We will use the DNA-binding activity of the catalytically 'dead' version of Cas9, dCas9, to regulate genes.

For activation to be possible, dCas9 needs to be fused to a RNA polymerase (RNAP) recruiting element. We fused the ω (omega) subunit of RNAP to dCas9 [1]. The ω subunit works as a RNAP recruiting element in E. coli when working in a strain in which RNAP lacks the ω subunit. We used JEN202, "an E. coli MG1655 mutant in which rpoZ, encoding for the ω subunit of RNAP, was replaced by a Spectinomycin resistance gene" [1], for all fluorescence measurements.

The bio-transistor for E. coli consists of a gene controlled by a synthetic promoter.

We synthesized promoters based on the constitutive promoter J23117 [1]. This promoter consists of BBa_J233117, preceded by a protospacer-adjacent motif (PAM) rich upstream regulating sequence (URS) [4].

To show that the bio-transistor works with different promoter sequences, we tested our system for the promoters J23117 and J23117_alt. The sequence for J23117_alt was randomly generated, except for the -35 and -10 regions that were conserved from J23117. Some of our team members wrote a program in C++ and then in Python that generated this type of random sequence while conserving part of an original sequence.
To observe the activity of a single bio-transistor, we used a gene encoding for green fluorescent protein (GFP) as a reporter gene.

If dCas9-ω guided by an sgRNA binds on the promoter close to the transcription start site, the binding of RNAP to DNA is sterically inhibited, and transcription is repressed. We chose to use sgRNAs complementary to sequences 14 or 18 bp upstream from the transcription start site (TSS). We will call these the inhibiting sites -14 and -18.
If dCas9-ω guided by an sgRNA binds at an optimal distance upstream from the promoter, the ω subunit recruits RNAP and transcription is activated. We chose to use sgRNAs complementary to the sequence 71 bp upstream from the TSS. [1]
Note that the activating sgRNA and the -18 inhibiting sgRNA are complementary to the bottom strand of the promoter, whereas the -14 inhibiting sgRNA is complementary to the top strand.
When generating sequences for the sgRNAs and the binding sites, it is important that they are not present in the host organism's genome to avoid regulating genes other than the targeted one.

If both one inhibiting and the activating sgRNA for the same promoter are present in the same cell, we foresaw 2 possible situations. We simulated both with our model: either dCas9-ω can bind both the activating and the inhibiting sites, in this case the model predicts that inhibition will tend to be stronger, or dCas9-ω cannot bind both sites due to steric hindrance in which case the overall effect should tend to be activating.
For more information on these situations, check out our modeling page, or see how this turned out experimentally in our results!

We constructed plasmid pdCas9-ω, encoding for dCas9-ω regulated by a Tetracylcine-inducible promoter, from pdCas9-bacteria [2] and pWJ66 [1]. We synthesized 'sgRNA expressing cassettes' (IDT) controlled by the constitutive promoter pBad and inserted these into pdCas9-ω. We inserted either 1 or 2 sgRNAs (combinations of 1 activating and 2 inhibiting, affecting the same promoter).
We used pWJ89 [1], GFP controlled by the J23117 promoter, [1] as a 'reporter-transistor'. We constructed a 2nd 'reporter-transistor' called pWJ89_alt, GFP controlled by the J23117_alt promoter, from pWJ89 and J23117_alt (synthesized by IDT).
Find out more about the construction of these plasmids in our Lab Notebook!

We transformed JEN202 cells with one of the 'reporter-transistors' and pdCas9-ω with sgRNAs complementary to the activating and/or inhibiting regions of the promoter of the transistor, and measured the fluorescence of these cells with a plate reader or by flow cytometry. After trying several concentrations of Anhydrotetracycline (ATc), we decided to do all further experiments with 1 ng/mL ATc.
Take a look at our results here!

In a biological circuit, the production of the sgRNAs would most likely be induced, instead of constitutive. To test this with our bio-transistors, we changed the promoter of the 'sgRNA expressing cassette' to make it inducible. We did this by inserting a 'sgRNA expressing cassette', controlled by constitutive promoter pBad, in a plasmid containing AraC, placing the pBad promoter next to AraC. When placed next to one another, AraC is a repressor of pBad, and pBad can be induced by adding Arabinose to the medium [5]. Thus, the production of this sgRNAs becomes inducible with Arabinose.

We constructed three plasmids in this fashion with sgRNAs complementary to the J23117_alt promoter: one with the activating sgRNA controlled by pBad/AraC, one with one of the inhibiting sgRNAs controlled by pBAd/AraC, and one with the same inhibiting sgRNA controlled by pBad/Arac and the activating sgRNA controlled by pBad (constitutive).
Find out more about the construction of these plasmids in our Lab Notebook!

To make biological circuits with our bio-transistors, we will have to link several bio-transistors. To test whether the "signal" is strong enough for this to be possible with our system, we modified the transistor with promoter J23117 to express the sgRNA complementary to the activating site of J23117_alt, instead of GFP. In this way, when the first transistor (J23117) is activated by dCas9-ω bound to the corresponding sgRNA, another sgRNA will be expressed. This sgRNA, in complex with dCas9-ω, will bind the activating site of the second transistor (J23117_alt) which will activate the transcription of GFP. In an ideal situation, we would like to see that fluorescence levels obtained like this are close to levels obtained when simply activating one transistor.

