Combined CRISPRi and Antisense RNA Toggle Switch

A core concept of synthetic biology is controlling gene expression, often achieved through inducers and protein repressors to create feedback loops and switches. 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. Using GFP and RFP in our circuit as a proof of concept, RFP is suppressed under the first inducible promoter while GFP is produced. Under the second inducible promoter, the dCas9 is unable to bind to mrfp, derepressing mrfp and suppressing GFP.

The problem of conventional switches

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: 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.[1]

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[2]. 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, as a result the genome does not have to be engineered directly.	dCas9 protein’s targeting is specified by their sgRNAs, and off-targeting is extremely rare.	customizable sgRNA makes it possible for dCas9 to target an almost unlimited number of genes[3], thus allowing very high flexibility.	response time might also be shorter because RNA translation is not necessary

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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.