream of a promoter, overlapping a promoter, downstream of a promoter, or within regions that influence transcriptional initiation or elongation. Placement of guide RNA target sites relative to a promoter (e.g. a functional expression construct control promoter) transcription start site can be selected to support transcriptional activation, transcriptional repression, or modulation of functional expression construct promoter accessibility mediated by control expression construct regulatory elements.

[0271] Guide RNA target sites used by control expression construct regulatory elements can be selected based on sequence characteristics, promoter context, and desired regulatory outcome. Design considerations can include proximity to the transcription start site, orientation relative to the promoter, spacing between multiple guide RNA target sites, and compatibility with the CRISPR transcriptional regulatory machinery interacting with the control expression construct regulatory elements.

[0272] Multiple guide RNA target sites can be included within the functional expression construct (e.g., in a single promoter region of the same) to support cooperative regulation, graded regulatory responses, redundancy, or robustness. Guide RNA target sites can be identical or distinct and can be arranged to support binding of multiple control expression construct regulatory elements simultaneously or sequentially.

[0273] In configurations in which control expression construct regulatory elements mediate transcriptional activation of the functional expression construct, guide RNA target sites can be selected to position recruited transcriptional activator complexes in regions that promote transcriptional initiation or enhancer-like activity. In configurations in which control expression construct regulatory elements mediate transcriptional repression of the functional expression construct, guide RNA target sites can be selected to position recruited transcriptional inhibitor complexes in regions that interfere with transcriptional initiation, transcriptional elongation, or promoter accessibility in the functional expression construct.

[0274] Functional expression construct regulatory signal target sites can be selected to support multiplexed regulation by multiple control expression construct regulatory elements. In such configurations, distinct guide RNA target sites are designed to interact with distinct guide RNAs encoded by control expression construct regulatory element regions without crossreactivity.

[0275] In systems that include multiple state switches, different sets of control expression construct regulatory signal target sites can be associated with different state switches to support selective activation and repression across regulatory states. Orthogonal sets of guide RNA target sites can be used to support independent regulation of multiple promoters by different control expression construct regulatory elements within the same system.

[0276] Through selection and arrangement of regulatory signal target sites responsive to control expression construct regulatory elements, the system supports coordinated selfactivation, cross-repression, and regulation of functional expression constructs while preserving specificity and scalability across multiple regulatory states.Functional Element Regions

[0277] Each functional expression construct includes a functional element region that encodes one or more functional elements. The functional element region in each switch provides the output layer of the programmable genetic toggle switch system and produces functional elements whose expression state corresponds to the regulatory state established by the control expression constructs of the system.

[0278] Functional element regions are regulated by control expression construct regulatory elements through interaction with functional expression construct regulatory element signal target sites as described in greater detail elsewhere herein. Through this regulation, expression of functional elements is activated in one regulatory state and repressed in another regulatorystate depending on what state switch they are associated in if that sate switch is activated or not.

[0279] The functional element region can encode a single functional element or multiple functional elements. Where multiple functional elements are encoded, the functional element region can encode the functional elements as separate transcriptional units, as a single transcriptional unit producing multiple products, or as linked sequences that are processed into individual functional elements.

[0280] A functional element region can encode one functional element, two functional elements, three functional elements, four functional elements, or more than four functional elements. In some configurations, a functional element region encodes between one and ten functional elements, between two and eight functional elements, or between two and five functional elements. In some embodiments, a functional element region can encode 1-10, 20, 30, 40, 50, or more functional elements.

[0281] Different classes of functional elements can be produced from the same functional element region. For example, a functional element region can produce a reporter functional element and an effector functional element, a protein of interest and a regulatory RNA, or multiple reporter functional elements producing distinguishable outputs. Through such combinations, the programmable genetic toggle switch system supports coordinated and complex functional outputs corresponding to regulatory state selection.Functional Elements

[0282] Functional elements encoded by a functional element region can belong to a variety of functional classes depending on the intended application of the system. Exemplary classes of functional elements include, without limitation, reporter functional elements, effector functional elements, regulatory functional elements, proteins of interest, and RNA molecules of interest.

[0283] Functional elements can function independently or in combination with other functional elements encoded by the same or different functional expression constructs or with elements endogenous to a cell or other environment in which the system is expressed. The selection of functional element class(es) incorporated in the switch and / or system can be coordinated with the regulatory or other architecture of the system to provide desired outputs.Reporter Functional Elements

[0284] Reporter functional elements provide a detectable output corresponding to the regulatory state of the system. Reporter functional elements can encode proteins, peptides, or RNA molecules that produce an optical, luminescent, colorimetric, or otherwise measurable signal.

[0285] Exemplary reporter functional elements include optically active proteins (e.g., fluorescent proteins), luminescent proteins, chromogenic proteins, enzymatic reporters, and RNA-based reporters. Fluorescent reporter proteins can include proteins emitting light in different spectral ranges, including green, red, yellow, cyan, blue, or far-red wavelengths. Luminescent reporter proteins can include luciferase-based reporters or related systems. Enzymatic reporters can include enzymes that generate detectable products.

[0286] Reporter functional elements can be used individually or in combinations to provide single-state or multi-state readouts. In systems including multiple state switches, different reporter functional elements can be associated with different regulatory states to provide visually or instrumentally distinguishable outputs.Effector Functional Elements

[0287] Effector functional elements are or encode molecules that alter cellular behavior or execute a biological function in response to the regulatory state of the system. Effector functional elements can include proteins, peptides, or polynucleotides (e.g., DNA, RNAs). In some embodiments, the effector functional element can regulate, modulate, modify, or otherwise influence one or more biological and / or cellular process, function, activity, state, or any combination thereof. Example processes, functions, activities, states, and the like include genetic expression, protein expression, epigenetic state, signal transduction pathways, metabolism, growth, differentiation, survival, and / or the like.

[0288] Exemplary effector functional elements include enzymes, genetic regulators, epigenetic regulators, signaling proteins, transcriptional and translational regulators, metabolic enzymes, transport proteins, structural proteins, or proteins that modulate cellular stress responses. Effector functional elements can also include polynucleotides (including, but not limited to RNAs) and genetic modifying systems or components thereof that interfere with or modulate expression of endogenous genes.

[0289] Exemplary genetic modifying systems include programmable nuclease systems such as zinc finger nucleases and CRISPR systems. Effector functional elements can include components of CRISPR systems that function to regulate, modify, or edit endogenous orexogenous genetic sequences in response to the regulatory state of the programmable genetic toggle switch system. In such configurations, expression of CRISPR system components is controlled by functional expression constructs and is therefore coupled to the regulatory state selected by the control expression constructs.

[0290] CRISPR systems encoded as effector functional elements can include a Class 1 CRISPR system or components thereof and / or a Class 2 CRISPR system or a component thereof. Class 1 CRISPR systems utilize multi-subunit effector complexes, while Class 2 CRISPR systems utilize single, large effector proteins. Functional expression constructs can encode one or more components of either class.

[0291] Class 1 CRISPR system components that can be effector functional elements can include Cas proteins associated with multi-subunit effector complexes. Exemplary Class 1 Cas effector proteins include Cas3, Cas5, Cas6, Cas7, Cas8, CaslO, and functional variants thereof. In some embodiments, effector functional elements can include one or more Cas proteins that assemble into a Cas effector complex in the cell. In some embodiments, the effector functional elements can be or include subsets of Cas proteins sufficient to support CRISPR-mediated activity in combination with other Cas proteins expressed from separate constructs in the programmable genetic toggle switch system of the present disclosure or outside of the programmable genetic toggle switch system of the present disclosure.

[0292] Class 2 CRISPR system components can be included in the programmable genetic toggle switch system of the present disclosure as effector functional elements. In such embodiments, the effector functional elements can include one or more Class 2 Cas effector proteins. Exemplary Class 2 Cas effector proteins include Cas9 proteins, Casl2 proteins, Casl3 proteins, and functional variants thereof. In some embodiments, the Class 2 Cas effector is modified such that it is a nickase, or is catalytically inactive (dead (“dCas”)). In some embodiments, the Cas9 can be a nuclease, nickase, or a catalytically inactive DNA-binding protein (dCas9). In some embodiments, the Casl2 protein is a nuclease, nickase, or is catalytically inactive (dCas12). In some embodiments, the Casl2 can function as DNA-targeting nuclease with single-stranded or double-stranded cleavage activity. In some embodiments, the Cas is a Casl3 protein. In some embodiments, the Casl3 can function as RNA-targeting nucleases and can modulate abundance or activity of a target RNA.

[0293] Cas effector proteins included in the system as effector functional elements can include wild-type proteins, catalytically modified variants, engineered variants with alteredspecificity or activity, base-editing components, prime-editing components, or fusion proteins incorporating additional functional domains. Expression of such Cas proteins as functional elements allows the toggle switch system to control CRISPR-mediated activities, such as DNA or RNA modulation or modification (including collateral DNA or RNA cleavage) in a statedependent manner.

[0294] In some embodiments, the effector functional elements include guide RNAs, single guide RNAs, CRISPR RNAs, trans-activating CRISPR RNAs, or guide RNA arrays configured to interact with and / or complex with Class 1 or Class 2 Cas proteins. Guide RNAs included in the system as effector functional elements can be configured to target endogenous genes, regulatory regions, non-coding regions, or introduced genetic elements, such as any within the cell or other environment in which the system is expressed in. Guide RNA arrays can be processed into individual guide RNAs and can include guides targeting one or more genomic loci.

[0295] CRISPR system components encoded as effector functional elements can function independently or in combination. For example, a functional expression construct can encode a Cas effector protein, while guide RNAs are encoded by the same functional expression construct, a different functional expression construct, or a separate expression construct present in the system. In this manner, the toggle switch system can control one or more components of a CRISPR system while other components are constitutively expressed or regulated independently.

[0296] Through state-dependent expression of Class 1 or Class 2 CRISPR system components, the programmable genetic toggle switch system can selectively regulate endogenous gene expression, modify genomic or transcriptomic sequences, modulate RNA abundance, and / or execute other CRISPR-mediated functions in response to switching between regulatory states.

[0297] Effector functional elements can be selected to produce a functional response only in a defined regulatory state, such as when one particular state switch is activated, allowing state-dependent execution of cellular functions.Payload Functional Elements

[0298] Functional elements can include payload functional elements. As used herein, “payload functional elements” refers to functional elements that perform a biological, biochemical, or regulatory function upon delivery a cell or organism. In some embodiments,delivery of a payload functional element occurs when expressed by the functional expression construct from wich they are encoded. Payload functional elements are produced from functional element regions regulated by functional expression constructs and are expressed in a state-dependent manner in response to the regulatory state established by the control expression constructs.

[0299] Payload functional elements can include proteins, peptides, RNA molecules, or other biomolecules that execute an intended activity, including modulation of gene expression, alteration of cellular phenotype, execution of a metabolic function, production of a therapeutic or industrial product, or initiation of a signaling or differentiation program. Payload functional elements can act on endogenous cellular pathways, introduced genetic elements, or extracellular targets.

[0300] Payload functional elements can be distinct from reporter functional elements and can be selected independently of any detectable output. In some configurations, payload functional elements are produced only in a selected regulatory state, while production is suppressed in other regulatory states. In other configurations, different payload functional elements are produced in different regulatory states, allowing the programmable genetic toggle switch system to direct mutually exclusive or selectively exclusive biological functions.

[0301] Multiple payload functional elements can be produced from a single functional element region or from multiple functional expression constructs associated with the same state switch. Payload functional elements can function independently or cooperatively and can be combined with reporter or regulatory functional elements to provide coordinated functional output and state verification.

[0302] In some embodiments, payload functional elements include regulatory functional elements, proteins of interest, and RNA molecules of interest. In some embodiments, effector functional elements and reporter functional elements can be payload functional elements.Table C describes additional payload functional elements.Table 1. Exemplary ’ayload Functional ElementsPayload functional Exemplary payload functional Exemplary functions or outcomes element class elementsTherapeutic Cytokines, growth factors, Modulation of immune payloads hormones, antibodies, antibody responses, treatment of disease fragments, enzymes with states, restoration of biological therapeutic activity functionGenome regulation CRISPR system components, Regulation, modification, or payloads Cas proteins, guide RNAs, editing of endogenous or guide RNA arrays, transcription exogenous genetic sequences factorsRNA regulatory Regulatory RNAs, antisense Modulation of gene expression, payloads RNAs, RNA interference RNA stability, or translation molecules, RNA aptamersMetabolic payloads Metabolic enzymes, pathway Alteration of metabolic flux,regulators, transport proteins biosynthesis of metabolites,production of industrial or research compounds Developmental or Lineage-specific transcription Control of cell fate, differentiation factors, signaling molecules differentiation state, or payloads developmental programs Signaling payloads Ligands, receptors, signaling Activation or suppression of adapters intracellular or intercellular signaling pathwaysSynthetic biology Logic gate components, Construction of higher-order payloads orthogonal regulators, modular genetic circuits or programmable effectors cellular behaviorsIndustrial or Enzymes, secretion factors, Enhancement of production bioprocess payloads stress-response proteins yields, stability, or process robustnessResearch or Affinity tags, biosensors, Monitoring, tracking, or diagnostic payloads molecular probes interrogation of biologicalprocesses

[0303] Payload functional elements listed in Table 1 are exemplary and not limiting. Payload functional elements can be selected based on the intended application of the programmable genetic toggle switch system and can differ between regulatory states. In systems including multiple state switches, different payload functional elements can be produced in different regulatory states, allowing mutually exclusive or selectively exclusive execution of biological functions.

[0304] Multiple payload functional elements from the same or different classes can be produced within a single regulatory state. Payload functional elements can functionindependently or cooperatively and can be combined with reporter or regulatory functional elements to provide coordinated functional output and state verification.Regulatory Functional Elements

[0305] Functional elements can include regulatory functional elements. As used in this context herein “regulatory elements” are those functional elements that regulate expression or activity of other genes, pathways, or molecular processes downstream of the programmable genetic toggle switch system. Regulatory functional elements are produced from functional element regions whose expression is driven by a functional expression construct promoter and controlled by control expression construct regulatory elements.

[0306] Regulatory functional elements can include transcription factors, RNA-binding proteins, regulatory RNAs, or other molecules that modulate and / or regulate gene expression, RNA stability, translation, or protein activity. When produced in response to a selected regulatory state, regulatory functional elements propagate or reinforce the regulatory outcome established by the control expression constructs.

[0307] Regulatory functional elements can function as secondary regulators that extend the effects of state switching to additional genetic circuits or cellular processes. Through this arrangement, the programmable genetic toggle switch system can operate as a higher-order regulatory module within a broader regulatory network, that can include a cell in which the system is expressed.Proteins of Interest

[0308] Functional elements can include proteins of interest (“POP’). As used herein, POIs refer to as proteins selected based on a desired functional, structural, biochemical, or regulatory property of the protein. Proteins of interest are produced from functional element regions under the control of functional expression construct promoter which is regulated by control expression construct regulatory elements, and thus and are expressed in a state-dependent manner.

[0309] Proteins of interest can include therapeutic proteins, industrial enzymes, research reagents, antigens, structural proteins, signaling proteins, synthetic proteins, protein scaffolds, or other proteins selected for production or study. Proteins of interest can be produced as full-length proteins, protein fragments, fusion proteins, or proteins modified to include tags, localization signals, or regulatory domains.

[0310] State-dependent production of proteins of interest allows the programmable genetic toggle switch system to control protein availability, activity, or localization in response to switching between regulatory states.RNA Molecules of Interest

[0311] Functional elements can include RNA molecules of interest, defined as RNA molecules selected to perform a functional, regulatory, or structural role within a cell or organism. RNA molecules of interest are produced from functional element regions regulated by functional expression constructs.

[0312] RNA molecules of interest can include messenger RNAs, non-coding RNAs, regulatory RNAs, guide RNAs for programmable genetic modifying systems (including but not limited to CRISPR systems), antisense RNAs, RNA aptamers, or other RNA species. RNA molecules of interest can function directly as regulatory or functional molecules or can serve as intermediates for downstream processes.

[0313] State-dependent production of RNA molecules of interest allows the programmable genetic toggle switch system to regulate RNA abundance, RNA activity, or RNA-mediated pathways in response to changes in regulatory state.CRISPR Transcriptional Regulatory Machinery

[0314] The programmable genetic toggle switch systems described herein can include CRISPR activation and inhibition (CRISPR A / I) transcriptional regulatory machinery that interacts with control expression construct regulatory elements and functional expression construct regulatory element signal target sites to regulate transcription.

[0315] CRISPR A / I transcriptional regulatory machinery includes one or more CRISPR effector proteins configured to modulate transcription without introducing double-stranded breaks in target nucleic acids. The CRISPR A / I transcriptional regulatory machinery functions in concert with guide RNAs produced by expression of the control expression construct regulatory element regions to direct transcriptional activation or transcriptional repression at defined target sites.CRISPR Transcriptional Activator Complexes

[0316] CRISPR A / I transcriptional regulatory machinery can include one or more CRISPR transcriptional activator complexes. CRISPR transcriptional activator complexes include a CRISPR effector protein and one or more transcriptional activation domains that promote transcription when recruited to a regulatory element signal target site.

[0317] CRISPR transcriptional activator complexes can include catalytically inactive or modified Cas proteins that retain nucleic acid binding activity while lacking nuclease activity. Such Cas proteins can be operatively linked to one or more transcriptional activation domains. Exemplary transcriptional activation domains include VP64, VP160, VP192, p65, Rta, combinations thereof, or other activation domains capable of promoting transcription.

[0318] CRISPR transcriptional activator complexes can be recruited to control expression construct promoters or functional expression construct promoters through interaction with guide RNAs encoded by control expression construct regulatory element regions. Recruitment of a CRISPR transcriptional activator complex to an activator signal target site increases transcription from the associated promoter.CRISPR Transcriptional Inhibitor Complexes

[0319] CRISPR A / I transcriptional regulatory machinery can include one or more CRISPR transcriptional inhibitor complexes. CRISPR transcriptional inhibitor complexes include a CRISPR effector protein and one or more transcriptional inhibition or repression domains that suppress transcription when recruited to a regulatory element signal target site.

[0320] CRISPR transcriptional inhibitor complexes can include catalytically inactive or modified Cas proteins operatively linked to transcriptional repression domains. Exemplary repression domains include KRAB, SID, MeCP2, or other domains capable of suppressing transcription. In some configurations, transcriptional inhibition is achieved through steric interference with transcriptional machinery rather than through an appended repression domain.

