The CASwitch: a synthetic biology solution for high-performance inducible gene expression systems in biotechnology

Abstract

Inducible gene expression systems can be used to control the expression of a gene of interest by means of a small-molecule. One of the most common designs involves engineering a small-molecule responsive transcription factor (TF) and its cognate promoter, which often results in a compromise between minimal uninduced background expression (leakiness) and maximal induced expression. Here, we focussed on an alternative strategy using quantitative synthetic biology to mitigate leakiness while maintaining high expression, without modifying neither the TF nor the promoter. Through mathematical modelling and experimental validations, we designed the CASwitch, a mammalian synthetic gene circuit based on combining two well-known network motifs: the Coherent Feed-Forward Loop (CFFL) and the Mutual Inhibition (MI). The CASwitch combines the CRISPR-Cas endoribonuclease CasRx with the state-of-the-art Tet-On3G inducible gene system to achieve high performances. To demonstrate the potentialities of the CASwitch, we applied it to three different scenarios: enhancing a whole-cell biosensor, controlling expression of a toxic gene and inducible production of Adeno-Associated Virus (AAV) vectors.

A general strategy to engineer high-performance mammalian Whole-Cell Biosensors

“…This cleavage removes the polyA signal thus leading to mRNA degradation. Since the CasRx remains bound to the DR sequence, it is unable to recognise and cleave additional FLuc mRNAs; hence, a mutual inhibition is established between the CasRx and the FLuc mRNA, as previously reported [16]. In the MI implementation, depicted in Figure 5.b, the CasRx is constitutively expressed, while in the CIL implementation in Figure 5.c, the CasRx is driven by the synthetic promoter pCMV/2B.…”

“…The design of high-performance biosensors aims at improving the five features of the input-output response schematised in Figure 1. We thus selected gene network motifs that could yield optimal features [10, 16, 17, 18, 19] and then combined them into more complex gene networks to build high performance biosensors, as depicted in Figure 2. The performance of each gene network was assessed by comprehensive mathematical and computational analyses.…”

“…Transcription Factor amplified biosensor (TF) . The simplest modification to the basic biosensor is to add an exogenous transcription factor ( X ) between the analyte-responsive promoter and the reporter gene Z to amplify the output [16, 20, 21], as shown in Figure 2.b.…”

“…Mutual Inhibition (MI) and Coherent Inhibitory Loop (CIL) biosensors . These two gene network motifs (Figure 2.d,e) are able to reduce leaky gene expression of the reporter Z and to improve sensitivity and fold change [16, 17]. In both networks, the analyte-dependent promoter drives the transcription-factor X that activates species Z , while species W and Z mutually repress each other at the post-transcriptional level.…”

“…The experimental implementations of the MI and CIL biosensors are reported in Figure 5.b,c. They were obtained by modifying their original implementations [16]. Specifically, the VPR-dCas9-N8 protein, driven by the pMRE promoter, acts as species X .…”