We constructed this in one plasmid that we called pWJ89_alt_Z4-to-X4 by inserting the J23117 promoter followed by an 'sgRNA expressing cassette' (synthesized by IDT) into pWJ89_alt, the plasmid that contains GFP controlled by J23117_alt.
You can find out more about the construction of this plasmid in our Lab Notebook!

In digital circuits, transistors are assembled to form logic gates [6], which can then be linked to form complex circuits. Based on this, we decided to assemble our bio-transistors to form a (bio)logic gate. Below is our design for the NAND gate [7].

The design of our NAND gate contains 3 bio-transistors. The inputs, A and B, are in the form of sgRNAs called A0, A1, B0 and B1.
Note that in our model the sequence A1 is not present in the transistors. dCas9-ω will bind to A1 but it will not find a binding site on the DNA.
The output, C, is the transcription of GFP for C=0, and RFP for C=1.

We did the following design with the hypothesis that when activating and inhibiting sites of the same promoter are bound by dCas9-ω, the overall effect will be inhibition. We also suppose that, when neither the activating nor the inhibiting sites are bound, the basal transcription level is similar to the inhibited level.
Note that many other designs of this gate are possible, and that with similar systems we could also reproduce other gates, such as NOT, AND, OR, NOR, XOR, etc.

Each gRNA «cassette» is designed as in fig.1 :
5' - DsRed2 – polyA - HH ribozyme – gRNA SDS – gRNA scaffold – HDV ribozyme – 3'.

• DsRed2 is a fluorescent protein. It acts as a reporter gene for the production of gRNAs [1].
• The polyA tail is a sequence of 50 adenines. This particular sequence aims to replace the MATLAT1 triple helix in mammalian cells [3]. It increases translational efficiency [4] and contributes to efficient mRNA progression away from the gene [5].
• The hammerhead (HH) ribozyme and the hepatitis delta virus (HDV) ribozyme both carry out self-cleavage after transcription, [2] and [3].
 

This design, under promoter ADH1, allows to recruit RNA polymerase II. This RNA polymerase allows production of multiple gRNAs from a single transcript, contrary to RNA polymerase III [3].

The ability to process information is fundamental to life. Cells use complex gene regulatory networks to effectively respond to the myriad of signals they receive from both the outside environment and their internal metabolism. This information processing capability enables them to move around, communicate, reproduce - in one word, survive.

Since the early 2000s, researchers have sought to harness this capability by re-engineering cellular signal processing pathways for various biotechnology applications. By implementing rational, controllable logic elements in cells, researchers aim to transform living systems into engineered "machines" that may perform functions ranging from industrial production to biomedical therapies, bioremediation and energy production [1].

Up to date, various strategies and genetic parts have been used to implement information processing systems in cells. Many of these designs use the regulation of DNA expression as the adjustable signal in the circuit. A good example is the “repressilator” built by Elowitz et al. in 2000 that uses three orthogonal repressor-promoter pairs : LacI, tetR and λ cI to produce an oscillating signal [2]. Since then, multiple other repressors and activators (zinc-fingers, TALEs) have been used to target specific DNA sequences. Alternative strategies to engineer synthetic biological circuits are based on the control of RNA stability, RNA translation or protein-protein interactions as the basis for signal transmission.

The challenge today is to transition from the creation of small cellular genetic “devices” to cells that efficiently perform logical functions observed in electronic circuits. This issue calls for innovative strategies and rational design [3]. The engineering-driven approach of synthetic biology which includes parts standardization, modular components, modeling and systematic strategies to create biological circuits with reliable and predictable behaviors - holds part of the solution. The other key to the problem is to increase the number of orthogonal genetic components that may be used for signal processing [4]. The characterization of new parts that do not cross-talk with the cellular machinery nor with other parts of the genetic circuit promises to improve the robustness and efficiency of future circuits.

Retroactivity is not the only limitation of current cellular signal processing circuits. Such circuits are slow: their response time can be measured in hours or even days. Furthermore, they are often unreliable and offer low signal-to-noise ratios. Low output signal may make connecting multiple circuits impossible. Another limiting factor is the fact that the transcription factors used in the circuit may be toxic for the cell and that the circuit itself may monopolize the cell’s resources. Finally, circuit parts may behave unexpectedly in new genetic contexts [5]. These issues underline the huge task at hand when one dreams of transferring computing from a world of silicon into the realm of biology.

CRISPR stands for clustered, regularly interspaced, short palindromic repeat arrays. It plays the role of an immune system for bacteria by targeting and degrading foreign DNA. The CRISPR systems uses the Cas9 (CRISPR-associated) nuclease to introduce double-strand breaks to DNA sequences that are complementary to its guide RNA (gRNA). Catalytically dead Cas9 (dCas9) lacks the ability to cleave DNA and may act as programmable transcription regulator - by either preventing the binding of the RNA polymerase (RNAP) to the targeted DNA - or as an activator when it is fused to a RNAP recruiting element (the omega subunit of RNAP in E. coli and VP64 in Yeast) [7].

The main advantage of this system is that many different orthogonal pairs of synthetic promoters and targeting gRNAs can be designed. In addition, synthetic promoters may be built with numerous regulating binding sites in order to receive a variety of inputs, both activating and inhibiting. These properties make CRISPR a useful addition to the synthetic biologist’s toolbox. Its versatility may help build more complex and extendable genetic circuits, as has been already shown in recent publications. Indeed, this technology has been used to create simple logic circuits - such as NOR gates and 3-gate circuits [8].