Cell-specific expression

Use gene editing technology to make a (re)programmable system for cell-type-specific expression of genetic payloads for gene therapy.

Practical and scalable multi-sensor cell-specific expression techniques

1) miRNA signature logic (“who am I?”) → CRISPR gate

  • What: Read endogenous miRNA patterns with miR-OFF (detarget a transgene where a miRNA is high) and miR-ON (activate where it’s high), then drive a CRISPR layer.

  • How to wire:

    • Put miRNA target sites (e.g., miR-122 for liver, miR-142-3p for hematopoietic) in the 3′UTR of (i) your payload, (ii) anti-CRISPR proteins (Acr), or (iii) dCas9/base editor halves to implement AND/NOT gates. This is small-payload friendly (fits in AAV). PMC, ScienceDirect
  • Why good: Extremely portable, widely used in vivo (AAV, mRNA). Cas9-ON via miRNA-repressed Acr is a great general switch (low background, strong selectivity). PMC

2) gRNA-programmable promoters → deep/wide logic without new proteins

  • What: Use CRISPRa/CRISPRi-responsive synthetic promoters/operators (promoters studded with gRNA target sites) controlled by dCas9-VPR/SAM or KRAB. You can stack many orthogonal promoters and gRNAs to implement multi-input logic with just a handful of proteins. Nature
  • Why good: Very reprogrammable (swap gRNAs, not proteins), scalable to multi-layer logic; validated in mammalian cells and safe-harbor landing pads (see below). Nature
  • Related: A general blueprint for multi-input CRISPRa promoters (deep/wide circuits) was formalized, showing systematic design for AND-like and multi-branch networks (while that study optimized prokaryotic/cell-free parts, the architecture is directly portable). PNAS

3) Synthetic TF toolkits (ZF-based) for orthogonal, druggable control

  • What: Libraries like COMET (44 activators, 12 repressors, 83 cognate promoters) and synZiFTRs (compact, human-derived, drug-controllable zinc-finger TFs). They give you many orthogonal inputs you can map to sensors. Nature, Science
  • Why good: Mature, well-characterized parts; easy to compose Boolean logic and titratable outputs; synZiFTRs are designed with clinical practicality in mind (compact, humanized, small-molecule control). Science

4) RNA-only, highly multiplexable logic with endoRNase switches (PERSIST)

  • What: PERSIST uses CRISPR endoRNases (e.g., Csy4, CasE/Cas6) as RNA-level ON/OFF switches; nine orthogonal enzymes support all 16 two-input Boolean functions; circuits resist epigenetic silencing and are good for mRNA delivery. PMC, PubMed
  • Why good: Great for layering many inputs (miRNAs, endogenous RNAs, inducible RNAs) before a CRISPRa/TF stage. PMC

5) Extracellular marker + intracellular state AND-gates (synNotch + CRISPR/TF)

  • What: synNotch receptors convert a surface antigen cue into a user-defined transcriptional output (e.g., gRNA, TF, recombinase). Combine with miRNA logic to get (antigen) AND (miRNA profile) targeting. Oxford Academic
  • Why good: Adds an environmental/cell-cell interaction input; widely used in engineered T cells and generalizable. Oxford Academic

6) Memory & intersectional targeting with recombinases

  • What: Use Cre/Flp/Dre/Bxb1/φC31 to “write” the result of a sensor computation (e.g., flip a STOP, install a payload). Modern sets like BLADE provide clean, composable recombinase logic gates. Courses at UW
  • Why good: Stable memory of transient cues; works beautifully with enhancers/promoters and safe-harbor landing pads (Bxb1, etc.). PMC

7) Split effectors as AND-gates

  • What: Express split-Cas9/base editors/dCas9 halves under different sensors; reconstitution (inteins, rapamycin dimerizers, light, etc.) gives a powerful AND gate with low leak. Oxford Academic, PMC, Nature

“Starter blueprints” you can adapt

A) All-genetic AND/NOT for cell identity (portable across systems)

  • Inputs: 2–5 miRNAs (identity markers) + 1 drug (clinician control).
  • Logic layer: miRNA-repressed Acr (NOT), miRNA-gated dCas9 halves (AND).
  • Core: CRISPRa responsive promoters (2–6 orthogonal promoters) driving payload(s).
  • Output: Editor/effector under a final gRNA-programmable promoter. PMC, Nature

B) Antigen × state intersection

  • synNotch (antigen) → expresses gRNA A; miR logic licenses dCas9-VPR; final CRISPRa promoter needs gRNA A & B (k-of-n logic). Add PERSIST RNAs to expand inputs without new proteins. Oxford Academic, PMC

C) “Sense → Decide → Write” with memory

  • Sensors (miRNA, synNotch, PERSIST) → recombinase (Bxb1/Cre) flips in/out a payload cassette at a landing pad; optional CRISPRoff/on locks the state epigenetically. PMC, ScienceDirect

Where enhancers/promoters come from (and how to target cell types)