[0321] CRISPR transcriptional inhibitor complexes can be recruited to control expression construct promoters or functional expression construct promoters through interaction with guide RNAs encoded by control expression construct regulatory element regions. Recruitment of a CRISPR transcriptional inhibitor complex to a repressor signal target site suppresses transcription from the associated promoter.Organization and Expression of CRISPR A / I Machinery

[0322] CRISPR A / I transcriptional regulatory machinery can be encoded by one or more expression constructs present in the system. In some configurations, CRISPR transcriptional activator complexes and CRISPR transcriptional inhibitor complexes are encoded on the same expression construct. In other configurations, CRISPR transcriptional activator complexes and CRISPR transcriptional inhibitor complexes are encoded on separate expression constructs.

[0323] CRISPR A / I transcriptional regulatory machinery can be expressed constitutively or under transcriptional control. Expression of CRISPR A / I transcriptional regulatory machinery can be independent of the regulatory state of the toggle switch system or can be regulated in a state-dependent manner.

[0324] CRISPR A / I transcriptional regulatory machinery can be shared across multiple control expression constructs and functional expression constructs within the same system. Through this shared machinery, guide RNAs encoded by different control expression construct regulatory element regions can direct distinct regulatory outcomes using common CRISPR A / I components.Interaction of CRISPR A / I Machinery with Control and Functional Expression Constructs

[0325] CRISPR A / I transcriptional regulatory machinery can interact with control expression construct regulatory elements and functional expression construct regulatory element signal target sites to implement self-activation, cross-repression, and regulation of functional output.

[0326] Guide RNAs encoded by control expression construct regulatory element regions direct CRISPR A / I transcriptional regulatory machinery to specific activator signal target sites or repressor signal target sites associated with control expression construct promoters and functional expression construct promoters. The regulatory effect at a given target site is determined by the guide RNA and the CRISPR transcriptional regulatory complex recruited to that site.

[0327] Through coordinated recruitment of CRISPR transcriptional activator complexes and CRISPR transcriptional inhibitor complexes, the programmable genetic toggle switch system establishes and maintains discrete regulatory states and translates those regulatory states into functional output.Modular and Orthogonal CRISPR A / I Implementations

[0328] CRISPR A / I transcriptional regulatory machinery can be implemented in modular and orthogonal configurations. Different CRISPR effector proteins, activation domains, repression domains, or guide RNA scaffolds can be used within the same system to support independent or orthogonal regulatory pathways.

[0329] In systems including multiple state switches, CRISPR A / I transcriptional regulatory machinery can support selective recruitment to different target sites based on guide RNAidentity, guide RNA scaffold structure, or associated effector protein. This modularity supports scalable implementation of multi-state toggle switch systems and complex regulatory logic.Additional Components

[0330] In addition to the control expression constructs, functional expression constructs, and CRISPR A / I transcriptional regulatory machinery described herein, the programmable genetic toggle switch system can include one or more additional components that support, modify, stabilize, or expand the system function. Additional components can be encoded by expression constructs that are separate from the core system constructs or can be incorporated into one or more of the core system constructs, depending on the desired system architecture.

[0331] Additional components can function at the level of transcription, RNA processing, RNA stability, translation, protein stability, cellular localization, or environmental responsiveness. Inclusion of additional components allows tuning of system performance, robustness, scalability, and compatibility with different cellular contexts.

[0332] Expression of additional components can be driven by promoters selected from constitutive, inducible, repressible, optogenetically regulated, synthetic, or hybrid promoters, including promoters that function independently of state switching logic and promoters that respond to regulatory signals generated by the control expression constructs.RNA Processing and Stability Components

[0333] Additional components can include RNA processing or RNA stability components that act on transcripts produced by control expression constructs or functional expression constructs. Such components can include endoribonucleases, exonucleases, RNA-binding proteins, RNA chaperones, or RNA-modifying enzymes.

[0334] RNA processing and / or stability components can be included in and / or encoded by a dedicated expression construct separate from the control expression constructs and / or functional expression constructs. In other configurations, the RNA processing and / or stability components can be included and / or encoded by an expression construct in the system such as the control expression construct and / or functional expression construct. In some embodiments RNA processing or stability components can be included in and / or encoded by the same construct(s) as the CRISPR A / I machinery. RNA processing and / or stability components can be encoded within the same vector or transcriptional unit as CRISPR A / I machinery or within a functional expression construct. In some embodiments, expression of the RNA processing and / or staibilty and / or CRISPR A / I machinery is decoupled from state-switching logic.

[0335] RNA processing components can act to process guide RNA arrays, regulate transcript abundance, modulate transcript half-life, or coordinate expression of multiple RNA species produced by the system.

[0336] In some embodiments, the RNA processing component comprises an endoribonuclease. In some embodiments, the endoribonuclease is Cys4.Protein Stability and Turnover Components

[0337] Additional components can include protein stability or protein turnover components that regulate the abundance or persistence of proteins produced by the system. Such components can include proteases, ubiquitin ligases, degron-recognizing factors, or other proteins that influence protein degradation pathways.

[0338] Protein stability components can be encoded by separate expression constructs or can be incorporated into a core expression construct such as the control expression construct, the functional expression construct, and / or CRISPR A / I machinery construct as fusion partners, tags, or regulatory domains associated with functional elements. Through these components, the system can control the duration or reversibility of functional element activity following state switching.Localization and Compartmentalization Components

[0339] Additional components can include localization or compartmentalization components that influence the intracellular or extracellular distribution of system components. Such components can include nuclear localization signals, nuclear export signals, organelle targeting signals, secretion signals, membrane-targeting domains, or scaffolding proteins.

[0340] Localization components can be encoded as part of functional expression constructs, as part of CRISPR A / I machinery constructs, and / or control expression constructs, or as separate expression constructs. Localization of regulatory machinery or functional elements can be selected to support efficient transcriptional regulation, targeted functional output, or spatial separation of regulatory processes.Environmental Sensing and Signal Transduction Components

[0341] Additional components can include environmental sensing or signal transduction components that respond to external or internal stimuli. Such components can include light-responsive proteins, temperature-sensitive regulators, ligand-binding proteins, chemically responsive transcription factors, redox-sensitive domains, or osmosensitive elements.

[0342] Environmental sensing components can be encoded within control expression constructs to provide input signals that initiate switching between regulatory states. Alternatively, environmental sensing components can be encoded by separate expression constructs that interact with control expression constructs and / or CRISPR A / I machinery to modulate system behavior.Orthogonal and Insulating Components

[0343] Additional components can include orthogonal regulatory components or insulating elements that reduce crosstalk with host cellular machinery or between different parts of the system. Such components can include orthogonal transcription factors, orthogonal promoters, transcriptional insulators, RNA insulators, or scaffold proteins.

[0344] Orthogonal and insulating components can be encoded by separate expression constructs or incorporated into control expression constructs and / or functional expression constructs to preserve modularity and predictable behavior of the system.Multi-Switch Systems

[0345] In some embodiments, the programmable genetic toggle switch system further includes three or more state switches, including a first state switch, a second state switch, and a nthstate switch. For ease of discussion only the third state switch used in the embodiments herein is representative of an nthstate switch in a multiplexed toggle switch architecture and logic. One of skill in the art will appreciate that it is demonstrative of the architecture configurations and implementations that can be extended to the nthswitch added to the system.

[0346] Each state switch comprises a control expression construct and optionally a functional expression construct, as described herein with respect to the first and second state switches. The third control expression construct comprises a promoter operably linked to a regulatory element encoding region and includes one or more regulatory signal target sites configured to respond to regulatory elements produced by other state switches. Likewise, the optional third functional expression construct comprises a functional promoter operably linked to a functional element region and includes one or more regulatory signal target sites.

[0347] The third state switch is configured to exhibit self-activation, cross-repression, and reciprocal regulation with respect to the first and second state switches. In particular, upon activation of the third state switch, the third control expression construct produces one or more regulatory elements that positively regulate transcription of the third control expression construct, thereby stabilizing activation of the third state switch. In addition, the third controlexpression construct produces one or more regulatory elements configured to repress transcription of the control expression constructs and / or functional expression constructs of the first state switch, the second state switch, or both.

[0348] Conversely, the first and second state switches are further configured to regulate the third state switch. In some embodiments, the control expression constructs of the first and second state switches include additional regulatory element encoding regions that produce regulatory elements configured to repress transcription of the third control expression construct and / or the third functional expression construct. Correspondingly, the third control expression construct and / or third functional expression construct include regulatory signal target sites responsive to regulatory elements produced by the first and second state switches.

[0349] Through this bidirectional regulatory configuration, activation of the third state switch suppresses activation and / or functional output of the first and second state switches, and activation of the first or second state switches suppresses activation and / or functional output of the third state switch. The third state switch thereby operates as a mutually exclusive or selectively exclusive state relative to the first and second state switches, depending on the specific regulatory connections implemented.Logic and Design Principles for an n-Switch Toggle System

[0350] In some embodiments, the toggle switch system is extensible to n state switches, where n is any integer greater than or equal to two. Each state switch i (where i = 1 … n) comprises an i-th control expression construct and optionally an i-th functional expression construct, and each state switch is configured according to a shared architectural logic that enables scalable multiplexing.Self-activation

[0351] For each state switch i, the i-th control expression construct is configured to encode one or more regulatory elements that positively regulate transcription of the i-th control expression construct. This self-activation stabilizes the active state of switch i once initiated and provides bistability or multistability within the system.Cross-repression between switches

[0352] Each state switch i is further configured to repress one or more other state switches j (where j ≠ i). In particular, the i-th control expression construct encodes one or more regulatory elements that repress transcription of the j-th control expression construct and / or the j-th functional expression construct. Correspondingly, the j-th constructs include regulatorysignal target sites responsive to regulatory elements produced by the i-th control expression construct.

[0353] The pattern of cross-repression can be configured as: (1) fully mutually exclusive, wherein activation of any state switch represses all other state switches; or (2) Selectively exclusive, wherein activation of a given state switch represses only a defined subset of other state switches, permitting co-activation of permitted combinations of switches or functional components.Reciprocal regulation and symmetry

[0354] For each regulatory interaction from switch z to switch j, the reciprocal capacity for regulation from switch j to switch z may be included, omitted, or modified, allowing the system to be configured as symmetric or asymmetric. In some embodiments, each state switch contains within its control expression construct regulatory element encoding regions sufficient to produce regulatory elements capable of regulating multiple other state switches, thereby enabling dense or sparse regulatory networks.Functional-layer regulation

[0355] Regulatory logic may be applied independently at the level of control expression constructs and functional expression constructs. In some embodiments, cross-repression occurs exclusively at the control expression construct level, while in other embodiments crossrepression occurs at both the control and functional expression construct levels. This layered design allows exclusive control of state identity, functional output, or both.Scalability to arbitrary n

[0356] Because each state switch is constructed from a modular set of components — including promoters, regulatory element encoding regions, and regulatory signal target sites — the same design logic can be iteratively applied to introduce additional state switches without altering the fundamental architecture of the system. Each additional state switch is integrated by: (i) including self-activating regulatory elements; (ii) including regulatory elements configured to repress one or more existing switches; (iii) Including regulatory signal target sites responsive to regulatory elements produced by one or more existing switches. Accordingly, the toggle switch system supports exclusive activation of a single state switch, exclusive activation of a defined subset of state switches, or exclusive activation of selected functional components, for any number of state switches, using the same regulatory logic extended to the nth state switch.Vectors

[0357] Also provided herein are one or vectors that can contain one or more of the programmable genetic toggle switch systems constructs or component thereof described herein. In some aspects, the vector can contain one or more polynucleotides encoding one or more elements of programmable genetic toggle switch systems constructs or component thereof described herein described herein. The vectors can be useful in producing bacterial, fungal, yeast, plant cells, animal cells, and transgenic animals that can express one or more components of the programmable genetic toggle switch systems constructs or component thereof described herein described herein. Within the scope of this disclosure are vectors containing one or more of the polynucleotide sequences described herein. One or more of the polynucleotides that are part of the programmable genetic toggle switch systems constructs or component thereof described herein described herein can be included in a vector or vector system. The vectors and / or vector systems can be used, for example, to express one or more of the polynucleotides in a cell, such as a producer cell or host cell, to produce a programmable genetic toggle switch system described elsewhere herein. Other uses for the vectors and vector systems described herein are also within the scope of this disclosure. In general, and throughout this specification, the term “vector” refers to a tool that allows or facilitates the transfer of an entity from one environment to another. In some contexts which will be appreciated by those of ordinary skill in the art, “vector” can be a term of art to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. A vector can be a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment. Generally, a vector is capable of replication when associated with the proper control elements.

[0358] Vectors include, but are not limited to, nucleic acid molecules that are singlestranded, double-stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g. circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art. One type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, wherein virally-derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g. retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses (AAVs)). Viralvectors also include polynucleotides carried by a virus for transfection into a host cell. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as “expression vectors.” Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.

[0359] Recombinant expression vectors can be composed of a nucleic acid (e.g. a polynucleotide) of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory elements, which can be selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed. Within an expression vector, “operably linked” and “operatively-linked” are used interchangeably herein and further defined elsewhere herein. In the context of a vector or expression construct, the term “operably linked” is intended to mean that the nucleotide sequence of interest is linked (either directly or indirectly) to one or more regulatory element(s) in a manner that allows for expression or control of expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell). Operatively linked includes regulatory linkage where a promoter, for example, contains regulatory signal binding sites and thus the promoter would be operatively linked to the regulatory elements that bind or otherwise interact with the regulatory signal binding sites so as to regulate activity of that promoter. These and other aspects of the vectors and vector systems are described elsewhere herein.

[0360] In some aspects, the vector can be a bicistronic vector. In some aspects, a bicistronic vector can be used for one or more elements of the programmable genetic toggle switch system described herein. Where the element of the programmable genetic toggle switch system is an RNA, its expression can be driven by a Pol III promoter, such as a U6 promoter. In some aspects, the two are combined.Cell-based Vector Amplification and Expression

[0361] Vectors can be designed for expression of one or more elements of the programmable genetic toggle switch system described herein (e.g. nucleic acid transcripts,proteins, enzymes, and combinations thereof) in a suitable host cell. In some aspects, the suitable host cell is a prokaryotic cell. Suitable host cells include, but are not limited to, bacterial cells, yeast cells, insect cells, and mammalian cells. The vectors can be viral-based or non-viral based. In some aspects, the suitable host cell is a eukaryotic cell. In some aspects, the suitable host cell is a suitable bacterial cell. Suitable bacterial cells include, but are not limited to bacterial cells from the bacteria of the species Escherichia coli. Many suitable strains of E. coli are known in the art for expression of vectors. These include, but are not limited to Pirl, Stbl2, Stbl3, Stbl4, TOP 10, XL1 Blue, and XL 10 Gold. In some aspects, the host cell is a suitable insect cell. Suitable insect cells include those from Spodoptera frugiperda. Suitable strains of S. frugiperda cells include, but are not limited to Sf9 and Sf21. In some aspects, the host cell is a suitable yeast cell. In some aspects, the yeast cell can be from Saccharomyces cerevisiae. In some aspects, the host cell is a suitable mammalian cell. Many types of mammalian cells have been developed to express vectors. Suitable mammalian cells include, but are not limited to, HEK293, Chinese Hamster Ovary Cells (CHOs), mouse myeloma cells, HeLa, U2OS, A549, HT1080, CAD, P19, NIH 3T3, L929, N2a, MCF-7, Y79, SO-Rb50, HepG G2, DIKX-X11, J558L, Baby hamster kidney cells (BHK), and chicken embryo fibroblasts (CEFs). Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).

[0362] In some aspects, the vector can be a yeast expression vector. Examples of vectors for expression in yeast Saccharomyces cerevisiae include pYepSecl (Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kuijan and Herskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al., 1987. Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (InVitrogen Corp, San Diego, Calif.). As used herein, a "yeast expression vector" refers to a nucleic acid that contains one or more sequences encoding an RNA and / or polypeptide and may further contain any desired elements that control the expression of the nucleic acid(s), as well as any elements that enable the replication and maintenance of the expression vector inside the yeast cell. Many suitable yeast expression vectors and features thereof are known in the art; for example, various vectors and techniques are illustrated in in Yeast Protocols, 2nd edition, Xiao, W., ed. (Humana Press, New York, 2007) and Buckholz, R. G. and Gleeson, M. A. (1991) Biotechnology (NY) 9(11): 1067-72. Yeast vectors can contain, without limitation, a centromeric (CEN) sequence, an autonomous replicationsequence (ARS), a promoter, such as an RNA Polymerase III promoter, operably linked to a sequence or gene of interest, a terminator such as an RNA polymerase III terminator, an origin of replication, and a marker gene (e.g., auxotrophic, antibiotic, or other selectable markers). Examples of expression vectors for use in yeast may include plasmids, yeast artificial chromosomes, 2p plasmids, yeast integrative plasmids, yeast replicative plasmids, shuttle vectors, and episomal plasmids.

[0363] In some aspects, the vector is a baculovirus vector or expression vector and can be suitable for expression of polynucleotides and / or proteins in insect cells. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39). rAAV (recombinant Adeno-associated viral) vectors are preferably produced in insect cells, e.g., Spodoptera frugiperda Sf9 insect cells, grown in serum-free suspension culture. Serum-free insect cells can be purchased from commercial vendors, e.g., Sigma Aldrich (EX-CELL 405).

[0364] In some embodiments, the vector is a mammalian expression vector. In some aspects, the mammalian expression vector is capable of expressing one or more polynucleotides and / or polypeptides in a mammalian cell. Examples of mammalian expression vectors include, but are not limited to, pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6: 187-195). The mammalian expression vector can include one or more suitable regulatory elements capable of controlling expression of the one or more polynucleotides and / or proteins in the mammalian cell. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, simian virus 40, and others disclosed herein and known in the art. More detail on suitable regulatory elements are described elsewhere herein.

[0365] For other suitable expression vectors and vector systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1989.

[0366] In some embodiments, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissuespecific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promotersinclude the albumin promoter (liver-specific; Pinkert, et al., 1987. Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame and Eaton, 1988. Adv. Immunol. 43: 235-275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) and immunoglobulins (Baneiji, etal., 1983. Ce / / 33: 729-740; Queen and Baltimore, 1983. Cell 33: 741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Set. USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al., 1985. Science 230: 912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U. S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, e.g., the murine hox promoters (Kessel and Gruss, 1990. Science 249: 374-379) and the a-fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3: 537-546). With regards to these prokaryotic and eukaryotic vectors, mention is made of U. S. Patent 6,750,059, the contents of which are incorporated by reference herein in their entirety. Other aspects can utilize viral vectors, with regards to which mention is made of U. S. Patent application 13 / 092,085, the contents of which are incorporated by reference herein in their entirety. Tissue-specific regulatory elements are known in the art and in this regard, mention is made of U. S. Patent 7,776,321, the contents of which are incorporated by reference herein in their entirety. In some embodiments, a regulatory element can be operably linked to one or more elements of programmable genetic toggle switch system so as to drive expression of the one or more elements of the programmable genetic toggle switch system described herein. Specific regulatory elements in connection with the expression constructs of the programmable genetic toggle switch system are also described in greater detail elsewhere herein.

