Team:Hong Kong HKUST/Description
Inspiration: Team HKUST 2016
Our inspiration was derived from the HKUST iGEM team of 2016’s tristable switch
Troika. Switches used in their team, and most of the other genetic circuits, are based on proteins and operon pairs. We found that we could try and improve the reliability, diversity, and sensitivity of currently existing biological switches using CRISPR interference (CRISPRi).
With further reading and research, we developed a novel solution to iGEM: to attain bistability (or even multistability) using CRISPRi mechanism.
What do you want to know about Biscuit?
Why current methods need to be improved
Biological switches allow the turning of protein expression on and off at will via a genetic circuit. Bistability can be achieved by incorporating a negative feedback loop in the circuit. By building more complex circuits, multistable gene expression may also be accomplished. The basic logic is illustrated in the circuit diagram below.
Classic Collins switch[1]: repressor 1 inhibits transcription from Promoter 1 and is induced by Inducer 1, vice versa
Until recently, a majority of switches achieve bistability by using two sets of promoters and repressor proteins. Promoters used are likely protein repressors paired with their operons (such as Krüppel associated box (KRAB), methionine repressors and the MarR family of transcriptional regulators). Using the above diagram as an example, each inducer induces one state, which represses the other state.
However, engineering protein regulators is an extremely huge task, and one that might not even pay off. Protein structures and activities are very hard to predict as even mutations that are remotely located from the active site can alter their activities. Altering endogenous pathways requires direct engineering of the genome, which is always problematic. Lastly, introducing these toggle switches means expressing foreign, toxic, proteins in the organism, risking the cell’s well-being.
Why our switch is better
 |
 |
 |
 |
| Simple | Specific | Versatile | Fast |
|---|---|---|---|
| allows regulation endogenous pathways only by introducing a plasmid that carries dCas9 and RNAs that target genomic DNA, the genome does not have to be engineered directly. RNA is also easier to design and has a greater degree of engineered orthogonality.[3] |
dCas9 protein’s targeting is specified by their sgRNAs, and off-targeting is extremely rare.[4] | customizable sgRNA makes it possible for dCas9 to target an almost unlimited number of genes, thus allowing very high flexibility.[3] | response time might also be shorter because RNA translation is not necessary[3] |
Mechanism of our switch
CRISPRi is our OFF button
CRISPR Cas9 systems are widely used in gene editing to knockout and modify genes. Cas9 proteins recognize the sequence with the help of a PAM sequence and short-guide RNA (sgRNA). Upon recognition, the endonuclease domain in the Cas9 protein would generate a double-stranded break at the bound target DNA.
CRISPR interference (CRISPRi) utilizes a catalytically inactivated form of Cas9 protein (dCas9), which does not cleave the target DNA after binding to it. Instead, dCas9 inhibits expression by sterically hindering the transcription machinery. As there is no change to the DNA, CRISPRi allows targeted but reversible knockdown rather than knockout.
In our toggle switch, CRISPRi is used as the mechanism of repression to replace traditional regulatory proteins. While traditional repressor proteins pair with their corresponding operon[5], CRISPR dCas9 is guided to its designated target DNA by the corresponding customized sgRNA for repression. The benefits of the CRISPRi switch over traditional switches are discussed in the above paragraph.
Switch it back ON with asRNA
A switch would not be so useful if it is not reversible and cannot be switched to the other state(s) once toggling for the first time. Hence reversibility is almost essential in a toggle switch. We incorporate synthetic antisense RNAs (asRNAs) in our switch to reverse the suppression after repressing gene expression using CRISPRi. It does this by complementary binding to the sgRNA while it is complexed with dCas9, or prevent free sgRNA from binding to dCas9. A study published in 2016 has demonstrated tunable levels of derepression are achievable in this way, reaching up to 95% derepression[6].
Our Model Circuit
To bring our project to life, we have designed a circuit that drives the Escherichia coli (E.coli) strain DH5-alpha to alternately produce green fluorescent protein (GFP) and red fluorescent protein (RFP) depending on the inducer added to our system.
Proposed Toggle Switch Construct
Overview of Circuitry
Our proposed toggle switch with CRSIPRi and asRNA induced by arabinose and IPTG. In such a construct, sgRNA corresponding to the two states (inhibiting GFP or RFP) are transcribed under inducible promoters. asRNAs are synthesized under the regulation of the opposite inducible promoters to reverse the repression effect brought about by the corresponding sgRNA and dCas9.