  • Enhancer discovery & delivery: PESCA (parallel enhancer single-cell assay), AAV-STARR-seq, scMPRA and new enhancer-AAV toolboxes give vetted, neuron and brain-region selective enhancers that you can pair with the logic above. bioRxiv, ResearchGate, Cell, ScienceDirect
  • Synthetic promoter libraries: Designed, short synthetic promoters (<250 bp) tuned to stimuli provide compact, modular readouts that scale in mammalian cells. PMC

Emerging or reprogrammable techniques

  • Bridge RNA–guided recombinases (IS110) — RNA-programmable recombination (insertions, inversions, excisions) with a single protein + RNA, potentially a compact way to “write” enhancer/promoter states downstream of sensors. Nature, PMC
  • RNA-sensing guide RNAs — gRNAs that activate only upon detecting specific RNAs (e.g., dual-toehold or miRNA-sensing sgRNAs) offer direct RNA→CRISPR wiring to expand input channels without new proteins. eLife, Nature
  • Programmable, humanized synTFs — synZiFTRs for small molecule in-human reprogramming; Science

Stable and predictable integration

Use landing pads (e.g., Bxb1 at AAVS1/ROSA26) to drop in circuits once, then swap modules via RMCE. This tames position effects, supports large libraries, and keeps expression stable over time — ideal for iterating sensor combinations. PMC, ScienceDirect

Reprogrammable cell-type targeting & logic

miRNA sensors & CRISPR control

  • Cell-type-specific CRISPR with miR-Cas9 switches. Oxford Academic
  • Cas9-ON via miRNA-repressed anti-CRISPR (in vivo, multi-ortholog). PMC

CRISPRa/CRISPRi-responsive promoters in mammalian cells

  • CRISPR-based synthetic transcription platform with orthogonal operator/promoter libraries (predictable tuning, stable chromosomal expression). Nature

Synthetic TF toolkits (large orthogonal palettes)

  • COMET (44 activators, 12 repressors, 83 promoters; Boolean logic). Nature
  • synZiFTRs (compact, human-derived, drug-regulated). Science

RNA-only logic (composability & anti-silencing)

  • PERSIST — 9 orthogonal endoRNases; full 2-input Boolean set; long-term stability. PMC

Extracellular antigen sensing

  • synNotch for combinatorial antigen recognition; wire to CRISPR/TF layers. Oxford Academic

Enhancer discovery & cell-type AAVs

  • PESCA (parallel enhancer single-cell assay). bioRxiv
  • Enhancer-AAV toolboxes for cortical/neuronal subtypes. Cell, bioRxiv
  • AAV-STARR-seq in brain (thousands of enhancer candidates). ResearchGate
  • scMPRA for cell-type-specific cis-activity. bioRxiv

Split-Cas and inducible editors (compact AND-gates)

  • Split dCas9 logic (3-input AND; Suntag integration). PMC
  • Rapamycin/light-inducible split-Cas9/base editors. PMC, Science, Nature

Landing pads & RMCE

  • Bxb1/φC31 landing pads for reproducible expression and library scale-up. PMC, ScienceDirect

NEW mechanisms (compact & programmable)

  • Bridge RNA–guided recombination (IS110) — RNA-programmed genome rearrangements. Nature

Practical build tips

  • Start with identity sensors like miRNA MREs to gate Acr (for OFF where you don’t want editing) and/or dCas9 halves (for AND where you do). This is for multi-input logic in vivo. PMC
  • Do logic with gRNAs, not proteins. Use CRISPRa/CRISPRi-responsive promoters/operators to combine many inputs; adding a new input is just a new gRNA/operator. Nature
  • Keep circuits portable: Prefer RNA-level gates (PERSIST) and ZF TFs (COMET/synZiFTRs) when payload budget or anti-silencing matters; both scale well. PMC, Nature
  • Stabilize with landing pads (Bxb1), then swap modules by RMCE for rapid rewiring. PMC
  • Find strong, specific enhancers for hard cell types (brain): use PESCA/AAV-STARR-seq/scMPRA hits and layer logic on top (miRNA/synNotch) for extra precision. bioRxiv, ResearchGate

For highly reprogrammable, multi-sensor, cell-specific expression, the most buildable stack today is:

miRNA & extracellular sensors (synNotch) → RNA/TF logic (PERSIST, COMET/synZiFTR, CRISPRa/i-responsive promoters) → optional memory (recombinases, CRISPRoff) → payload, delivered via AAV/mRNA and stabilized with Bxb1 landing pads. This stack is modular, scalable (add inputs as gRNAs), and supported by well-used papers and toolkits. PMC, Nature

Other cell-specific expression strategies

Here are several approaches to create scalable, multi-sensor cell-specific expression systems using modern gene editing technologies:

  • combinatorial recombinase logic gate systems (Cre/lox, Flp/FRT, STOP cassettes)
  • CRISPR-based transcriptional logic
  • CellREADR
  • RNA logic circuits using toehold switches and riboregulators
  • cell-type specific enhancers
  • just go read existing promoter/enhancer libraries for cell-type fingerprinting, like neurons, excitatory neuron, inhibitory neuron, etc...

other stuff

allele specific sensors? "genotype sensors".

polygenic allele sensors? IQ sensor?

what about racial genotypes-- with 3 or 4 specific alleles you should be able to identify a specific racial genotype maybe?