[0367] Vectors may be introduced and propagated in a prokaryote or prokaryotic cell. In some aspects, a prokaryote is used to amplify copies of a vector to be introduced into a eukaryotic cell or as an intermediate vector in the production of a vector to be introduced into a eukaryotic cell (e.g. amplifying a plasmid as part of a viral vector packaging system). In some aspects, a prokaryote is used to amplify copies of a vector and express one or more nucleic acids, such as to provide a source of one or more proteins for delivery to a host cell or host organism.

[0368] In some aspects, the vector can be a fusion vector or fusion expression vector. In some aspects, fusion vectors add a number of amino acids to a protein encoded therein, such as to the amino terminus, carboxy terminus, or both of a recombinant protein. Such fusionvectors can serve one or more purposes, such as: (i) to increase expression of recombinant protein; (ii) to increase the solubility of the recombinant protein; and (iii) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. In some aspects, expression of polynucleotides (such as non-coding polynucleotides) and proteins in prokaryotes can be carried out in Escherichia coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion polynucleotides and / or proteins. In some aspects, the fusion expression vector can include a proteolytic cleavage site, which can be introduced at the junction of the fusion vector backbone or other fusion moiety and the recombinant polynucleotide or protein to enable separation of the recombinant polynucleotide or protein from the fusion vector backbone or other fusion moiety subsequent to purification of the fusion polynucleotide or protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Example fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N. J.) that fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al., (1988) Gene 69:301-315) and pET lid (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).

[0369] In some embodiments, one or more vectors driving expression of and / or encoding one or more elements of programmable genetic toggle switch system herein are introduced into a host cell such that expression of the elements of the engineered delivery system described herein direct formation of a CRISPR system or other programmable genetic modifying system).

[0370] In some aspects, two or more of the elements of the programmable genetic toggle switch system expressed from the same or different regulatory element(s) and / or part of the same or different transcriptional units and / or expression constructs, can be combined in a single vector or be contained on multiple vectors.Vector Features

[0371] The vectors can include additional features that can confer one or more functionalities to the vector, the polynucleotide to be delivered, a virus particle produced there from, or polypeptide expressed thereof. Such features include, but are not limited to, regulatory elements, selectable markers, molecular identifiers (e.g. molecular barcodes), stabilizingelements, and the like. It will be appreciated by those skilled in the art that the design of the expression vector and additional features included can depend on such factors as the choice of the host cell to be transformed, the level of expression desired, etc.Regulatory Elements

[0372] In aspects, the polynucleotides and / or vectors thereof described herein (such as the programmable genetic toggle switch system of the present disclosure) can include one or more regulatory elements that can be operatively linked to one or more polynucleotides of the programmable genetic toggle switch system. The term “regulatory element” is intended to include promoters, enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences). Such regulatory elements are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). A tissue-specific promoter can direct expression primarily in a desired tissue of interest, such as muscle, neuron, bone, skin, blood, specific organs (e.g., liver, pancreas), or particular cell types (e.g., lymphocytes). Regulatory elements may also direct expression in a temporal-dependent manner, such as in a cell-cycle dependent or developmental stage-dependent manner, which may or may not also be tissue or cell-type specific. In some embodiments, a vector comprises one or more pol III promoter (e.g., 1, 2, 3, 4, 5, or more pol III promoters), one or more pol II promoters (e.g., 1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), or combinations thereof. Examples of pol III promoters include, but are not limited to, U6 and Hl promoters. Examples of pol II promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) (see, e.g., Boshart et al, Cell, 41:521-530 (1985)), the SV40 promoter, the dihydrofolate reductase promoter, the P-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1α promoter. Also encompassed by the term “regulatory element” are enhancer elements, such as WPRE; CMV enhancers; the R-U5’ segment in LTR of HTLV-I (Mol. Cell. Biol., Vol.8(1), p. 466-472, 1988); SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit P-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31, 1981).

[0373] In some aspects, the regulatory sequence can be a regulatory sequence described in U. S. Pat. No. 7,776,321, U. S. Pat. Pub. No. 2011 / 0027239, and PCT publication WO 2011 / 028929, the contents of which are incorporated by reference herein in their entirety. In some aspects, the vector can contain a minimal promoter. In some aspects, the minimal promoter is the Mecp2 promoter, tRNA promoter, or U6. In a further embodiment, the minimal promoter is tissue specific.

[0374] To express a polynucleotide, the vector can include one or more transcriptional and / or translational initiation regulatory sequences, e.g. promoters, that direct the transcription of the gene and / or translation of the encoded protein in a cell. In some aspects a constitutive promoter may be employed. Suitable constitutive promoters for mammalian cells are generally known in the art and include, but are not limited to SV40, CAG, CMV, EF-la, P-actin, RSV, and PGK. Suitable constitutive promoters for bacterial cells, yeast cells, and fungal cells are generally known in the art, such as a T-7 promoter for bacterial expression and an alcohol dehydrogenase promoter for expression in yeast.

[0375] In some aspects, the regulatory element can be a regulated promoter. " Regulated promoter" refers to promoters that direct gene expression not constitutively, but in a temporally- and / or spatially-regulated manner, and includes tissue-specific, tissue-preferred and inducible promoters. Regulated promoters include conditional promoters and inducible promoters. In some aspects, conditional promoters can be employed to direct expression of a polynucleotide in a specific cell type, under certain environmental conditions, and / or during a specific state of development. Suitable tissue specific promoters can include, but are not limited to, liver specific promoters (e.g. APOA2, SERPIN Al (hAAT), CYP3A4, and MIR122), pancreatic cell promoters (e.g. INS, IRS2, Pdxl, Alx3, Ppy), cardiac specific promoters (e.g. Myh6 (alpha MHC), MYL2 (MLC-2v), TNI3 (cTnl), NPPA (ANF), Slc8al (Next)), central nervous system cell promoters (SYN1, GFAP, INA, NES, MOBP, MBP, TH, FOXA2 (HNF3 beta)), skin cell specific promoters (e.g. FLG, K14, TGM3), immune cell specific promoters, (e.g. ITGAM, CD43 promoter, CD14 promoter, CD45 promoter, CD68 promoter), urogenital cell specific promoters (e.g. Pbsn, Upk2, Sbp, Ferll4), endothelial cell specific promoters (e.g. ENG), pluripotent and embryonic germ layer cell specific promoters (e.g. Oct4, NANOG, Synthetic Oct4, T brachyury, NES, SOX17, FOXA2, MIR122), and muscle cell specific promoter (e.g. Desmin). Other tissue and / or cell specific promoters are generally known in the art and are within the scope of this disclosure.

[0376] Inducible / conditional promoters can be positively inducible / conditional promoters (e.g. a promoter that activates transcription of the polynucleotide upon appropriate interaction with an activated activator, or an inducer (compound, environmental condition, or other stimulus) or a negative / conditional inducible promoter (e.g. a promoter that is repressed (e.g. bound by a repressor) until the repressor condition of the promotor is removed (e.g. inducer binds a repressor bound to the promoter stimulating release of the promoter by the repressor or removal of a chemical repressor from the promoter environment). The inducer can be a compound, environmental condition, or other stimulus. Thus, inducible / conditional promoters can be responsive to any suitable stimuli such as chemical, biological, or other molecular agents, temperature, light, and / or pH. Suitable inducible / conditional promoters include, but are not limited to, Tet-On, Tet-Off, Lac promoter, pBad, AlcA, LexA, Hsp70 promoter, Hsp90 promoter, pDawn, XVE / OlexA, GVG, and pOp / LhGR.

[0377] Where expression in a plant cell is desired, the components of the programmable genetic toggle switch system described herein are typically placed under control of a plant promoter, i.e. a promoter operable in plant cells. The use of different types of promoters is envisaged.

[0378] A constitutive plant promoter is a promoter that is able to express the open reading frame (ORF) that it controls in all or nearly all of the plant tissues during all or nearly all developmental stages of the plant (referred to as "constitutive expression"). One non-limiting example of a constitutive promoter is the cauliflower mosaic virus 35S promoter. Different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. In particular embodiments, one or more of the programmable genetic toggle switch system components are expressed under the control of a constitutive promoter, such as the cauliflower mosaic virus 35S promoter issue-preferred promoters can be utilized to target enhanced expression in certain cell types within a particular plant tissue, for instance vascular cells in leaves or roots or in specific cells of the seed. Examples of particular promoters for use in the programmable genetic toggle switch system are found in Kawamata et al., (1997) Plant Cell Physiol 38:792-803; Yamamoto et al., (1997) Plant J 12:255-65; Hire et al, (1992) Plant Mol Biol 20:207-18, Kuster et al, (1995) Plant Mol Biol 29:759-72, and Capana et al., (1994) Plant Mol Biol 25:681 -91.

[0379] Examples of promoters that are inducible and that can allow for spatiotemporal control of gene editing or gene expression may use a form of energy. The form of energy mayinclude but is not limited to sound energy, electromagnetic radiation, chemical energy and / or thermal energy. Examples of inducible systems include tetracycline inducible promoters (Tet-On or Tet-Off), small molecule two-hybrid transcription activations systems (FKBP, ABA, etc), or light inducible systems (Phytochrome, LOV domains, or cryptochrome)., such as a Light Inducible Transcriptional Effector (LITE) that direct changes in transcriptional activity in a sequence-specific manner. The components of a light inducible system may include one or more elements of the programmable genetic toggle switch system described herein, a light-responsive cytochrome heterodimer (e.g. from Arabidopsis thaliana), and a transcriptional activation / repression domain. In some aspects, the vector can include one or more of the inducible DNA binding proteins provided in PCT publication WO 2014 / 018423 and US Publications, 2015 / 0291966, 2017 / 0166903, 2019 / 0203212, which describe e.g. aspects of inducible DNA binding proteins and methods of use and can be adapted for use with the present invention.

[0380] In some aspects, transient or inducible expression can be achieved by including, for example, chemical -regulated promotors, i.e. whereby the application of an exogenous chemical induces gene expression. Modulation of gene expression can also be obtained by including a chemical-repressible promoter, where application of the chemical represses gene expression. Chemical-inducible promoters include, but are not limited to, the maize ln2-2 promoter, activated by benzene sulfonamide herbicide safeners (De Veylder et al., (1997) Plant Cell Physiol 38:568-77), the maize GST promoter (GST-11-27, WO93 / 01294), activated by hydrophobic electrophilic compounds used as pre-em ergent herbicides, and the tobacco PR-1 a promoter (Ono et al., (2004) Biosci Biotechnol Biochem 68:803-7) activated by salicylic acid. Promoters which are regulated by antibiotics, such as tetracycline-inducible and tetracycline-repressible promoters (Gatz et al., (1991 ) Mol Gen Genet 227:229-37; U. S. Patent Nos. 5,814,618 and 5,789,156) can also be used herein.

[0381] In some aspects, the vector or system thereof can include one or more elements capable of translocating and / or expressing an programmable genetic toggle switch system or component thereof to / in a specific cell component or organelle. Such organelles can include, but are not limited to, nucleus, ribosome, endoplasmic reticulum, golgi apparatus, chloroplast, mitochondria, vacuole, lysosome, cytoskeleton, plasma membrane, cell wall, peroxisome, centrioles, etc.Selectable Markers and Tags

[0382] One or more of the programmable genetic toggle switch system can be operably linked, fused to, or otherwise modified to include a polynucleotide that encodes or is a selectable marker or tag, which can be a polynucleotide or polypeptide. In some aspects, the polypeptide encoding a polypeptide selectable marker can be incorporated in the programmable genetic toggle switch system polynucleotide such that the selectable marker polypeptide. In some aspects, the selectable marker or tag is a polynucleotide barcode or unique molecular identifier (UMI).

[0383] It will be appreciated that the polynucleotide encoding such selectable markers or tags can be incorporated into a polynucleotide encoding one or more components of the programmable genetic toggle switch system described herein in an appropriate manner to allow expression of the selectable marker or tag. Such techniques and methods are described elsewhere herein and will be instantly appreciated by one of ordinary skill in the art in view of this disclosure. Many such selectable markers and tags are generally known in the art and are intended to be within the scope of this disclosure.

[0384] Suitable selectable markers and tags include, but are not limited to, affinity tags, such as chitin binding protein (CBP), maltose binding protein (MBP), glutathione-S-transferase (GST), poly(His) tag; solubilization tags such as thioredoxin (TRX) and poly(NANP), MBP, and GST; chromatography tags such as those consisting of polyanionic amino acids, such as FLAG-tag; epitope tags such as V5-tag, Myc-tag, HA-tag and NE-tag; protein tags that can allow specific enzymatic modification (such as biotinylation by biotin ligase) or chemical modification (such as reaction with Fl AsH-EDT2 for fluorescence imaging), DNA and / or RNA segments that contain restriction enzyme or other enzyme cleavage sites; DNA segments that encode products that provide resistance against otherwise toxic compounds including antibiotics, such as, spectinomycin, ampicillin, kanamycin, tetracycline, Basta, neomycin phosphotransferase II (NEO), hygromycin phosphotransferase (HPT)) and the like; DNA and / or RNA segments that encode products that are otherwise lacking in the recipient cell (e.g., tRNA genes, auxotrophic markers); DNA and / or RNA segments that encode products which can be readily identified (e.g., phenotypic markers such as P-galactosidase, GUS; fluorescent proteins such as green fluorescent protein (GFP), cyan (CFP), yellow (YFP), red (RFP), luciferase, and cell surface proteins); polynucleotides that can generate one or more new primer sites for PCR (e.g., the juxtaposition of two DNA sequences not previously juxtaposed), DNAsequences not acted upon or acted upon by a restriction endonuclease or other DNA modifying enzyme, chemical, etc.; epitope tags (e.g. GFP, FLAG- and His-tags), and, DNA sequences that make a molecular barcode or unique molecular identifier (UMI), DNA sequences required for a specific modification (e.g., methylation) that allows its identification. Other suitable markers will be appreciated by those of skill in the art.

[0385] Selectable markers and tags can be operably linked to one or more components of the programmable genetic toggle switch system described herein via suitable linker, such as a glycine or glycine serine linker.Cell-free Vector and Polynucleotide Expression

[0386] In some aspects, the polynucleotide encoding one or more features of the programmable genetic toggle switch system can be expressed from a vector or suitable polynucleotide in a cell-free in vitro system. In other words, the polynucleotide can be transcribed and optionally translated in vitro. In vitro transcription / translation systems and appropriate vectors are generally known in the art and commercially available. Generally, in vitro transcription and in vitro translation systems replicate the processes of RNA and protein synthesis, respectively, outside of the cellular environment. Vectors and suitable polynucleotides for in vitro transcription can include T7, SP6, T3, promoter regulatory sequences that can be recognized and acted upon by an appropriate polymerase to transcribe the polynucleotide or vector.

[0387] In vitro translation can be stand-alone (e.g. translation of a purified polyribonucleotide) or linked / coupled to transcription. In some aspects, the cell-free (or in vitro) translation system can include extracts from rabbit reticulocytes, wheat germ, and / or E. coli. The extracts can include various macromolecular components that are needed for translation of exogenous RNA (e.g. 70S or 80S ribosomes, tRNAs, aminoacyl-tRNA, synthetases, initiation, elongation factors, termination factors, etc.). Other components can be included or added during the translation reaction, including but not limited to, amino acids, energy sources (ATP, GTP), energy regenerating systems (creatine phosphate and creatine phosphokinase (eukaryotic systems)) (phosphoenol pyruvate and pyruvate kinase for bacterial systems), and other co-factors (Mg2+, K+, etc.). As previously mentioned, in vitro translation can be based on RNA or DNA starting material. Some translation systems can utilize an RNA template as starting material (e.g. reticulocyte lysates and wheat germ extracts). Some translation systems can utilize a DNA template as a starting material (e.g. E coli-basedsystems). In these systems transcription and translation are coupled and DNA is first transcribed into RNA, which is subsequently translated. Suitable standard and coupled cell-free translation systems are generally known in the art and are commercially available.Codon Optimization of Vector Polynucleotides

[0388] As described elsewhere herein, the polynucleotide encoding one or more aspects of the programmable genetic toggle switch system described herein can be codon optimized. In some aspects, one or more polynucleotides contained in a vector (“vector polynucleotides”) described herein that are in addition to an optionally codon optimized polynucleotide encoding aspects of the programmable genetic toggle switch system described herein can be codon optimized. In general, codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g., about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit particular bias for certain codons of a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the “Codon Usage Database” available at www.kazusa.orjp / codon / and these tables can be adapted in a number of ways. See Nakamura, Y., et al. “Codon usage tabulated from the international DNA sequence databases: status for the year 2000” Nucl. Acids Res. 28:292 (2000). Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, PA), are also available. In some embodiments, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence encoding a DNA / RNA-targeting Cas protein corresponds to the most frequently used codon for a particular amino acid. As to codon usage in yeast, reference is made to the online Yeast Genome database available at http: / / www.yeastgenome.org / community / codon_usage.shtml, or Codon selection in yeast, Bennetzen and Hall, J Biol Chem. 1982 Mar 25;257(6):3026-31. As to codon usage in plantsincluding algae, reference is made to Codon usage in higher plants, green algae, and cyanobacteria, Campbell and Gowri, Plant Physiol. 1990 Jan; 92(1): 1-11.; as well as Codon usage in plant genes, Murray et al, Nucleic Acids Res. 1989 Jan 25;17(2):477-98; o Selection on the codon bias of chloroplast and cyanelle genes in different plant and algal lineages, Morton BR, J Mol Evol. 1998 Apr;46(4):449-59.