For rapid visualization of state-switching, RFP and GFP are chosen as reporter proteins. The behavior of the construct, as a proof of concept design, relies much on the mathematical model based on data from characterizations of individual components, but the desired dynamics of the switch is illustrated below.
| Inducer | Synthesis | Visual Effect | |
|---|---|---|---|
| State 0 | No inducer | dCas9, GFP, RFP | Orange |
| State 1 | Arabinose (Ara) | dCas9, sgRNA_RFP, asRNA_GFP, GFP | Green |
| State 2 | IPTG | dCas9, sgRNA_GFP, asRNA_RFP, RFP | Red |
State 0 -- initial state
dCas9, GFP, and RFP are constitutively produced
State 1 - Arabinose causes inhibition of RFP production
sgRNA_RFP is produced when arabinose is added. Then, the sgRNA forms a complex with the constitutively expressed dCas9 (which is always present in the system). This complex is guided by the sgRNA to specifically target the mrfp protein coding region, thereby suppressing the gene.
As a result, only green light is emitted.
State 2 - IPTG reactivates RFP while suppressing GFP
Adding IPTG causes asRNA_RFP and sgRNA_GFP to be produced.
As illustrated before, sgRNA will form a complex with dCas9. However, as sgRNA_GFP is produced in this state, the complex will suppress GFP instead of RFP.
Meanwhile, the asRNA_RFP produced will target the dCas9/sgRNA_RFP complex currently bound to the RFP gene.
asRNA causes the dCas9 complex to dissociate by tightly binding to the sgRNA.
This will cause RFP production to resume, while also suppressing GFP.
Circuit components and functions
Constitutively expressed Fluorescent Proteins
As a proof of concept design, two commonly used fluorescent proteins are chosen as a reporter gene for the two output states. Green Fluorescent Protein (GFP) under the expression construct BBa_K608002-BBa_E0040-BBa_B0015 was designated as output state 1 while Red Fluorescent Protein (RFP) of construct BBa_K608002-BBa_E1010-BBa_B0015 was designated output state 2.
Notice that both fluorescent proteins are expressed under the same constitutive promoter and RBS, which allows the expression level of both fluorescent proteins to be similar. However, as mentioned in our human practice page, Professor Sun Fei pointed out that the same promoters may cause competition between themselves. Thus, an experiment was designed to characterize such promoter interference.
Constitutively expressed dCas9
The dCas9 will be expressed by BBa_K608002 (medium promoter BBa_J23110 and Medium RBS BBa_B0032). As previous research has shown that the overexpression of dCas9 may cause cytotoxicity to host cell, weaker strength of promoter and RBS have been chosen to prevent such an outcome.
sgRNA and asRNA transcription by inducible promoter
The two sgRNAs are expressed under the regulation of inducible promoters pBAD (Arabinose inducible promoter) and pLac (IPTG inducible promoter). The sgRNA is transcribed and then bound to the dCas9 protein, providing precise guidance for dCas9.
The two asRNAs are expressed under the regulation of their respective inducible promoter. pBAD (Arabinose inducible promoter) and LacL (IPTG inducible promoter) promoter are selected for the following purpose. These repressor based inducible promoters are well-characterized for their strength and transcription start site (TSS), which allow us to plug the RNA transcription template sequence directly behind the TSS. Standard Promoter library of pBAD is also available with different strength of expression to allow for fine-tuning of expressed strength.
References:
[1] T. S. Gardner, C. R. Cantor, and J. J. Collins, “Construction of a genetic toggle switch in Escherichia coli,” vol. 403, no. 6767, pp. 339–342, 2000.
[2] F. Jee Loon, C. Chi Bun, C. Matthew Wook, and L. Susanna Su Jan, “The imminent role of protein engineering in synthetic biology,” vol. 30, no. 3, 2012.
[3] L. Qi and A. Arkin, “A versatile framework for microbial engineering using synthetic non-coding RNAs.,” May 2014.
[4] W. Xuebing, K. Andrea J., and S. Phillip A., “Target specificity of the CRISPR-Cas9 system,” vol. 2, no. 2, Jun. 2014.
[5] T. S. Gardner, C. R. Cantor, and J. J. Collins, “Construction of a genetic toggle switch in Escherichia coli,” vol. 403, no. 6767, pp. 339–342, 2000.
[6] Y. J. Lee, A. Hoynes-Oconnor, M. C. Leong, and T. S. Moon, “Programmable control of bacterial gene expression with the combined CRISPR and antisense RNA system,” Nucleic Acids Research, vol. 44, no. 5, pp. 2462–2473, Feb. 2016.
[7] M.H. Hanewich-Hollatz, Z. Chen, L. M. Hochrein, J. Huang, and N. A. Pierce, "Conditional Guide RNAs: Programmable Conditional Regulation of CRISPR/Cas Function in Bacterial and Mammalian Cells via Dynamic RNA Nanotechnology" ACS Central Science 2019 5 (7), 1241-1249, Jun. 2019.