Part:BBa_K3017070
Combined CRISPRi and antisense RNA Toggle Switch - Model circuit
This construct is a proof of concept of a biological switch, novel to iGEM community, designed by our team. With reference to a related research[1], our team has combined the CRISPRi system with RNA regulators to achieve a toggle switch. The switch utilizes the catalytically inactive form of Cas9 (dCas9) to achieve targeted and reversible repression of genes via specific single-guide RNAs (sgRNAs). Alternatively, the transcription of antisense RNA (asRNAs) reverses the effect of the dCas9 modulated repression on the desired genes. This method of regulation would allow for the ability to fine-tune and easily customize the execution of highly complex genetic circuits.
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. 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.
Circuit dynamics
| 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/ lactose | 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 (BBa_K3017002) 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.
State 2 - IPTG reactivates RFP while suppressing GFP
Adding IPTG causes asRNA_RFP(BBa_K3017004) and sgRNA_GFP(BBa_K3017001) to be produced. As illustrated before, sgRNA will form a complex with dCas9. However, as sgRNA_GFP (BBa_K3017003) is produced in this state, the complex represses gfp instead of mrfp. Meanwhile, the asRNA_RFP(BBa_K3017004) produced will target the dCas9/sgRNA_RFP complex currently bound to the mrfp. asRNA causes the dCas9 complex to dissociate by tightly binding to the sgRNA.
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.
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
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, CRISPR dCas9 is guided to its designated target DNA by the corresponding customized sgRNA for repression. 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% depression.
The two sgRNAs are expressed under the regulation of inducible promoters pBAD (Arabinose inducible promoter) and LacL (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 strength and transcription start site (TSS), which allow us to plug the RNA 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:
References:
[1] 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.
[2] C. Anders, O. Niewoehner, A. Duerst, and M. Jinek, “Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease,” Nature, vol. 513, no. 7519, pp. 569–573, 2014.
[3] S. H. Sternberg, S. Redding, M. Jinek, E. C. Greene, and J. A. Doudna, “DNA Interrogation by the CRISPR RNA-Guided Endonuclease Cas9,” Biophysical Journal, vol. 106, no. 2, 2014.
[4] T. Karvelis, G. Gasiunas, A. Miksys, R. Barrangou, P. Horvath, and V. Siksnys, “crRNA and tracrRNA guide Cas9-mediated DNA interference inStreptococcus thermophilus,” RNA Biology, vol. 10, no. 5, pp. 841–851, 2013.
[5] T. Møller, T. Franch, P. Højrup, D. R. Keene, H. P. Bächinger, R. G. Brennan, and P. Valentin-Hansen, “Hfq,” Molecular Cell, vol. 9, no. 1, pp. 23–30, 2002.
[6] G. M. Cech, A. Szalewska-Pałasz, K. Kubiak, A. Malabirade, W. Grange, V. Arluison, and G. Węgrzyn, “The Escherichia Coli Hfq Protein: An Unattended DNA-Transactions Regulator,” Frontiers in Molecular Biosciences, vol. 3, 2016.
[7]N. Brzozowska et al., “Characterizing Genetic Circuit Components in E. coli towards a Campylobacter jejuni Biosensor,” p. 290155, 2018.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 852
Illegal PstI site found at 2274
Illegal PstI site found at 2478
Illegal PstI site found at 2508
Illegal PstI site found at 3720 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NheI site found at 5543
Illegal NheI site found at 6921
Illegal NheI site found at 7093
Illegal NheI site found at 7116
Illegal NheI site found at 8733
Illegal PstI site found at 852
Illegal PstI site found at 2274
Illegal PstI site found at 2478
Illegal PstI site found at 2508
Illegal PstI site found at 3720 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 313
Illegal BamHI site found at 5482
Illegal BamHI site found at 6860 - 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 852
Illegal PstI site found at 2274
Illegal PstI site found at 2478
Illegal PstI site found at 2508
Illegal PstI site found at 3720 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 852
Illegal PstI site found at 2274
Illegal PstI site found at 2478
Illegal PstI site found at 2508
Illegal PstI site found at 3720
Illegal NgoMIV site found at 1140
Illegal NgoMIV site found at 2244
Illegal NgoMIV site found at 2317
Illegal NgoMIV site found at 2802
Illegal NgoMIV site found at 3711
Illegal AgeI site found at 5317
Illegal AgeI site found at 6695
Illegal AgeI site found at 7702
Illegal AgeI site found at 7814
Illegal AgeI site found at 9342
Illegal AgeI site found at 9454 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 5299
Illegal SapI site found at 6677
| None |