[0389] The vector polynucleotide can be codon optimized for expression in a specific celltype, tissue type, organ type, and / or subject type. In some aspects, a codon optimized sequence is a sequence optimized for expression in a eukaryote, e.g., humans (i.e. being optimized for expression in a human or human cell), or for another eukaryote, such as another animal (e.g. a mammal or avian) as is described elsewhere herein. Such codon optimized sequences are within the ambit of the ordinary skilled artisan in view of the description herein. In some aspects, the polynucleotide is codon optimized for a specific cell type. Such cell types can include, but are not limited to, epithelial cells (including skin cells, cells lining the gastrointestinal tract, cells lining other hollow organs), nerve cells (nerves, brain cells, spinal column cells, nerve support cells (e.g. astrocytes, glial cells, Schwann cells etc.), muscle cells (e.g. cardiac muscle, smooth muscle cells, and skeletal muscle cells), connective tissue cells ( fat and other soft tissue padding cells, bone cells, tendon cells, cartilage cells), blood cells, stem cells and other progenitor cells, immune system cells, germ cells, and combinations thereof. Such codon optimized sequences are within the ambit of the ordinary skilled artisan in view of the description herein. In some aspects, the polynucleotide is codon optimized for a specific tissue type. Such tissue types can include, but are not limited to, muscle tissue, connective tissue, connective tissue, nervous tissue, and epithelial tissue. Such codon optimized sequences are within the ambit of the ordinary skilled artisan in view of the description herein. In some aspects, the polynucleotide is codon optimized for a specific organ. Such organs include, but are not limited to, muscles, skin, intestines, liver, spleen, brain, lungs, stomach, heart, kidneys, gallbladder, pancreas, bladder, thyroid, bone, blood vessels, blood, and combinations thereof. Such codon optimized sequences are within the ambit of the ordinary skilled artisan in view of the description herein.

[0390] In some embodiments, a vector polynucleotide is codon optimized for expression in particular cells, such as prokaryotic or eukaryotic cells. The eukaryotic cells may be those of or derived from a particular organism, such as a plant or a mammal, including but not limitedto human, or non-human eukaryote or animal or mammal as discussed herein, e.g., mouse, rat, rabbit, dog, livestock, or non-human mammal or primate.Non-Viral Vectors and Carriers

[0391] In some aspects, the vector is a non-viral vector or carrier. In some aspects, non-viral vectors can have the advantage(s) of reduced toxicity and / or immunogenicity and / or increased bio-safety as compared to viral vectors The terms of art “Non-viral vectors and carriers” and as used herein in this context refers to molecules and / or compositions that are not based on one or more component of a virus or virus genome (excluding any nucleotide to be delivered and / or expressed by the non-viral vector) that can be capable of attaching to, incorporating, coupling, and / or otherwise interacting with an programmable genetic toggle switch system polynucleotide of the present disclosure and can be capable of ferrying the polynucleotide to a cell and / or expressing the polynucleotide. It will be appreciated that this does not exclude the inclusion of a virus-based polynucleotide that is to be delivered. For example, if a gRNA to be delivered is directed against a virus component and it is inserted or otherwise coupled to an otherwise non-viral vector or carrier, this would not make said vector a “viral vector”. Non-viral vectors and carriers include naked polynucleotides, chemical -based carriers, polynucleotide (non-viral) based vectors, and particle-based carriers. It will be appreciated that the term “vector” as used in the context of non-viral vectors and carriers refers to polynucleotide vectors and “carriers” used in this context refers to a non-nucleic acid, polynucleotide molecule, or composition that be attached to or otherwise interact with, encapsulate, and / or associate with a polynucleotide to be delivered, such as an programmable genetic toggle switch system of the present disclosure.Naked Polynucleotides

[0392] In some aspects one or more programmable genetic toggle switch system polynucleotides described elsewhere herein can be included in a naked polynucleotide. The term of art “naked polynucleotide” as used herein refers to polynucleotides that are not associated with another molecule (e.g. proteins, lipids, and / or other molecules) that can often help protect it from environmental factors and / or degradation. As used herein, associated with includes, but is not limited to, linked to, adhered to, adsorbed to, enclosed in, enclosed in or within, mixed with, and the like. Naked polynucleotides that include one or more of the programmable genetic toggle switch system polynucleotides described herein can be delivered directly to a host cell and optionally expressed therein. The naked polynucleotides can haveany suitable two- and three-dimensional configurations. By way of non-limiting examples, naked polynucleotides can be single-stranded molecules, double stranded molecules, circular molecules (e.g. plasmids and artificial chromosomes), molecules that contain portions that are single stranded and portions that are double stranded (e.g. ribozymes), and the like. In some aspects, the naked polynucleotide contains only the programmable genetic toggle switch system polynucleotide(s) of the present invention. In some aspects, the naked polynucleotide can contain other nucleic acids and / or polynucleotides in addition to the programmable genetic toggle switch system polynucleotide(s) of the present invention. The naked polynucleotides can include one or more elements of a transposon system. Transposons and system thereof are described in greater detail elsewhere herein.Non-Viral Polynucleotide Vectors

[0393] In some aspects, one or more of the programmable genetic toggle switch system polynucleotides can be included in a non-viral polynucleotide vector. Suitable non-viral polynucleotide vectors include, but are not limited to, transposon vectors and vector systems, plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, AR(antibiotic resistance)-free plasmids and miniplasmids, circular covalently closed vectors (e.g. minicircles, minivectors, miniknots,), linear covalently closed vectors (“dumbbell shaped”), MIDGE (minimalistic immunologically defined gene expression) vectors, MiLV (micro-linear vector) vectors, Ministrings, mini-intronic plasmids, PSK systems (post-segregationally killing systems), ORT (operator repressor titration) plasmids, and the like. See e.g. Hardee et al. 2017. Genes. 8(2):65.

[0394] In some aspects, the non-viral polynucleotide vector can have a conditional origin of replication. In some aspects, the non-viral polynucleotide vector can be an ORT plasmid. In some aspects, the non-viral polynucleotide vector can have a minimalistic immunologically defined gene expression. In some aspects, the non-viral polynucleotide vector can have one or more post-segregationally killing system genes. In some aspects, the non-viral polynucleotide vector is AR-free. In some aspects, the non-viral polynucleotide vector is a minivector. In some aspects, the non-viral polynucleotide vector includes a nuclear localization signal. In some aspects, the non-viral polynucleotide vector can include one or more CpG motifs. In some aspects, the non-viral polynucleotide vectors can include one or more scaffold / matrix attachment regions (S / MARs). See e.g. Mirkovitch et al. 1984. Cell. 39:223-232, Wong et al.2015. Adv. Genet. 89:113-152, whose techniques and vectors can be adapted for use in thepresent invention. S / MARs are AT-rich sequences that play a role in the spatial organization of chromosomes through DNA loop base attachment to the nuclear matrix. S / MARs are often found close to regulatory elements such as promoters, enhancers, and origins of DNA replication. Inclusion of one or S / MARs can facilitate a once-per-cell-cycle replication to maintain the non-viral polynucleotide vector as an episome in daughter cells. In aspects, the S / MAR sequence is located downstream of an actively transcribed polynucleotide (e.g. one or more programmable genetic toggle switch system polynucleotides of the present dislcosure) included in the non-viral polynucleotide vector. In some aspects, the S / MAR can be a S / MAR from the beta-interferon gene cluster. See e.g. Verghese et al. 2014. Nucleic Acid Res. 42:e53; Xu et al. 2016. Sci. China Life Sci. 59:1024-1033; Jin et al. 2016. 8:702-711; Koirala et al.2014. Adv. Exp. Med. Biol. 801:703-709; and Nehlsen et al. 2006. Gene Ther. Mol. Biol.10:233-244, whose techniques and vectors can be adapted for use in the present invention.

[0395] In some aspects, the non-viral vector is a transposon vector or system thereof. As used herein, “transposon” (also referred to as transposable element) refers to a polynucleotide sequence that is capable of moving form location in a genome to another. There are several classes of transposons. Transposons include retrotransposons and DNA transposons. Retrotransposons require the transcription of the polynucleotide that is moved (or transposed) in order to transpose the polynucleotide to a new genome or polynucleotide. DNA transposons are those that do not require reverse transcription of the polynucleotide that is moved (or transposed) in order to transpose the polynucleotide to a new genome or polynucleotide. In some aspects, the non-viral polynucleotide vector can be a retrotransposon vector. In some aspects, the retrotransposon vector includes long terminal repeats. In some aspects, the retrotransposon vector does not include long terminal repeats. In some aspects, the non-viral polynucleotide vector can be a DNA transposon vector. DNA transposon vectors can include a polynucleotide sequence encoding a transposase. In some aspects, the transposon vector is configured as a non-autonomous transposon vector, meaning that the transposition does not occur spontaneously on its own. In some of these aspects, the transposon vector lacks one or more polynucleotide sequences encoding proteins required for transposition. In some aspects, the non-autonomous transposon vectors lack one or more Ac elements.

[0396] In some aspects a non-viral polynucleotide transposon vector system can include a first polynucleotide vector that contains the programmable genetic toggle switch system polynucleotide(s) of the present invention flanked on the 5’ and 3’ ends by transposon terminalinverted repeats (TIRs) and a second polynucleotide vector that includes a polynucleotide capable of encoding a transposase coupled to a promoter to drive expression of the transposase. When both are expressed in the same cell the transposase can be expressed from the second vector and can transpose the material between the TIRs on the first vector (e.g. the programmable genetic toggle switch system polynucleotide(s) of the present invention) and integrate it into one or more positions in the host cell’s genome. In some aspects the transposon vector or system thereof can be configured as a gene trap. In some aspects, the TIRs can be configured to flank a strong splice acceptor site followed by a reporter and / or other gene (e.g. one or more of the programmable genetic toggle switch system polynucleotide(s) of the present invention) and a strong poly A tail. When transposition occurs while using this vector or system thereof, the transposon can insert into an intron of a gene and the inserted reporter or other gene can provoke a mis-splicing process and as a result it in activates the trapped gene.

[0397] Any suitable transposon system can be used. Suitable transposon and systems thereof can include, Sleeping Beauty transposon system (Tcl / mariner superfamily) (see e.g. Ivics et al. 1997. Cell. 91(4): 501-510), piggyBac (piggyBac superfamily) (see e.g. Li et al.2013 110(25): E2279-E2287 and Yusa et al. 2011. PNAS. 108(4): 1531-1536), Tol2 (superfamily hAT), Frog Prince (Tcl / mariner superfamily) (see e.g. Miskey et al. 2003 Nucleic Acid Res. 31(23):6873-6881) and variants thereof.Chemical Carriers

[0398] In some aspects the programmable genetic toggle switch system polynucleotide(s) can be coupled to a chemical carrier. Chemical carriers that can be suitable for delivery of polynucleotides can be broadly classified into the following classes: (i) inorganic particles, (ii) lipid-based, (iii) polymer-based, and (iv) peptide based. They can be categorized as (1) those that can form condensed complexes with a polynucleotide (such as the programmable genetic toggle switch system polynucleotide(s) of the present invention), (2) those capable of targeting specific cells, (3) those capable of increasing delivery of the polynucleotide (such as the programmable genetic toggle switch system polynucleotide(s) of the present invention) to the nucleus or cytosol of a host cell, (4) those capable of disintegrating from DNA / RNA in the cytosol of a host cell, and (5) those capable of sustained or controlled release. It will be appreciated that any one given chemical carrier can include features from multiple categories. The term “particle” as used herein, refers to any suitable sized particles for delivery of theprogrammable genetic toggle switch system system components described herein. Suitable sizes include macro-, micro-, and nano-sized particles.

[0399] In some aspects, the non-viral carrier can be an inorganic particle. In some aspects, the inorganic particle, can be a nanoparticle. The inorganic particles can be configured and optimized by varying size, shape, and / or porosity. In some aspects, the inorganic particles are optimized to escape from the reticulo endothelial system. In some aspects, the inorganic particles can be optimized to protect an entrapped molecule from degradation., the Suitable inorganic particles that can be used as non-viral carriers in this context can include, but are not limited to, calcium phosphate, silica, metals (e.g. gold, platinum, silver, palladium, rhodium, osmium, iridium, ruthenium, mercury, copper, rhenium, titanium, niobium, tantalum, and combinations thereof), magnetic compounds, poarticles, and materials, (e.g. supermagnetic iron oxide and magnetite), quantum dots, fullerenes (e.g. carbon nanoparticles, nanotubes, nanostrings, and the like), and combinations thereof. Other suitable inorganic non-viral carriers are discussed elsewhere herein.

[0400] In some aspects, the non-viral carrier can be lipid-based. Suitable lipid-based carriers are also described in greater detail herein. In some aspects, the lipid-based carrier includes a cationic lipid or an amphiphilic lipid that is capable of binding or otherwise interacting with a negative charge on the polynucleotide to be delivered (e.g. such as an programmable genetic toggle switch system polynucleotide of the present invention). In some aspects, chemical non-viral carrier systems can include a polynucleotide such as the programmable genetic toggle switch system polynucleotide(s) of the present invention) and a lipid (such as a cationic lipid). These are also referred to in the art as lipoplexes. Other aspects of lipoplexes are described elsewhere herein. In some aspects, the non-viral lipid-based carrier can be a lipid nano emulsion. Lipid nano emulsions can be formed by the dispersion of an immisicible liquid in another stabilized emulsifying agent and can have particles of about 200 nm that are composed of the lipid, water, and surfactant that can contain the polynucleotide to be delivered (e.g. the programmable genetic toggle switch system polynucleotide(s) of the present invention). In some aspects, the lipid-based non-viral carrier can be a solid lipid particle or nanoparticle.

[0401] In some aspects, the non-viral carrier can be peptide-based. In some aspects, the peptide-based non-viral carrier can include one or more cationic amino acids. In some aspects, 35 to 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or 100 % of the amino acids are cationic.In some aspects, peptide carriers can be used in conjunction with other types of carriers (e.g. polymer-based carriers and lipid-based carriers to functionalize these carriers). In some aspects, the functionalization is targeting a host cell. Suitable polymers that can be included in the polymer-based non-viral carrier can include, but are not limited to, polyethylenimine (PEI), chitosan, poly (DL-lactide) (PLA), poly (DL-Lactide-co-glycoside) (PLGA), dendrimers (see e.g. US Pat. Pub. 2017 / 0079916 whose techniques and compositions can be adapted for use with the programmable genetic toggle switch system polynucleotides of the present invention), polymethacrylate, and combinations thereof.

[0402] In some aspects, the non-viral carrier can be configured to release an engineered delivery system polynucleotide that is associated with or attached to the non-viral carrier in response to an external stimulus, such as pH, temperature, osmolarity, concentration of a specific molecule or composition (e.g. calcium, NaCl, and the like), pressure and the like. In some aspects, the non-viral carrier can be a particle that is configured includes one or more of the programmable genetic toggle switch system polynucleotides describe herein and a environmental triggering agent response element, and optionally a triggering agent. In some aspects, the particle can include a polymer that can be selected from the group of polymethacrylates and polyacrylates. In some aspects, the non-viral particle can include one or more aspects of the compositions microparticles described in US Pat. Pubs. 20150232883 and 20050123596, whose techniques and compositions can be adapted for use in the present invention.

[0403] In some aspects, the non-viral carrier can be a polymer-based carrier. In some aspects, the polymer is cationic or is predominantly cationic such that it can interact in a chargedependent manner with the negatively charged polynucleotide to be delivered (such as the programmable genetic toggle switch system polynucleotide(s) of the present invention). Polymer-based systems are described in greater detail elsewhere herein.Viral Vectors

[0404] In some aspects, the vector is a viral vector. The term of art “viral vector” and as used herein in this context refers to polynucleotide based vectors that contain one or more elements from or based upon one or more elements of a virus that can be capable of expressing and packaging a polynucleotide, such as an programmable genetic toggle switch system polynucleotide of the present invention, into a virus particle and producing said virus particle when used alone or with one or more other viral vectors (such as in a viral vector system).Viral vectors and systems thereof can be used for producing viral particles for delivery of and / or expression of one or more components of the programmable genetic toggle switch system described herein. The viral vector can be part of a viral vector system involving multiple vectors. In some aspects, systems incorporating multiple viral vectors can increase the safety of these systems. Suitable viral vectors can include retroviral-based vectors, lentiviral-based vectors, adenoviral-based vectors, adeno associated vectors, helper-dependent adenoviral (HdAd) vectors, hybrid adenoviral vectors, herpes simplex virus-based vectors, poxvirus-based vectors, and Epstein-Barr virus-based vectors. Other aspects of viral vectors and viral particles produce therefrom are described elsewhere herein. In some aspects, the viral vectors are configured to produce replication incompetent viral particles for improved safety of these systems.

[0405] Suitable retroviral vectors for the programmable genetic toggle switch system can include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol. 66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992); Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol. 63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991); PCT / US94 / 05700). Selection of a retroviral gene transfer system may therefore depend on the target tissue.

[0406] Suitable lentiviral vectors include, but are not limited to, human immunodeficiency virus (HIV)-based lentiviral vectors, feline immunodeficiency virus (FIV)-based lentiviral vectors, simian immunodeficiency virus (SIV)-based lentiviral vectors, Moloney Murine Leukaemia Virus (Mo-MLV), Visna.maedi virus (VMV)-based lentiviral vector, carpine arthritis-encephalitis virus (CAEV)-based lentiviral vector, bovine immune deficiency virus (BIV)-based lentiviral vector, and Equine infectious anemia (EIAV)-based lentiviral vector. In some embodiments, an HIV-based lentiviral vector system can be used. In some embodiments, a FIV-based lentiviral vector system can be used. In some aspects, the lentiviral vector is an EIAV-based lentiviral vector or vector system.

[0407] In some aspects, the vector can be an adenoviral vector. In some aspects the vector can be a helper-dependent adenoviral vector or system thereof. These are also referred to in the art as “gutless” or “gutted” vectors and are a modified generation of adenoviral vectors (see e.g. Thrasher et al. 2006. Nature. 443: E5-7. In some aspects, the vector is a hybrid-adenoviral vector or system thereof. Hybrid adenoviral vectors are composed of the high transductionefficiency of a gene-deleted adenoviral vector and the long-term genome-integrating potential of adeno-associated, retroviruses, lentivirus, and transposon based-gene transfer. See e.g. Balague et al. 2000. Blood. 95:820-828; Morral et al. 1998. Hum. Gene Ther. 9:2709-2716; Kubo and Mitani. 2003. J. Virol. 77(5): 2964-2971; Zhang et al. 2013. PloS One. 8(10) e76771; and Cooney et al. 2015. Mol. Ther. 23(4):667-674), whose techniques and vectors described therein can be modified and adapted for use in the programmable genetic toggle switch system of the present disclosure.

[0408] In some embodiments, vector(s) can be an adeno-associated virus (AAV) vector. See, e.g., West et al., Virology 160:38-47 (1987); U. S. Pat. No. 4,797,368; WO 93 / 24641; Kotin, Human Gene Therapy 5:793-801 (1994); and Muzyczka, J. Clin. Invest. 94:1351 (1994). In some aspects, the AAV vector or system thereof is configured as a “gutless” vector, similar to that described in connection with a retroviral vector. In some aspects, the “gutless” AAV vector or system thereof can have the cis-acting viral DNA elements involved in genome amplification and packaging in linkage with the heterologous sequences of interest (e.g. the programmable genetic toggle switch system polynucleotide(s)).

[0409] In some aspects, the vector can be a Herpes Simplex Viral (HSV)-based vector or system thereof. HSV systems can include the disabled infections single copy (DISC) viruses, which are composed of a glycoprotein H defective mutant HSV genome. See e.g. 2009. Trobridge. Exp. Opin. Biol. Ther. 9:1427-1436, whose techniques and vectors described therein can be modified and adapted for use in the programmable genetic toggle switch system of the present disclosure.

[0410] In some aspects, the vector can be a poxvirus vector or system thereof. In some aspects, the poxvirus vector can result in cytoplasmic expression of one or more programmable genetic toggle switch system polynucleotides of the present dislcousre. In some aspects the capacity of a poxvirus vector or system thereof can be about 25 kb or more. In some aspects, a poxivirus vector or system thereof can include a one or more programmable genetic toggle switch system polynucleotides of the present disclosure.Vector Construction

[0411] The vectors described herein can be constructed using any suitable process or technique. In some aspects, one or more suitable recombination and / or cloning methods or techniques can be used to the vector(s) described herein. Suitable recombination and / or cloning techniques and / or methods can include, but not limited to, those described in U. S. Applicationpublication No. US 2004-0171156 Al. Other suitable methods and techniques are described elsewhere herein.

[0412] In some embodiments, the vector can have one or more insertion sites, such as a restriction endonuclease recognition sequence (also referred to as a “cloning site”). In some embodiments, one or more insertion sites (e.g., about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertion sites) are located upstream and / or downstream of one or more sequence elements of one or more vectors.

[0413] Vectors can be introduced into host cells using chemical, physical, or biological delivery methods. Such methods include transformation, transfection, electroporation, conjugation, infection, particle-based delivery, and combinations thereof. Following introduction, vectors can be maintained episomally, integrated into the genome, or transiently present depending on vector design and host cell properties.

[0414] Vectors described herein support expression of the programmable genetic toggle switch system in dividing and non-dividing cells and across single-cell, population-level, and multicellular contexts.CELLS AND ORGANISMS

[0415] The programmable genetic toggle switch systems described herein can be included in cells, cell populations, or organisms. In some embodiments, the organism is a non-human organism. Cells and organisms provide the biological context in which the control expression constructs, functional expression constructs, CRISPRA / I transcriptional regulatory machinery, and additional components interact to establish and maintain regulatory states and functional output.

[0416] Cells and organisms described herein can include prokaryotic cells, eukaryotic cells, or multicellular organisms. In preferred implementations, the system is present in eukaryotic cells, including yeast cells, fungal cells, plant cells, insect cells, and mammalian cells. Exemplary cells are described elsewhere herein, e.g., see section on Vectors. One of ordinary skill in the art will appreciate their use in this context as well.Bacterial and Yeast Cells

[0417] The programmable genetic toggle switch systems described herein can be present in bacterial cells. Bacterial cells provide a genetically tractable and well-characterized platform for implementation of programmable regulatory circuits, including mutually exclusive or selectively exclusive state switches. In bacterial cells, control expression constructs, functionalexpression constructs, and / or CRISPR-based regulatory machinery can be expressed from plasmid vectors, integrative vectors, or combinations thereof.

[0418] Bacterial cells support expression of synthetic promoters, inducible promoters, repressible promoters, and optogenetically regulated promoters used in the control and functional expression constructs. Bacterial cells further support guide RNA expression, guide RNA array processing, and CRISPR-mediated transcriptional regulation directed to bacterial promoters or genomic loci. In bacterial populations, regulatory states established by the toggle switch system can be maintained across cell divisions and propagated through continued selfactivation and cross-repression of the state switches.

[0419] Exemplary bacterial genera suitable for use as host cells include Escherichia, Bacillus, Pseudomonas, Streptomyces, Corynebacterium, Lactobacillus, Clostridium, Salmonella, Vibrio, Shewanella, Rhodobacter, and Acinetobacter. Exemplary Escherichia species include Escherichia coli, Escherichia albertii, Escherichia fergusonii, and Escherichia marmotae. Exemplary Bacillus species include Bacillus subtilis, Bacillus licheniformis, Bacillus megaterium, Bacillus amyloliquefaciens, and Bacillus pumilus. Exemplary Pseudomonas species include Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas syringae, and Pseudomonas stutzeri. Exemplary Streptomyces species include Streptomyces coelicolor, Streptomyces lividans, Streptomyces griseus, Streptomyces avermitilis, and Streptomyces venezuelae. Exemplary Corynebacterium species include Corynebacterium glutamicum, Corynebacterium efficiens, Corynebacterium diphtheriae, and Corynebacterium jeikeium. Exemplary Lactobacillus species include Lactobacillus plantarum, Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus acidophilus, and Lactobacillus reuteri. Exemplary Clostridium species include Clostridium acetobutylicum, Clostridium difficile, Clostridium perfringens, Clostridium beijerinckii, and Clostridium thermocellum. Exemplary Salmonella species include Salmonella enterica, Salmonella bongori, Salmonella typhi, and Salmonella typhimurium. Exemplary Vibrio species include Vibrio cholerae, Vibrio natriegens, Vibrio vulnificus, Vibrio parahaemolyticus, and Vibrio fischeri. Exemplary Shewanella species include Shewanella oneidensis, Shewanella putrefaciens, Shewanella baltica, and Shewanella loihica. Exemplary Rhodobacter species include Rhodobacter sphaeroides, Rhodobacter capsulatus, Rhodobacter blasticus, and Rhodobacter veldkampii. Exemplary Acinetobacter species include Acinetobacter baylyi, Acinetobacter baumannii, Acinetobacter calcoaceticus, and Acinetobacter pittii.

[0420] The programmable genetic toggle switch system can be included in yeast cells. Yeast cells provide a robust and genetically tractable platform for implementation of multistate genetic circuits, including mutually exclusive or selectively exclusive state switches.

[0421] Yeast cells can include species from the genera Saccharomyces, Pichia, Kluyveromyces, Hansenula, Yarrowia, Schizosaccharomyces, and related yeasts. In some embodiments, the yeast is a Yarrowia or Saccharomyces species. In some embodiments, the yeast is Yarrowia lipolytica, Yarrowia deformans, Yarrowia keelungensis, Yarrowia bubula, Yarrowia phangngensis, Yarrowia divulgata, Yarrowia porcina, or Yarrowia yakushimensis. In some embodiments, the yeast is Saccharomyces cerevisiae, Saccharomyces paradoxus, Saccharomyces bayanus, Saccharomyces pastorianus, Saccharomyces mikatae, Saccharomyces kudriavzevii, Saccharomyces uvarum, or Saccharomyces arboricola. In some embodiments, the yeast is Pichia pastoris, Pichia kudriavzevii, Pichia occidentalis, Pichia fermentans, Pichia norvegensis, or Pichia membranifaciens. In some embodiments, the yeast is Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces dobzhanskii, or Kluyveromyces wickerhamii. In some embodiments, the yeast is Hansenula polymorpha, Hansenula fabianii, Hansenula anomala, or Hansenula jadinii. In some embodiments, the yeast is Schizosaccharomyces pombe, Schizosaccharomyces japonicus, or Schizosaccharomyces octosporus.

[0422] In yeast cells, control expression constructs and / or functional expression constructs and / or CRISPR A / I machinery constructs can be carried on episomal vectors, integrative vectors, or combinations thereof. Constructs can be integrated at defined genomic loci or maintained extrachromosomally. control expression constructs and / or functional expression constructs and / or CRISPR A / I machinery constructs can be can be expressed constitutively or under transcriptional control appropriate for yeast expression systems.

[0423] Yeast cells support expression of optogenetically regulated promoters, inducible promoters, and synthetic promoters used in the control and functional expression constructs. Yeast cells further support guide RNA expression, guide RNA array processing, and CRISPR-based transcriptional regulation directed to yeast promoters and genomic loci.Other Eukaryotic Cells

[0424] In addition to yeast cells, the programmable genetic toggle switch system can be included and / or expressed in other eukaryotic cells, including fungal cells, plant cells, insect cells, and mammalian cells. Exemplary other eukaryotic cells cells are described in connectionwith Vectors elsewhere herein. One of skill in the art will appreciate them as suitable cells in this context as well.

[0425] In multicellular organisms, the system can be present in one or more tissues, cell types, or developmental stages. Regulatory state selection can be uniform across a population of cells or heterogeneous depending on delivery, stimulation, or regulatory design.Multicellular Organisms

[0426] The programmable genetic toggle switch system components can be present in multicellular organisms, including plants, animals, and engineered organisms. In such organisms, the system can function at the cellular level, tissue level, or organismal level. In some embodiments, the multicellular organism is a non-human multicellular organism.

[0427] In multicellular contexts, the system can be configured to respond to environmental cues, developmental signals, or externally applied stimuli to switch between regulatory states. Functional output can include production of proteins, RNAs, or other molecules in a statedependent manner.Environmental Context and Stability

[0428] Cells and organisms containing the programmable genetic toggle switch system can be maintained under a variety of environmental conditions, including different growth media, nutrient conditions, temperatures, osmolarities, and light conditions. One of ordinary skill in the art will be familiar with culturing of cells and cell populations including in commercial bioreactor scale settings.

[0429] Regulatory states established by the system can persist across environmental changes or can be altered in response to environmental stimuli, depending on system configuration. In yeast cells, regulatory state stability can be maintained during batch culture, continuous culture, or other growth regimes.KITS

[0430] Described here are kits for implementing, assembling, delivering, or using the programmable genetic toggle switch systems described herein. Kits described herein can be configured for construction and deployment of control expression constructs, functional expression constructs, CRISPR A / I transcriptional regulatory machinery, and additional components in cells, cell populations, or organisms. Any of the compounds, compositions, systems, and cells described herein or a combination thereof can be presented a combination kit. As used herein, the terms "combination kit" or "kit of parts" refers to the compounds,compositions, systems, cells and any additional components that are used to package, sell, market, deliver, and / or administer the combination of elements or a single element, such as the active ingredient, contained therein. Such additional components include, but are not limited to, packaging, syringes, blister packages, bottles, and the like. The separate kit components can be contained in a single package or in separate packages within the kit.

[0431] Kits can include nucleic acid components, vectors, reagents, and instructions sufficient to assemble and operate a programmable genetic toggle switch system in a desired host context. Kits can be configured for research, industrial, diagnostic, or synthetic biology applications.

[0432] Kits can include one or more control expression constructs that encode control expression construct promoters, regulatory element regions, and associated regulatory signal target sites. Such constructs can be provided as plasmids, linear nucleic acids, or other vector formats.

[0433] Kits can include one or more functional expression constructs that include functional expression construct promoters, functional expression construct regulatory element signal target sites, and functional element regions encoding functional elements such as reporters, effectors, regulatory elements, payload functional elements, or combinations thereof.

[0434] Kits can include CRISPR A / I transcriptional regulatory machinery components, including expression constructs encoding CRISPR transcriptional activator complexes, CRISPR transcriptional inhibitor complexes, or both. Such components can be provided on a single expression construct or on separate expression constructs.

[0435] Kits can further include additional component constructs, including constructs encoding RNA processing components, protein stability components, localization components, environmental sensing components, orthogonal regulators, or insulating elements.

[0436] Kits can include vectors suitable for expression in prokaryotic cells, eukaryotic cells, or both. Vector formats can include plasmid vectors, integrative vectors, episomal vectors, viral vectors, or non-viral vectors. Vectors can be configured to support expression in yeast cells, bacterial cells, or other host cells.

[0437] In some kits, multiple constructs are provided on separate vectors to support modular assembly or staged introduction into host cells. In other kits, multiple constructs are provided on a single vector to simplify delivery or maintenance.

[0438] Kits can be configured for use in specific host organisms. In some embodiments, kits are configured for use in yeast cells, including Saccharomyces species, Yarrowia species, Pichia species, Kluyveromyces species, Hansenula species, and Schizosaccharomyces species.

[0439] Host-specific kits can include promoters, regulatory elements, selectable markers, and vectors selected to function in the intended host. Kits can also include reagents or instructions tailored to transformation, integration, or maintenance of constructs in the selected host.

[0440] Kits can include components provided as pre-assembled constructs or as modular parts suitable for assembly by the user. Modular parts can include promoters, regulatory element regions, functional element regions, guide RNA sequences, or regulatory signal target site sequences that can be combined to generate customized toggle switch configurations.

[0441] Kits can support two-state systems, multi-state systems, or expandable systems in which additional state switches or functional outputs are added over time.

[0442] Kits can include written or electronic instructions describing assembly of constructs, introduction into host cells, stimulation of state switching, detection of functional output, or maintenance of regulatory states. Instructions can describe use of optical, chemical, thermal, or other stimuli to initiate or reverse switching between regulatory states.

[0443] Kits can support use of the programmable genetic toggle switch system in single cells, cell populations, or multicellular contexts.

[0444] Kits described herein provide modular and flexible tools for implementing programmable genetic toggle switch systems across a range of host organisms and applications. By supplying constructs, vectors, and supporting components in kit form, the systems described herein can be readily deployed, customized, and scaled for diverse uses.METHODS OF USE

[0445] Described herein are methods of using the programmable genetic toggle switch systems described herein to establish, switch between, and maintain discrete regulatory states in vitro, in cells, cell populations, and / or organisms. The methods can include coordinated interaction between control expression constructs, functional expression constructs, CRISPR A / I transcriptional regulatory machinery, and optional additional components.

[0446] In general, the methods involve placing a system that includes at least a first state switch and a second state switch into a cell or organism, applying a stimulus that activates one of the state switches, and allowing the activated state switch to self-activate and repress theother state switch. Through this process, a selected regulatory state is established and maintained, and functional elements associated with that regulatory state are produced.

[0447] In some embodiments, a programmable genetic toggle switch system is introduced into a cell, population of cells, or organism. The system includes control expression constructs configured for mutual repression and self-activation and functional expression constructs configured to produce functional elements in a state-dependent manner.

[0448] A first stimulus is applied that activates a first state switch. Activation of the first state switch results in positive regulation of its own control expression construct and repression of the control expression construct associated with a second state switch. As a result, the system transitions into a first regulatory state.

[0449] In another step, a second stimulus is applied that activates the second state switch. Activation of the second state switch results in positive regulation of its own control expression construct and repression of the control expression construct associated with the first state switch. As a result, the system transitions into a second regulatory state.

[0450] Once established, a regulatory state can be maintained through continued selfactivation of the active state switch and repression of the inactive state switch. Maintenance of the regulatory state can persist in the absence of continued stimulation or can be reinforced by sustained or repeated stimulation, depending on system configuration.

[0451] In some aspects, the method of switching between regulatory states in a programmable genetic toggle switch system of the present disclosure includes (a) applying a first stimulus or a second stimulus to the system, wherein the first stimulus selectively activates a first state switch and the second stimulus selectively activates a second state switch; (b) upon activation of the first state switch, inducing positive regulation of transcription of a first control expression construct and repression of transcription of a second control expression construct; or (c) upon activation of the second state switch, inducing positive regulation of transcription of the second control expression construct and repression of transcription of the first control expression construct; thereby switching the system into a first regulatory state or a second regulatory state that is maintained by self-activation of the activated state switch and repression of the non-activated state switch. In some aspects, the programmable genetic toggle switch system is present in a cell or population thereof or an organism, optionally a yeast.

[0452] In some aspects, the method further includes, after switching the system into the first regulatory state, applying the second stimulus to the system to deactivate the first stateswitch and activate the second state switch, thereby repressing transcription of the first control expression construct and inducing positive regulation of transcription of the second control expression construct so as to switch the system from the first regulatory state to the second regulatory state.

[0453] In some aspects, the first stimulus or the second stimulus is applied transiently, and wherein the regulatory state established by activation of the first state switch or the second state switch is maintained after removal of the transient stimulus. In some aspects, the first stimulus or the second stimulus is applied for a sustained duration, and wherein the regulatory state is maintained during the sustained application of the stimulus. In some aspects, the first stimulus and / or the second stimulus includes one or more of an optical stimulus, a thermal stimulus, a chemical stimulus, a pH stimulus, an osmolarity stimulus, an electrical stimulus, or a mechanical stimulus.

[0454] In some aspects, the first stimulus and the second stimulus are orthogonal stimuli, such that application of the first stimulus selectively activates the first state switch without activating the second state switch, and application of the second stimulus selectively activates the second state switch without activating the first state switch.

[0455] In some aspects, the first stimulus and the second stimulus are applied in combination or sequentially.

[0456] In some aspects, switching the system into the first regulatory state or the second regulatory state results in a corresponding change in expression of one or more functional elements encoded by a functional expression construct associated with the activated state switch and repression of expression of one or more functional elements encoded by a functional expression construct associated with the non-activated state switch.

[0457] In some aspects, the first regulatory state or the second regulatory state is maintained through one or more cell divisions following activation of the first state switch or the second state switch.

[0458] In some aspects, the first regulatory state or the second regulatory state is maintained upon exposure of the system to one or more environmental changes selected from changes in temperature, nutrient availability, pH, osmolarity, oxygen availability, growth conditions, or any combination thereof.

[0459] In systems that include more than two state switches, the method extends to selection among three or more regulatory states. In such configurations, a stimulus is appliedthat activates a selected state switch from among a plurality of state switches present in the system. Activation of the selected state switch results in positive regulation of its own control expression construct and repression of control expression constructs associated with one or more other state switches. Through this coordinated self-activation and cross-repression, the system transitions into a selected regulatory state corresponding to the activated state switch.

[0460] In multi-state systems, each state switch can be associated with a distinct stimulus or with a distinct combination of stimuli, allowing selective activation of individual state switches. Once a regulatory state is established, continued self-activation of the active state switch and repression of non-selected state switches maintains the selected regulatory state. Transition to a different regulatory state is achieved by application of a stimulus that activates a different state switch, resulting in repression of the previously active state switch and activation of the newly selected state switch.

[0461] In multi-state implementations, the method can include use of orthogonal stimuli to selectively activate different state switches without unintentionally activating other state switches. Orthogonal stimuli can differ in type, magnitude, timing, or modality, such that each stimulus preferentially activates a corresponding state switch. Exemplary orthogonal stimuli include optical stimuli of different wavelengths, chemical stimuli of different molecular identities or concentrations, thermal stimuli applied at different temperature ranges, or combinations thereof. Where optical stimuli are used, different state switches can be selectively activated using light of different wavelengths, intensities, durations, or temporal patterns, allowing wavelength-selective switching among regulatory states. Through use of orthogonal stimuli, the method supports reliable selection, maintenance, and transition among multiple regulatory states within the same cell, cell population, or organism.

[0462] Through this approach, the programmable genetic toggle switch system supports switching among multiple regulatory states using the same general methodological framework applied to two-state systems.Methods of Regulating Functional Output

[0463] In some methods, switching between regulatory states results in changes in expression of functional elements encoded by functional expression constructs. Functional elements associated with the active state switch are produced, while functional elements associated with the inactive state switch are suppressed.

[0464] Functional output can include production of reporter functional elements, effector functional elements, regulatory functional elements, payload functional elements, or combinations thereof. Through state switching, the methods allow mutually exclusive or selectively exclusive functional outputs to be generated within the same cell or organism.

[0465] In multi-state implementations, different functional elements can be produced in different regulatory states, allowing selection among multiple functional programs using the same underlying genetic system.Methods for Memory and State Persistence

[0466] In some methods, the programmable genetic toggle switch system functions as a genetic memory element. After application of a stimulus and establishment of a regulatory state, the regulatory state persists over time and across one or more cell divisions.

[0467] In cell populations, daughter cells inherit the regulatory state of the parent cell through continued activity of self-activating control expression constructs and repression of alternative state switches. The regulatory state can persist under steady-state growth conditions or across changes in environmental conditions, depending on system design.

[0468] Reversal of the regulatory state is achieved by application of a stimulus that activates a different state switch, thereby overwriting the existing regulatory state.Exemplary Applications of the SystemsSynthetic Biology and Circuit Design

[0469] The programmable genetic toggle switch systems of the present disclosure can be utilized to engineered programmable genetic circuits. In some embodiments, the systems are used to construct programmable genetic circuits with bistable or multistable behavior. The toggle switch systems function as logic elements, memory elements, or state selectors within larger synthetic biology frameworks.Gene Regulation and Genome Control

[0470] The programmable genetic toggle switch systems of the present disclosure can be utilized to regulate gene expression and or modify polynucleotides, such as the genome or transcriptome. In some embodiments, the programmable genetic toggle switch systems of the present disclosure are used to control expression of endogenous or exogenous genes through state-dependent production of CRISPR system components, transcription factors, or regulatory RNAs. Switching between states results in activation or repression of defined gene sets.Metabolic Engineering

[0471] The programmable genetic toggle switch systems of the present disclosure can be used to modify cellular metabolism. In some embodiments, the programmable genetic toggle switch systems of the present disclosure are used to control metabolic pathways. Different regulatory states correspond to different metabolic programs, allowing switching between growth phases, production phases, or stress-response phases within the same organism.Cell Fate and Di fferentiation Control

[0472] The programmable genetic toggle switch systems of the present disclosure can be used to modulate cell fate and / or differentiation. In eukaryotic cells, including yeast and multicellular organisms, the programmable genetic toggle switch systems of the present disclosure can be used to control cell state, differentiation programs, or developmental pathways by selectively producing regulatory or effector functional elements in different regulatory states.Biosensing and Environmental Response

[0473] The programmable genetic toggle switch systems of the present disclosure can be used as biosensors to provide information on the environment of the system. In some embodiments, the programmable genetic toggle switch systems of the present disclosure can be are used to sense and respond to environmental conditions. External stimuli such as light, chemical signals, temperature changes, or osmolarity changes can activate a state switch in the system causing a switch in regulatory states, resulting in detectable or functional responses.Industrial and Bioprocess Applications

[0474] The programmable genetic toggle switch systems of the present disclosure can be used in an industrial or bioprocess, such as industrial cell cultures. In some embodiments, the programmable genetic toggle switch systems of the present disclosure can be used to control production of enzymes, metabolites, or other products. State switching allows separation of growth and production phases or coordination of complex production workflows.

[0475] Further embodiments are illustrated in the following Examples which are given for illustrative purposes only and are not intended to limit the scope of the invention.EXAMPLES

[0476] Now having described the embodiments of the present disclosure, in general, the following Examples describe some additional embodiments of the present disclosure. Whileembodiments of the present disclosure are described in connection with the following examples and the corresponding text and figures, there is no intent to limit embodiments of the present disclosure to this description. On the contrary, the intent is to cover all alternatives, modifications, and equivalents included within the spirit and scope of embodiments of the present disclosure. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the probes disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C, and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.Example 1

[0477] Applicant has assembled expression cassettes of Yarrowia lipolytica optimized coding sequences for combinatorial tuning of a CRISPRa / i toggle switch system. This includes dCas9 and dCas12a fused to repression domains Mxil, TPL-H3-8, and TPL-H1 (ranked from strongest repressing to weakest) or activation domains VPR and VP 16, as well as Csy4 RNA endonuclease for guide RNA processing from arrays. Applicant has also assembled several toggle switch guide RNA array variants with different stabilities as well as control arrays allowing us to test the function of the CRISPRa / i components and the control guide RNA array expression by light-activated transcription factors. Overall, this strategy allows for construction of 48 toggle switch variants, as well as controls, which can be screened for the desired toggleswitch behavior — stable Venus reporter expression upon minimal duration blue light stimulation. Cells with this phenotype can be selected based on colony fluorescence using a fluorescence scanner. Applicant is developing this protocol using the EL222-pC120 reporter plasmids as proof of concept and is developing additional stimuli-responsive transcription factors regulating the pGal promoter. Screening many transformants will allow for some variation in expression level of the randomly integrated constructs to be captured. Transformants identified in the initial solid culture screen will be followed up with 96-well liquid culture assays (which Applicant has used as a standard for data collection), followed by additional dynamic characterization in continuous culture systems recently demonstrated by Applicant (FIG. 2). This initial screen aims to decrease the strength of repression by usingmore switchable transcriptional repression domains and improve response times using the two-state toggle constructs set forth in FIG. 2.

[0478] Applicant has also designed a combinatorial guide RNA array assembly strategy that will allow assembly of libraries of thousands to millions of toggle switch guide RNA array variants containing between 1 and 3 guide RNAs each targeting self-activation, other repression, and downstream reporter or metabolic array activation and repression (totaling 12 guide RNAs per array). These libraries will be assembled from oligo pools containing single guide RNAs on each oligonucleotide with golden gate cloning sites flanking this encoding the array position and finally barcode flanks allowing us to amplify individual libraries. This will allow additional tuning of these different feedback modes through recruiting multiple repressive or activating transcription factors. For example, two repressive dCas9 fusions may be necessary to repress an activated toggle switch array promoter. This approach can also allow for optimization of activation and repression sites across a series of synthetic toggle switch promoters and downstream reporter (or e.g., a metabolic array) promoters to build toggle switches that do not interfere with the native gene expression programs.Example 2

[0479] FIG. 11 shows a schematic of an optoswitch testing vector, which was also previously used by Applicant to confirm EL222-pC120-blue light activation) and can be used similarly to test additional constructs regulating the pGal or other inducible promoters. Any number of inducible promoter and transcription factor construct elements may be used with reference to the different sites in the Optoswitch Drop-in testing vector of FIG. 11. Constructs will be validated in Yarrowia by illuminating growing cells in Applicant’s minibioreactor turbidostat and assaying their fluorescence with flow cytometry. Working constructs will be indicated by at least 5-fold increase in steady-state reporter expression in response to continuous inducer stimulus exposure, relative to the corresponding steady state in absence of the inducer stimulus.Example 3

[0480] Applicant characterized the dynamic response of EL222-blue-light activation of a pC120::mCherry fluorescent reporter in our Pioreactors running in turbidostat mode. For this Applicant modulated the illumination duration with a single 465 nm peak blue LED. Applicant observed a decrease in fluorescence of the cultures that were not exposed to light or for short durations, 1 hour or less, across the 8 hours of data collection. The cultures Applicantmaintained at 1 OD900overnight prior to the experiment, the cultures are perhaps still adapting to the continuous culture conditions and diluting out some basal activation following seed culture transfer to the bioreactors and exposure to ambient light. For all illumination durations 2 hours or greater Applicant observed increases in mCherry reporter fluorescence above the un-illuminated control, especially 6-8 hours after the beginning of illumination. The continuous 8 hour illumination reactor showed substantial activation of 3-fold after 8 hours and a clear exponential trend that Applicant believes would lead to the approximately 100-fold activation that was observed following overnight illumination in the past. The 4 hour and 2 hour illumination reactors had higher mCherry fluorescence than the un-illuminated control after hour 5 but this is somewhat confounded by the decreasing mCherry signal in the control. Interestingly, the 4 hour illumination sample was still increasing in mCherry signal at hour 8 while the 2 hour illumination sample was decreasing. This can suggest that fluorescence peaks between 5 and 6 hours following illumination, with transcriptional responses peaking about 1-2 hours prior to this. This illumination pattern can be used to inform screening of Applicant’s toggle switch strain library and test wavelength specificity of the optogenetic systems using different wavelengths of light corresponding to the activation wavelength of the transcription factor component included in the construct.

[0481] These results at least demonstrate Applicant’s ability to define the transfer function of light activated transcription factors in continuous culture conditions. This demonstrates Applicant’s ability to characterize the TOGLE Switch strains as well as NOT-NOR logic gates in a highly reproducible manner controlling for cell growth rate and metabolism.Example 4

[0482] Applicant designed a two-state toggle switch, based on optoswitch activation of CRISPRa / i arrays, which in response to light activation would establish a self-activating feedback loop to maintain the on-state, and other-state-repression to maintain the off-state. This approach combines the classical mutual repression motif for bistable, toggle-switch logic with self-activation, which improves switching in eukaryotic systems, where more positive regulators of transcription are required for strong activation than in prokaryotes (Ajo-Franklin et al., 2007, Genes & Dev., Rational design of memory in eukaryotic cells, 21: 2271-2276). Each of the states is indicated by a fluorescent reporter that is regulated by the toggle switch arrays. To simplify initial designs Appicant directly used the optoswitch responsive promoters to drive the toggle-switch CRISPRa / i arrays. Applicant will build out combinatorial logic gatesin the NOT-NOR layer and will implement an additional input transcriptional layer. The toggle-switch arrays will perform the toggle-switch logic and activate or repress the corresponding state reporters.

[0483] To prevent potential growth defects from the toggle-switch guide RNAs interfering with native genes, Applicant chose heterologous and synthetic promoters which have minimal homology to the Yarrowia lipolytica genome. The synthetic pC120 and the Saccharomyces cerevisiae pGal promoters are orthogonal and have been shown to work in Yarrowia in the past (See e.g., Schwartz et al., CRISPR Activation, 2018, Multiplexed CRISPR Activation of Cryptic Sugar Metabolism enables Yarrowia Liplytica Growth on Cellobiose, 13(9)1700584 and Wange et al., Int. J. Mol. Sci., 2022, A Single-Component Blue Light-Induced System Based on EL222 in Yarrowia lipolytica, 23(11), 6344). Applicant also made use of a series of synthetic promoters of varying strength that were designed using a large language model trained on promoter activity in Yarrowia of a series of heterologous promoters from across fungi, and tested in Yarrowia (See e.g., Yuzbashev et al., 2023, Communications Biol., A DNA assembly toolkit to unlock the CRISPR / Cas9 potential for metabolic engineering, 6: 858). These synthetic promoters vary in expression strength from similar to the strongest available TEF promoter, down more than 100-fold lower. Several of these promoters, particularly those of high strength, were found to contain significant homology to the Yarrowia genome resulting in off-target binding in our guide RNA design and excluded. Applicant based initial designs on these synthetic promoters that have been demonstrated to function in Yarrowia, as opposed to developing a synthetic promoter system into which guide RNA target sequences could be inserted.

[0484] Applicant designed activating and repressing guide RNAs for the series of synthetic promoters based on maximal on-target activity and minimal off-target activity using Benchling design tools. Activating guide RNAs were designed to bind between -200 and -350 bp relative to the start codon (Shaw et la., 2022, Nat. Comm. Inducible Expression of large gRNA arrays for multiplexed CRISPRai applications, 13: 4984) and upstream relative to the strongest, most start codon-proximal predicted TATA box (See e.g., Adato et al., 2024, Bioinformatics, ElemenT 2023: an enhanced tool for detection and curation of core promoter elements, 40(3) btael 10). Repressing guides were designed to bind between the TATA box and translation start site. The highest on-target / off-target ratio guides within these regions were used for initialdesigns. Further validation and optimization of guide RNAs for activation and inhibition is in progress, described below.

[0485] Applicant also designed several controls to allow us to test the function of CRISPRai and provide comparisons for toggle switch behavior. Toggle-switch-only controls were made with arrays containing only the guide RNAs for self-activation and other-repression but driven by constitutive reporters and with the original toggle switch array promoters, pC120 and pGal driving EYFP and mCherry reporters, respectively (FIG. 10A). These controls allow for confirmation or denial if faulty guide RNAs are responsible for lack of toggle switch behavior. Reporter-only controls were also made that lack the toggle-switch self-activation and other repression guide RNAs. The reporter only controls (FIG. 10B) will provide an optimal comparison for validating toggle switch behavior, representing the same transcriptional activation cascade as a toggle-switch array, but without the guides RNAs affecting state latching through self-activation and other repression. Therefore, a functional toggle switch array should result in delayed decay of the reporter signal of the most recent activated state relative to a reporter-only array exposed to the same activation stimulus.

[0486] Based on the desired use of wildtype industrially viable strains of Yarrowia lipolytica, such as YB-392, throughout this Examples and other Examples utilizing these strains, Applicant used antibiotic selectable markers flanked by LoxP sites for genomic integration of vectors and subsequent marker recycling with Cre recombinase, unless otherwise discussed. To expedite strain building and also capture additional expression variation that may be beneficial to toggle-switch development we used random genomic integration throughout this initial Build phase. In future work we plan to use targeted integration to reduce the need for screening (See e.g., Schwartz et al., ACS Synthetic Biol., 2016, Standardized Markerless Gene Integration for Pathway Engineering in Yarrowia lipolytica. 6(3): 402-409).Construct Design 1

[0487] To develop Applicant CRISPRa / i light-activated toggle-switch architecture, Applicant initially aimed to split the CRISPRa / i, light-activation, and toggle-switch components between two vectors to be randomly integrated into the genome of our chosen YB-392 base strain of Y. lipolytica. One vector would contain all of the regulatory machinery combining CRISPRa / i and light activation (optoswitch) components), and the other would contain toggle-switch guide RNA arrays and fluorescent reporters of the state of the toggle switch. This would allow combinatorial optimization of these regulatory components andtoggle switch designs through sequential or co-transformation of these vectors. Applicant aimed to test all combinations of dCas9 and dCas12a fused to 3 different repression domains Mxil, TPL-H3-8, and TPL-H1 (ranked from strongest repressing to weakest, (Voytas et al., 2022, Plant Bio. A single helix repression domain is functional across diverse eukaryotes.119(41) e2206986119) or two different activation domains VPR and VP16 (again, ranked strongest to weakest based, See e.g., Schwartz et al., 2018, Biotechnol. J. Multiplexed CRISPR Activation of Cryptic Sugar Metabolism Enables Yarrowia Lipolytica Growth on Cellobiose, 13(9) 1700584). In the context of a toggle switch a balance between activation and repression is necessary, so our goal in testing these combinations is to find a balanced activation and repression pair that will allow dynamic switching and stable state maintenance. See FIG. 8.

[0488] FIG. 12 shows a schematic of constructs built based upon the initial designs in FIG.8 but with the green state construct built reflecting a different design in which the reporter activation guide for the red-state (an) was included in the green-state switch (top construct) and showing that it would have both activation and repression on the Red-state switch reporter construct. Initial toggle switch strains were built by transforming YB-392 with Toggle Switch vectors that are randomly integrated with a neourseothricin resistance (NatR) marker. These strains were selected based on moderate basal -level reporter expression of EYFP and mCherry, as presented in Example 1 and parental strains in FIG.9A-9B. These toggle-switch-containing strains were then co-transformed with CRISPRai machinery vectors and Optoswitch machinery vectors, both randomly integrated, with selectable markers for Zeocin resistance and hygromycin resistance, respectively. As previously discussed, and as highlighted in dark gray in the top construct, the reporter activation guide RNA for the red-state (an) was included in the green state switch array. Inclusion of the green-state activation guide RNA (ao) would facilitate activation of the green-state pGMed-P34: EYFP reporter that would allow observation of toggle switch behavior (see FIG. 12). Inclusion of the aR in the green-state array results in activation of the pC 120 green-state toggle switch array, but not activation of the intended EYFP reporter. However, the pC120-mCherry reporter (bottom construct, FIG. 12) included in the optoswitch machinery vector should be activated by both EL222 and the self-activating guide RNA from the pC120-regulated array and thus activated by the green-state switch construct containing the apci20. While the red-state toggle-switch reporter behavior pRMed-P97: mCherry may confound measurement of the activation of pC120-mCherry reporter, Applicant expects that in the presence of the pC120-regulated array containing apci20 the pC120:mCherry reporterwill have a longer duration of activation following a transition to dark from blue-light activation compared to strains without the pC120-regulated toggle-switch array.Construct Building

[0489] Upon initial assembly of the combined CRISPRa / i and Optoswitch vector it was observed that the EL222 expression cassette failed to assemble correctly under the specicic conditions it was assembled under, but the CRISPRa / i machinery was all intact for the 4 combinations of TPL-H1 / TPLH3-8 and VP16 / VPR repression and activation domains, respectively. Instead of optimizing assembly conditions, Applicant revised the system design to be a three vector system. This three vector system separates the CRISPRa / i and optoswitch machinery across separate vectors and integrations. This design strategy provides more flexibility in development of additional optoswitches in Example 5. Initially, Applicant used the EL222 and pC120:mCherry vector (see e.g., FIG. 12, bottom construct) to provide the necessary EL222 to drive activation of the green state upon blue light stimulation. As the green toggle switch array uses a pC120 promoter as well, the use of the pC120:mCherry vector can demonstrate activation of the green-state by comparing the decay of the mCherry reporter in the strain with just EL222 pC120:mCherry and a strain that also has the toggle switch array.

[0490] Initial assembly of the toggle switch vectors was successful for all 13 of the medium strength reporter containing vectors. This included those with wildtype and truncated, fastturnover guide RNAs, as well as reporter-only controls (with no toggle switch logic), and toggle-switch-only gRNA controls. Applicant integrated these vectors into YB-392 and screened the resulting clones for fluorescence of the medium strength reporters, which in the absence of CRISPRai, should be well above the basal fluorescence of YB-392. As the reporters are at the beginning and end of the vectors, selecting clones with fluorescence above YB-392 ensures that the full construct was integrated. See FIG.9A-9B. These reporters have since been assembled and are now successfully integrated in YB-392 already containing a functional integration of the vector containing EL222 and pC120:mCherry reporter.

[0491] The strains screened in FIG. 9A-9B were transformed simultaneously with the available CRISPRai plasmids and the EL222 pC120:mCherry Optoswitch vector (FIG. 10A-10B) Several transformants of each of these final strains with typical colony morphology and growth rate were selected for testing. Parental strains lacking CRISPRai and Optoswitch vectors were also included for comparison.Results from Test 1

[0492] Applicant measured light responsive fluorescent reporter expression of this initial set of transformants twice with slightly different experimental designs (Experiment 1 and Experiment 2, Table 2). In both cases transformants were grown overnight in 600 µL of YPAD media in 96-well deepwell plates at 350 RPM in a standard long-throw shaking incubator. In the first experiment. Cultures were grown overnight in the dark then split and subjected to blue light (“light” indicates blue light treatment throughout) and dark respectively for 10 hours. Following the 10 hour light treatment, the “light” plate was returned to dark. In the second experiment, duplicate plates were made after treatment and one duplicate was switched to the opposite condition (Light-to-Dark and Dark-to-Light). For each measurement, plates were transferred to a biosafety cabinet minimizing exposure to ambient light and 60 µL samples were diluted into 90 µL of water in a U-bottom microplate and fluorescence was measured via flow cytometry.Table 2. Variations in conditions between two replications of initial toggle switch strain testing.Variable Experiment 1 Experiment 2Inoculum Tip inoculation of relatively 5 cells / µLconsistent amountsDilution lOx prior to splitting for 2x when splitting to light light treatments, after and dark plates and 2x when treatments and every 10-16 switching light conditions hours followingTreatment One plate was exposed to One plate was exposed to blue light for 10 hours, one blue light for 10 hours, one kept in dark. Following light kept in dark. Following light treatment both plates were treatment the plates were moved to dark split and one replicate was switched to the opposite conditionTiming Measurements were taken Measurements were taken every 2-4 hours during every 2-4 hours during activation and then every 12- activation and 14 hours after16 hours for 70 hours total switching treatmentsEL222-pC120:mCherry control strain activation

[0493] Applicant initially analyzed the activation of the simple EL222 and light activated pC120:mCherry direct reporter in our experimental conditions as shown in FIG. 13A-13B. In both experiments the pC120:mCherry response reached saturation at about 6 hours with a maximum expression of >10-fold above YB-392 basal fluorescence.

[0494] Upon removing the light stimulus the mCherry reporter decayed more rapidly in Experiment 1, essentially returning to the YB-392 baseline within 14 hours. In Experiment 2, the L-to-D sample only returned halfway to this baseline. Applicant hypothesized that this difference is due to the decreased dilution rate and subsequently decreased rate of cell growth. In Experiment 2, cell density barely doubled after 14 hours of incubation following a 2x dilution after the 10 hour light treatment indicating a very slow growth rate and approximate doubling time of 7 hours. By comparison, Experiment 1 with a lOx dilution nearly reached the same density after 14 hours, a double time of just over 4 hours, which accounting for some lag post dilution is not outside the expected growth rate for Yarrowia. Also in support of the slow growth and metabolism in Experiment 2 conditions the Dark-to-Light EL222-pC120-mCherry samples only reached half maximal activation of mCherry in 14 hours.

[0495] The light conditions used in these plate-based screening assays are adequate for maximal activation of pC120 promoters by EL222. Decay of reporter fluorescence (and activation) are strongest when rapid growth rates are maintained. lOx dilutions twice daily can allow longer time courses of toggle switch behavior.Lack of EL222-pC 120:mCherry activation in toggle switch test strains

[0496] Since Applicant transformed all of the strains with the EL222 and pC120:mCherry reporter containing Optoswitch vector we can also use similar activation of this reporter to screen our clones for functional integration of the Optoswitch vector (FIG. 14). Two clones were observed to contain a functional mCherry response to light similar to the Optoswitch vector control strain.Example 5

[0497] Applicant has built low-strength reporter toggle switch vectors. Applicant has generated Green-state switch vectors with the correct ao guide RNA in place of the aR guide RNA. See for example, the top construct shown in FIG. 8, FIG. 10B as compared to the top construct shown in FIG. 12. Applicant isolated two clones containing a single green-state reporter activating guide RNA that will allow Applicant to evaluate of CRISPR activation from the CRISPRai machinery vectors. Evaluation will be completed in YB-392 containing the Optoswitch machinery vector. The YB-392 transformants will be screened for >2-fold fluorescence over the parental strain. Fluorescence will be measured by standard flow cytometry. Transformants with >2-fold fluorescence over the parental strain will be considered moderate expression from the red- and green-state toggle switch reporters. The strains selectedfrom this screening will then be transformed with the 3 CRISPRai vectors, and resulting clones will be screened for blue-light activation of the green-state reporter.

[0498] Applicant is generating switch-only control strains. Switch only control vectors are being transformed into YB-392 containing the Optoswitch machinery vector. This allows for screening of the transformants for integration of the Optoswitch machinery based on activation of the pC120: EYFP reporter in the presence of blue light. The selected strains will then be transformed with the CRISPRai vectors. Applicant will screen at least a dozen transformants of each strain can be screened in 96 well plates.

[0499] To prevent an overlap of reporters, Applicant is generating single-state, green-only toggle switch variants.Example 9

[0500] Applicant will also conduct CRISPRa / i functional testing using Csy4-processed gRNA arrays in Yarrowia lipolytica. Applicant is systematically testing the performance and modularity of CRISPR-based transcriptional activation and inhibition in E lipolytica using Csy4-processed guide RNA arrays, as a foundation for scalable genetic logic and state control in our TOGLE platform. Applicants initial experimental framework uses the EL222-pC120 optogenetic system as a well-characterized, light-responsive transcriptional input that we aim to modulate with CRISPRai (FIG. 5). Strains were constructed in the POlf background containing an integrated pC120::mCherry reporters and constitutively expressed EL222, enabling direct measurement of transcriptional activation dynamics. To test this in YB-392 Applicant will reassemble the guide RNA expression vector under a different selectable marker suitable for the strain. Testing will be facilitated by the CRISPRai test vector described above. In the POlf background, Applicant introduced constitutively expressed pC120-targeting CRISPRa or CRISPRi guide RNAs together with constitutive Csy4, allowing precise processing of sgRNAs from arrayed transcripts. Applicant is transforming CRISPRai plasmids into these strains to test the function.

[0501] Initial experiments focus on a single TATA-proximal sgRNA to establish baseline activation and repression capacity for our in-house CRISPRai variants, including comparisons to previously validated systems. Use of the POlf strain background allows direct benchmarking against the previously validated pCRISPRa and pCRISPRi plasmids, which are LEU2-selectable and provide an important reference for expected activation and repression magnitudes. At this stage, transformants containing all needed reporters, sgRNA constructs,Illand Csy4 expression cassettes have been generated. Applicant will introduce, screen, and validate CRISPRai machinery functionality in the PO If background.

[0502] To support multiplexed control and expansion to higher-order logic, sgRNAs in the initial test cassette were flanked by PaqCI sites, enabling straightforward assembly of multiguide arrays. Applicant has constructed both three guide RNA repression arrays and two guide RNA activation arrays to test simultaneous targeting of multiple regulatory sites and to assess Csy4 processing efficiency and guide stoichiometry effects.

[0503] Collectively, these experiments will establish a functional baseline for Csy4-processed CRISPRai arrays in E lipolytica.Example 6

[0504] Applicant will also optimize the toggle switches through a combinatorial library approach. As Applicant has described and demonstrated in the previous working Examples, there are many possible variables and a very large design space for toggle switch development. To efficiently explore this design space, Applicant will evaluate variants in parallel as Applicant has previously done, such as on a 96-well plate scale as previously demonstrated. To expand to pooled combinatorial library assembly of toggle switch variants, Applicant developed a series of 12 unique 4 bp overhangs within the 20 bp Csy4 target site that assemble with high-fidelity that can be used to assemble toggle switch guide RNA arrays of up to 12 guides from oligopools. In this way Applicant can explore the effects of multiple repressing and / or activating guide RNAs targeting the same promoters simultaneously as well as varying guide stability, target site, and many other variables in parallel.Example 7

[0505] Building on Applicants established EL222 / pC120 blue-light-responsive system, Applicant is adapting GAL4-based transcriptional architectures and optogenetic proteinprotein interaction modules that couple light sensing and transcriptional regulation in a yeast two-hybrid fashion. Applicant has generated new GAL4DBD-activation domain fusion constructs paired with pGALl -based fluorescent reporters, which will serve as constitutive positive controls for GAL-dependent transcriptional activation. The Gal promoter was used in the initial demonstrations of CRISPRa in Yarrowia, representing a demonstrated positive control (See e.g., Schwartz et al., Systems Biotechnol. J. 2018, Multiplexed CRISPR Activation of Cryptic Sugar Metabolism Enables Yarrowia Lipolytica Growth on Cellobiose, 13(9): 1700584). These constructs are being evaluated in Y. lipolytica alongside S. cerevisiaereference strains to assess cross-species robustness and to resolve key design parameters such as fusion orientation, nuclear localization, and promoter context that are important for reliable optogenetic function.

[0506] Applicant has designed 38 different optoswitch variant plasmids and controls, generated 21 of these plasmids, and are screening transformants of 18 of the generated plasmids. Applicant will be completing the rest in similar fashion. Appicant will focus evaluation on wavelength specificity and orthogonality, with reporter activation assessed under UV, blue, green, and red illumination conditions. Preliminary screening results confirm strong blue-light specificity for EL222-mediated activation, consistent with prior results, and establishes a benchmark against which additional optogenetic systems can be compared.

[0507] Collectively, this work in these working Examples is defining a modular design space for light-responsive transcriptional control in Y. lipolytica, integrating constitutive and optogenetic activators, multiple wavelength inputs, and standardized reporters. These results directly support the development of higher-order optogenetic toggle switches and multi-state regulatory architectures by identifying transcription factor systems with favorable dynamic range, specificity, and compatibility with CRISPRa / i and recombinase-based memory modules central to the TOGGLE program.Example 8

[0508] Table 3 shows strains and genotypes used in relation to Examples 1-7.Table 3. Strain List for Examples 1-11YWL Short name Genotype1139 GFPswitch-only YB-392 | | [Al] (pWL1022) NATLox-(pWL998) S / l: pMYT039-pC120_2- RFPswitch-only dCas12- YFP_3-TXPR2_4_-(pWL995) 1 / 2: pMYT041-2_pP46-Yali sgRNA GFP VP16 dCas9-TPLH3-8 | switch only-(pWL977) TLip2EL_4-pMYT061_nan_23_Spacer_Cassette- (pWL996) 3 / 4: pMYT045-2_pP45-Yali sgRNA RFP switch only- (pWL978) TSynthGuo_4-(pWL997) 4 / E: pMYT046-pGALl_2- YlmCherry_3-TLip2 _4_, | | ZEOlox dCas12-VP16 dCas9-TPLH3-8 | pCBYL096-EL222-C120-mCherry1138 GFPswitch-only YB-392 | | [Al] (pWL1022) NATLox-(pWL998) S / l: pMYT039-pC120_2- RFPswitch-only dCas12- YFP_3-TXPR2_4_-(pWL995) 1 / 2: pMYT041-2_pP46-Yali sgRNA GFP VP16 dCas9-TPLHl | switch only-(pWL977) TLip2EL_4-pMYT061_nan_23_Spacer_Cassette- (pWL996) 3 / 4: pMYT045-2_pP45-Yali sgRNA RFP switch only- (pWL978) TSynthGuo_4-(pWL997) 4 / E: pMYT046-pGALl_2- YlmCherry_3-TLip2 _4_, | | ZEOlox dCas12-VP16 dCas9-TPLHl | pCBYL096-EL222-C120-mCherry1137 GFPswitch-only YB-392 | | [Al] (pWL1022) NATLox-(pWL998) S / l: pMYT039-pC120_2- RFPswitch-only dCas12- YFP_3-TXPR2_4_-(pWL995) 1 / 2: pMYT041-2_pP46-Yali sgRNA GFP VPR dCas9-TPLHl | switch only-(pWL977) TLip2EL_4-pMYT061_nan_23_Spacer_Cassette- (pWL996) 3 / 4: pMYT045-2_pP45-Yali sgRNA RFP switch only- (pWL978) TSynthGuo_4-(pWL997) 4 / E: pMYT046-pGALl_2- YlmCherry_3-TLip2 _4_, | | ZEOlox dCas12-VPR dCas9-TPLHl | pCBYL096-EL222-C120-mCherry1136 GFPswitch-only dCas12- YB-392 | | [A2]: (pWL1022) NATLox-(pWL998) S / l: pMYT039- VP16 dCas9-TPLH3-8 | pC120_2-YFP_3-TXPR2_4_-(pWL995) 1 / 2: pMYT041-2_pP46-Yali sgRNA GFP switch only-(pWL977) TLip2EL_4- pMYT061_nan_23_Spacer_Cassette-pMYT063_nan_34_Spacer_Cassette- (pWL997) 4 / E: pMYT046-pGALl_2-YlmCheny_3-TLip2 _4_, 1 1 ZEOlox dCas12-VP16 dCas9-TPLH3-8 | pCBYL096-EL222-C120-mCheny1135 GFPswitch-only dCas12- YB-392 | | [A2]: (pWL1022) NATLox-(pWL998) S / l: pMYT039- VP16 dCas9-TPLHl | pC120_2-YFP_3-TXPR2_4_-(pWL995) 1 / 2: pMYT041-2_pP46-Yali sgRNA GFP switch only-(pWL977) TLip2EL_4- pMYT061_nan_23_Spacer_Cassette-pMYT063_nan_34_Spacer_Cassette- (pWL997) 4 / E: pMYT046-pGALl_2-YlmCheny_3-TLip2 _4_, 1 1 ZEOlox dCas12-VP16 dCas9-TPLHl | pCBYL096-EL222-C120-mCheny1134 GFPswitch-only dCas12- YB-392 | | [A2]: (pWL1022) NATLox-(pWL998) S / l: pMYT039- VPR dCas9-TPLHl | pC120_2-YFP_3-TXPR2_4_-(pWL995) 1 / 2: pMYT041-2_pP46-Yali sgRNA GFP switch only-(pWL977) TLip2EL_4- pMYT061_nan_23_Spacer_Cassette-pMYT063_nan_34_Spacer_Cassette- (pWL997) 4 / E: pMYT046-pGALl_2-YlmCheny_3-TLip2 _4_, 1 1 ZEOlox dCas12-VPR dCas9-TPLHl | pCBYL096-EL222-C120-mCheny1133 RFPswitch-only dCas12- YB-392 | | [A3]: (pWL1022) NATLox_YaLi-(pWL998) S / l: pMYT039- VP16 dCas9-TPLH3-8 | pC 120 2-YFP 3 -TXPR2_4_-pMYT059_nan_l 2_Spacer_Cassette- pMYT061_nan_23_Spacer_Cassette-(pWL996) 3 / 4: pMYT045-2_pP45- Yali sgRNA RFP switch only-(pWL978) TSynthGuo_4-(pWL997) 4 / E: pMYT046-pGALl_2-YhnCherry_3-TLip2 _4_, | | ZEOlox dCas12-VP16 dCas9-TPLH3-8 | pCBYL096-EL222-C120-mCheny1132 RFPswitch-only dCas12- YB-392 | | [A3]: (pWL1022) NATLox_YaLi-(pWL998) S / l: pMYT039- VP16 dCas9-TPLHl | pC 120 2-YFP 3 -TXPR2_4_-pMYT059_nan_l 2_Spacer_Cassette- pMYT061_nan_23_Spacer_Cassette-(pWL996) 3 / 4: pMYT045-2_pP45- Yali sgRNA RFP switch only-(pWL978) TSynthGuo_4-(pWL997) 4 / E: pMYT046-pGALl_2-YlmCherry_3-TLip2 _4_, | | ZEOlox dCas12-VP16 dCas9-TPLHl | pCBYL096-EL222-C120-mCheny1131 RFPswitch-only dCas12- YB-392 | | [A3]: (pWL1022) NATLox_YaLi-(pWL998) S / l: pMYT039- VPR dCas9-TPLHl | pC 120 2-YFP 3 -TXPR2_4_-pMYT059_nan_l 2_Spacer_Cassette- pMYT061_nan_23_Spacer_Cassette-(pWL996) 3 / 4: pMYT045-2_pP45- Yali sgRNA RFP switch only-(pWL978) TSynthGuo_4-(pWL997) 4 / E: pMYT046-pGALl_2-YlmCherry_3-TLip2 _4_, | | ZEOlox dCas12-VPR dCas9-TPLHl | pCBYL096-EL222-C120-mCheny1130 no gRNA 1 1 dCas12-VP16 YB-392 1 1 [A4]: (pWL1022) NATLox_YaLi-(pWL998) S / l: pMYT039- dCas9-TPLH3-8 | pC 120 2-YFP 3 -TXPR2_4_-pMYT059_nan_l 2_Spacer_Cassette-pMYT061_nan_23_Spacer_Cassette-pMYT063_nan_34_Spacer_Cassette- (pWL997) 4 / E: pMYT046-pGALl_2-YlmCheny_3-TLip2 _4_, | | ZEOlox dCas12-VP16 dCas9-TPLH3-8 | pCBYL096-EL222-C120-mCheny1129 no gRNA 1 1 dCas12-VP16 YB-392 1 1 [A4]: (pWL1022) NATLox_YaLi-(pWL998) S / l: pMYT039- dCas9-TPLHl | pC 120 2-YFP 3 -TXPR2_4_-pMYT059_nan_l 2_Spacer_Cassette- pMYT061_nan_23_Spacer_Cassette-pMYT063_nan_34_Spacer_Cassette- (pWL997) 4 / E: pMYT046-pGALl_2-YlmChen...

Claims

CLAIMSWhat is claimed is:

1. A programmable genetic toggle switch system comprising:a first state switch comprising a first control expression construct; anda second state switch comprising a second control expression constructwherein the first control expression construct is configured, upon activation, to positively regulate transcription of the first control expression construct and repress transcription of the second control expression construct, andwherein the second control expression construct is configured, upon activation, to positively regulate transcription of the second control expression construct and repress transcription of the first control expression construct.

2. The system of claim 1, wherein the first state switch further comprises a first functional expression construct, wherein the second state switch further comprises a second functional expression construct, or both.

3. The system of claim 1 or 2, wherein the first control expression construct is configured, upon activation, to positively regulate the first functional expression construct and repress the second functional expression construct.

4. The system of claim 2 or 3, wherein the second control expression construct is configured, upon activation, to positively regulate the second functional expression construct and repress the first functional expression construct.

5. The system of any one of claims 1-4, wherein the first control expression construct comprisesa first control expression construct promoter,first control expression construct regulatory signal target sites, and first control expression construct regulatory element region, wherein the first control expression construct regulatory element region encodes one or more first control expression construct regulatory elements, andwherein the first control expression construct promoter is operatively coupled to the first control expression construct regulatory signal target sites and the first control expression construct regulatory element region.

6. The system of any one of claims 1-5, wherein the second control expression construct comprisesa second control expression construct promoter,second control expression construct regulatory signal target sites, and a second control construct regulatory element region, wherein the second control expression construct regulatory element region encodes one or more second control construct regulatory elements, andwherein the second control expression construct promoter is operatively coupled to the second control expression construct regulatory signal target sites and the second control expression construct regulatory element region.

7. The system of any one of claims 5-6, wherein the first control expression construct regulatory signal target sites comprise a first control expression construct promoter activator signal target site and a first control expression construct promoter repressor signal target site.

8. The system of any one of claims 5-7, wherein the first control expression construct regulatory element region encodes a first control expression construct promoter activator and a second control expression construct promoter repressor.

9. The system of claim 8, wherein the first control expression construct promoter activator is configured to bind or interact with first control expression construct promoter activator signal target site so as to activate the first control expression construct promoter.

10. The system of any one of claims 5-9, wherein the second control expression construct regulatory signal target sites comprise a second control expression construct promoteractivator signal target site and a second control expression construct promoter repressor signal target site.

11. The system of any one of claims 5-10, wherein the second control expression construct regulatory element region encodes a second control expression construct promoter activator and a first control expression construct promoter repressor.

12. The system of claim 11, wherein the second control expression construct promoter activator is configured to bind or interact with second control expression construct promoter activator signal target site so as to activate the second control expression construct promoter.

13. The system of any one of claims 11-12, wherein the first control expression construct promoter repressor is configured to bind or interact with the first control expression construct promoter repressor signal target site so as to repress activity of the first control expression construct promoter.

14. The system of any one of claims 8-13, wherein the second control expression construct promoter repressor is configured to bind or interact with the second control expression construct promoter repressor signal target site so as to repress activity of the second control expression construct promoter.

15. The system of any one of claims 2-14, wherein the first functional expression construct comprises a first functional element region that encodes one or more first functional elements.

16. The system of claim 15, wherein the first functional expression construct further comprises a first functional expression construct activator signal target site.

17. The system of any one of claims 15-16, wherein the first functional expression construct comprises a first functional expression construct repressor signal target site.

18. The system of any one of claims 15-17, wherein the first functional expression construct comprises a first functional expression construct promoter, wherein the first functional expression construct promoter is operatively coupled to the first functional element region.

19. The system of any one of claims 2-18, wherein the second functional expression construct comprises a second functional element region that encodes one or more second functional elements.

20. The system of claim 19, wherein the second functional expression construct further comprises a second functional expression construct activator signal target site.

21. The system of any one of claims 19-20, wherein the second functional expression construct comprises a second functional expression construct repressor signal target site.

22. The system of any one of claims 19-21, wherein the second functional expression construct comprises a second functional expression construct promoter, wherein the second functional expression construct promoter is operatively coupled to the second functional element region.

23. The system of any one of claims 5-22, wherein the first control expression construct regulatory element region encodes a first functional expression construct activator.

24. The system of any one of claims 5-23, wherein the first control expression construct regulatory element region encodes a second functional expression construct repressor.

25. The system of any one of claims 5-24, wherein the second control expression construct regulatory element region encodes a second functional expression construct activator.

26. The system of any one of claims 5-25, wherein the second control expression construct regulatory element region encodes a first functional expression construct repressor.

27. The system of any one of claims 15-26, wherein the one or more first functional elements comprises one or more regulatory functional elements, effector functional elements, reporter functional elements, payload functional elements, or any combination thereof.

28. The system of any one of claims 19-27, wherein the one or more second functional elements comprises one or more regulatory functional elements, effector functional elements, reporter functional elements, payload functional elements, or any combination thereof.

29. The system of any one of claims 27-28, wherein the one or more regulatory functional elements comprise sequence-directed regulatory elements, regulatory RNAs, regulatory proteins.

30. The system of claim 29, wherein the sequence-directed regulatory elements are RNA sequence-directed regulatory elements, optionally guide RNAs.

31. The system of any one of claims 27-30, wherein the reporter functional element is an optically active protein.

32. The system of any one of claims 15-31, wherein at least one of the one or more first functional elements is different than at least one of the one or more second functional elements.

33. The system of any one of claims 15-32, wherein all of the one or more first functional elements is different than the one or more second functional elements.

34. The system of any one of claims 5-33, wherein the one or more first control expression construct regulatory elements are sequence-directed regulatory elements.

35. The system of any one of claims 6-34, wherein the one or more second control expression construct regulatory elements are sequence-directed regulatory elements.

36. The system of any one of claims 34-35, wherein the sequence-directed regulatory elements are RNA sequence-directed regulatory elements.

37. The system of claim 36, wherein the RNA sequence-directed regulatory elements are guide RNAs.

38. The system of claim 37, wherein the guide RNAs comprise one or more activation guide RNA configured to recruit a CRISPR-based transcriptional activator complex, an inhibition guide RNA configured to recruit a CRISPR-based transcriptional inhibitor complex, or both.

39. The system of any one of claims 7-38, wherein the one or more first control expression construct regulatory elements comprise(i) a first control expression construct activation guide RNA configured to specifically bind the first control expression construct promoter activator signal target site and recruit a CRISPR transcriptional activator complex,(ii) a second control expression construct repression guide RNA configured to specifically bind the second control expression construct promoter repressor signal target site and recruit a CRISPR transcriptional inhibitor complex,(iii) a first functional expression construct activation guide RNA configured to specifically bind the first functional expression construct activator signal target site and recruit a CRISPR transcriptional activator complex,(iv) a second functional expression construct repression guide RNA configured to specifically bind the second functional expression construct repressor signal target site and recruit a CRISPR transcriptional inhibitor complex, orany combination thereof.

40. The system of any one of claims 10-39, wherein the one or more second control expression construct regulatory elements comprise(i) a second control expression construct activation guide RNA configured to specifically bind the second control expression construct promoter activator signal target site and recruit a CRISPR transcriptional activator complex,(ii) a first control expression construct repression guide RNA configured to specifically bind the first control expression construct promoter repressor signal target site and recruit a CRISPR transcriptional inhibitor complex,(iii) a second functional expression construct activation guide RNA configured to specifically bind the second functional expression construct activator signal target site and recruit a CRISPR transcriptional activator complex,(iv) a first functional expression construct repression guide RNA configured to specifically bind the first functional expression construct repressor signal target site and recruit a CRISPR transcriptional inhibitor complex, orany combination thereof.

41. The system of claim 39, wherein the first control expression construct activation guide RNA and the first functional expression construct activation guide RNA comprise guide RNA scaffold sequences that are distinct from guide RNA scaffold sequences of the second control expression construct repression guide RNA and the second functional expression construct repression guide RNA, such that recruitment of a CRISPR transcriptional activator complex or a CRISPR transcriptional inhibitor complex is determined by the scaffold sequence of the guide RNA.

42. The system of claim 40, wherein the second control expression construct activation guide RNA and the second functional expression construct activation guide RNA comprise guide RNA scaffold sequences that are distinct from guide RNA scaffold sequences of the first control expression construct repression guide RNA and the first functional expression construct repression guide RNA, such that recruitment of a CRISPR transcriptional activator complex or a CRISPR transcriptional inhibitor complex is determined by the scaffold sequence of the guide RNA.

43. The system of any one of claims 1-40, further comprising a third state switch comprising a third control expression construct, wherein the third state controlexpression construct is configured, upon activation, to positively regulate transcription of the third control expression construct and repress transcription of the first control expression construct, the second control expression construct, or both.

44. The system of claim 43, wherein the third state switch further comprises a third functional expression construct.

45. The system of any one of claims 43-44, wherein the third control expression construct is configured, upon activation, to positively regulate the third functional expression construct and repress transcription of the first functional expression construct, second functional expression construct, or both.

46. The system of any one of claims 43-45, Wherein the third control expression construct comprisesa third control expression construct promoter,third control expression construct regulatory signal target sites, and a third control expression construct regulatory element region, wherein the third control expression construct regulatory element region encodes one or more third control expression construct regulatory elements,wherein the third control expression construct promoter is operatively coupled to the third control expression construct regulatory signal target sites and third first control expression construct regulatory element region.

47. The system of claim 46, wherein the third control expression construct regulatory signal target sites comprise a third control expression construct promoter activator signal target site and a third control expression construct promoter repressor signal target site.

48. The system of any one of claims 46-47, wherein the third control expression construct regulatory element region encodes a third control expression construct promoter activator and first control expression construct promoter repressor, a second control expression construct promoter repressor, or both.

49. The system of claim 48, wherein the third control expression construct promoter activator is configured to bind or interact with third control expression construct promoter activator signal target site so as to activate the third control expression construct promoter.

50. The system of any one of claims 48-49, wherein the third control expression construct promoter repressor is configured to bind or interact with the first control expression construct promoter repressor signal target site so as to repress activity of the first control expression construct promoter and wherein the second control expression construct promoter repressor signal target site so as to repress activity of the second control expression construct promoter.

51. The system of any one of claims 43-50, wherein the third functional expression construct comprises a third functional element region that encodes one or more third functional elements.

52. The system of claim 51, wherein the third functional expression construct further comprises a third functional expression construct activator signal target site.

53. The system of any one of claims 51-52, wherein the third functional expression construct comprises a third functional expression construct repressor signal target site.

54. The system of any of claims 51-53, wherein the third functional expression construct comprises a third functional expression construct promoter, wherein the third functional expression construct promoter is operatively coupled to the third functional element region.

55. The system of any one of claims 46-54, wherein the third control expression construct regulatory element region encodes a third functional expression construct activator.

56. The system of any one of claims 46-55, wherein the third control expression construct regulatory element region encodes a first functional expression construct repressor, a second functional expression construct repressor, or both.

57. The system of any one of claims 52-56, wherein the one or more third functional elements comprises one or more regulatory functional elements, effector functional elements, reporter functional elements, payload functional elements, or any combination thereof.

58. The system of claim 57, wherein the one or more regulatory functional elements comprise sequence-directed regulatory elements, regulatory RNAs, regulatory proteins.

59. The system of claim 58, wherein the sequence-directed regulatory elements are RNA sequence-directed regulatory elements, optionally guide RNAs.

60. The system of any one of claims 58-59, wherein the reporter functional element is an optically active protein.

61. The system of any one of claims 52-60, wherein at least one of the one or more third functional elements is different than at least one of the one or more first functional elements, at least one of the one or more second functional elements, or both.

62. The system of any one of claims 52-61, wherein all of the one or more third functional elements is different than the one or more first functional elements, the one or more second functional elements, or both.

63. The system of any one of claims 46 to 62, wherein the one or more third control expression construct regulatory elements are sequence-directed regulatory elements.

64. The system of claim 63, wherein the sequence-directed regulatory elements are RNA sequence-directed regulatory elements.

65. The system of claim 64, wherein the RNA sequence-directed regulatory elements are guide RNAs.

66. The system of claim 65, wherein the guide RNAs comprise one or more activation guide RNA configured to recruit a CRISPR-based transcriptional activator complex, an inhibition guide RNA configured to recruit a CRISPR-based transcriptional inhibitor complex, or both.

67. The system of any one of claims 47-66, wherein the one or more third control expression construct regulatory elements comprise(i) a third control expression construct activation guide RNA configured to specifically bind the first control expression construct promoter activator signal target site and recruit a CRISPR transcriptional activator complex,(ii) a first control expression construct repression guide RNA configured to specifically bind the first control expression construct promoter repressor signal target site and recruit a CRISPR transcriptional inhibitor complex, a second control expression construct repression guide RNA configured to specifically bind the second control expression construct promoter repressor signal target site and recruit a CRISPR transcriptional inhibitor complex, or both,(iii) a third functional expression construct activation guide RNA configured to specifically bind the third functional expression construct activator signal target site and recruit a CRISPR transcriptional activator complex,(iv) a first functional expression construct repression guide RNA configured to specifically bind the first functional expression construct repressor signal target site and recruit a CRISPR transcriptional inhibitor complex, a second functional expression construct repression guide RNA configured to specifically bind the second functional expression construct repressor signal target site and recruit a CRISPR transcriptional inhibitor complex, or both, orany combination thereof.

68. The system of claim 67, wherein the third control expression construct activation guide RNA and the third functional expression construct activation guide RNA comprise guide RNA scaffold sequences that are distinct from guide RNA scaffold sequences of the first and the second control expression construct repression guide RNAs and the first and the second functional expression construct repression guide RNA, such that recruitment of a CRISPR transcriptional activator complex or a CRISPR transcriptional inhibitor complex is determined by the scaffold sequence of the guide RNA.

69. The system of any one of claims 43-68, wherein the first control expression construct is further configured, upon activation to repress transcription of the third control expression construct, repress transcription of the third functional expression construct, or both.

70. The system of any one of claims 43-69, wherein the second control expression construct is further configured, upon activation to repress transcription of the third control expression construct, repress transcription of the third functional expression construct, or both.

71. The system of any one of claims 43-70, wherein the first control expression construct regulatory element region encodes a third control expression construct promoter repressor, wherein the second control expression regulatory element region encodes a third control expression construct promoter repressor, or both.

72. The system of claim 71, wherein the third control expression construct promoter repressor is configured to interact with the third control expression construct promoter repressor signal target site so as to repress activity of the third control expression construct promoter.

73. The system of any one of claims 43-72, wherein the first control expression construct regulatory element region encodes a third functional expression constructpromoter repressor, wherein the second control expression regulatory element region encodes a third functional expression construct promoter repressor, or both.

74. The system of claim 73, wherein the third functional expression construct promoter repressor is configured to interact with the third functional expression construct promoter repressor signal target site so as to repress activity of the third functional expression construct promoter75. The system of any one of claims 1-74, further comprising one or more expression constructs encoding one or more components of CRISPR transcriptional regulatory machinery configured to interact with the activation guide RNAs and the repression guide RNAs.

76. The system of claim 75, wherein the CRISPR transcriptional regulatory machinery comprises a CRISPR transcriptional activator complex and a CRISPR transcriptional inhibitor complex.

77. The system of any one of claims 75-76, wherein the CRISPR transcriptional activator complex and the CRISPR transcription inhibitor complex each comprise a dead Cas (dCas) effector.

78. The system of claim 77, wherein the dCas effector of the CRISPR transcriptional activator complex is different than the dCas effector of the CRISPR inhibitor complex.

79. The system of claim 78, wherein the dCas effector of the CRISPR inhibitor complex is a dCas9.

80. The system of claim 77 or 78, wherein the dCas effector of the CRISPR activator complex is a dCas12.

81. The system of any one of claims 1-80, further comprising an expression construct encoding an endoribonuclease, optionally wherein the endoribonuclease is configured to process one or more RNA transcripts encoded by the system82. The system of any one of claims 1-81, wherein the expression constructs are contained in one or more vectors.

83. An isolated polynucleotide or set of isolated polynucleotides encoding the system of any one of claims 1-82.

84. A vector or set of vectors comprising nucleic acid sequences encoding the system of any one of claims 1-82.

85. A cell or population thereof or an organism comprising the system of any one of claims 1-82, the isolated polynucleotide or set of isolated polynucleotides of claim 83, and / or the vector or set of vectors of claim 84.

86. The cell or population thereof or the organism of claim 85, wherein the cell or organism is a prokaryotic cell, a eukaryotic cell, or a multicellular organism.

87. The cell or population thereof or the organism of claim 86, wherein the multicellular organism is not a human.

88. The cell or population thereof or the organism of any one of claims 85-87, wherein the cell or organism is a yeast.

89. A host cell line comprising the system of any one of claims 1-82.

90. A method of switching between regulatory states in a programmable genetic toggle switch system of any one of claims 1-82, the method comprising:(a) applying a first stimulus or a second stimulus to the system, wherein the first stimulus selectively activates a first state switch and the second stimulus selectively activates a second state switch;(b) upon activation of the first state switch, inducing positive regulation of transcription of a first control expression construct and repression of transcription of a second control expression construct; or(c) upon activation of the second state switch, inducing positive regulation of transcription of the second control expression construct and repression of transcription of the first control expression construct;thereby switching the system into a first regulatory state or a second regulatory state that is maintained by self-activation of the activated state switch and repression of the non-activated state switch.

91. The method of claim 90, wherein the programmable genetic toggle switch system is present in a cell or population thereof or an organism, optionally a yeast.

92. The method of any one of claims 90-91 further comprising, after switching the system into the first regulatory state, applying the second stimulus to the system to deactivate the first state switch and activate the second state switch, thereby repressing transcription of the first control expression construct and inducing positive regulation of transcription of the second control expression construct so as to switch the system from the first regulatory state to the second regulatory state.

93. The method of any one of claims 90-92, wherein the first stimulus or the second stimulus is applied transiently, and wherein the regulatory state established by activation of the first state switch or the second state switch is maintained after removal of the transient stimulus.

94. The method of any one of claims 90-93, wherein the first stimulus or the second stimulus is applied for a sustained duration, and wherein the regulatory state is maintained during the sustained application of the stimulus.

95. The method of any one of claims 90-94, wherein the first stimulus and / or the second stimulus comprises one or more of an optical stimulus, a thermal stimulus, a chemical stimulus, a pH stimulus, an osmolarity stimulus, an electrical stimulus, or a mechanical stimulus.

96. The method of any one of claims 90-95, wherein the first stimulus and the second stimulus are orthogonal stimuli, such that application of the first stimulus selectively activates the first state switch without activating the second state switch, and application of the second stimulus selectively activates the second state switch without activating the first state switch.

97. The method of any one of claims 90-96, wherein the first stimulus and the second stimulus are applied in combination or sequentially.

98. The method of any one of claims 90-97, wherein switching the system into the first regulatory state or the second regulatory state results in a corresponding change in expression of one or more functional elements encoded by a functional expression construct associated with the activated state switch and repression of expression of one or more functional elements encoded by a functional expression construct associated with the non-activated state switch.

99. The method of any one of claims 90-98, wherein the first regulatory state or the second regulatory state is maintained through one or more cell divisions following activation of the first state switch or the second state switch.

100. The method of any one of claims 90-99, wherein the first regulatory state or the second regulatory state is maintained upon exposure of the system to one or more environmental changes selected from changes in temperature, nutrient availability, pH, osmolarity, oxygen availability, growth conditions, or any combination thereof.

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