Modular and signal-responsive transcriptional regulation using CRISPRi-aided genetic switches in Escherichia coli

Abstract

Background: Precise and dynamic transcriptional regulation is a cornerstone of synthetic biology, enabling the construction of robust genetic circuits and programmable cellular systems. However, existing regulatory tools are often limited by issues such as leaky transcription and insufficient tunability, particularly in high-expression or complex genetic contexts. This study aimed to develop a CRISPRi-aided genetic switch platform that overcomes these limitations and expands the functionality of transcriptional regulation tools in synthetic biology.

Results: We established a versatile CRISPRi-aided genetic switch platform by integrating transcription factor-based biosensors with the Type V-A FnCas12a CRISPR system. Exploiting the RNase activity of FndCas12a, this system processes CRISPR RNAs (crRNAs) directly from biosensor-responsive mRNA transcripts, enabling precise, signal-dependent transcriptional regulation. To mitigate basal transcription and enhance regulatory precision, transcriptional terminator filters were incorporated, reducing leaky expression and increasing the dynamic range of target gene regulation. The platform demonstrated exceptional adaptability across diverse applications, including ligand-inducible genetic switches for transcriptional control, signal amplification circuits for enhanced output, and metabolic genetic switches for pathway reprogramming. Notably, the metabolic genetic switch dynamically repressed the endogenous gapA gene while compensating with orthologous gapC expression, effectively redirecting metabolic flux to balance cell growth.

Conclusions: The CRISPRi-aided genetic switch provides a powerful and flexible toolkit for synthetic biology, addressing the limitations of existing systems. By enabling precise and tunable transcriptional regulation, it offers robust solutions for a wide array of biotechnological applications, including pathway engineering and synthetic gene networks.

Keywords: CRISPR interference; Genetic switch; Metabolic genetic switch; Signal amplifier; Transcriptional regulation.

Fig. 1

Ligand-inducible CRISPRi-aided genetic switches targeting an episomal gene. (A, B) Schematic representations of an L-arabinose-inducible genetic switch (A) and an IPTG-inducible genetic switch (B). Both systems regulate the transcription of the rfp gene linked to a messenger crRNA cassette targeting the gfp gene (crRNA(T1)). FndCas12a expression was controlled by the P_rhaBAD promoter, induced with 1 mM L-rhamnose, to enable pre-crRNA processing and CRISPRi activity. The absence of FndCas12a was achieved by omitting L-rhamnose. E. coli DH5α strains harboring the genetic circuits were cultivated with specific inducers (4 mM L-arabinose for the P_BAD promoter and 0.25 mM IPTG for the P_TRC promoter). RFP fluorescence, GFP fluorescence, and cell growth (OD₆₀₀) were monitored using an Infinite 200 PRO microplate reader. Control values (cultures without inducers) are shown as open circles. Data are presented as the mean ± standard deviation (SD) from three biological replicates

Fig. 2

Impact of terminator filters on the performance of IPTG-inducible CRISPRi-aided genetic switches targeting an episomal reporter gene. (A, B, C) Schematic representations of genetic circuits: (A) rfp gene under the P_TRC promoter, (B) IPTG-inducible rfp-crRNA(T1) circuit, and (C) rfp gene linked to a T25 terminator and crRNA(T1). FndCas12a expression was controlled by the P_rhaBAD promoter, induced with 1 mM L-rhamnose, to facilitate pre-crRNA processing and CRISPRi activity. E. coli DH5α strains harboring these genetic circuits were cultivated in the presence of 0.25 mM IPTG. RFP fluorescence, GFP fluorescence, and cell growth (OD₆₀₀) were monitored using an Infinite 200 PRO microplate reader. Control values (cultures without IPTG) are indicated as open circles. Data are presented as the mean ± standard deviation (SD) from three biological replicates

Fig. 3

Application of CRISPRi-aided genetic switches targeting a chromosomal reporter gene. (A, B, C) L-Arabinose-inducible genetic switches targeting a chromosomal reporter gene (P_J23100-gfp). (A) P_BAD-rfp, (B) P_BAD-rfp-crRNA(T1), and (C) P_BAD-rfp-T244-crRNA(T1) genetic circuits were tested. (D, E, F) IPTG-inducible genetic switches targeting a chromosomal reporter gene. (D) P_TRC-rfp, (E) P_TRC-rfp-crRNA(T1), and (F) P_TRC-rfp-T25-T244-crRNA(T1) genetic circuits were tested. FndCas12a expression was controlled by the P_rhaBAD promoter, induced with 1 mM L-rhamnose, to facilitate pre-crRNA processing and CRISPRi activity. E. coli DH5α strains harboring the genetic circuits were cultivated with specific inducers (4 mM L-arabinose for the P_BAD promoter and 0.25 mM IPTG for the P_TRC promoter). RFP fluorescence, GFP fluorescence, and cell growth (OD₆₀₀) were monitored using an Infinite 200 PRO microplate reader. Control values (cultures without inducers) are shown as open circles. Data are presented as the mean ± standard deviation (SD) from three biological replicates

Fig. 4

Genetic signal amplification using CRISPRi-aided genetic switches targeting the TetR repressor. (A) Schematic representation of an aTC-inducible gfp gene linked to a crRNA cassette targeting the tetR gene (crRNA(TetR)). Various terminator filters (illustrated in panel B) were inserted upstream of the crRNA(TetR) cassette to regulate its expression. FndCas12a was constitutively expressed under the control of the P_J23114 promoter. (B) Heat map showing GFP fluorescence (a.u.) as a function of aTC concentration (0, 16, 31, 63, 125, and 250 nM) for 24 h. The performance of the genetic signal amplifier was evaluated in E. coli DH5α strains harboring the respective circuits. ON/OFF ratios for all groups are provided in Table S5. (C) GFP fluorescence (a.u.) for the P_TET-gfp-T244-crRNA(TetR) circuit co-transformed with either P_J23114-FndCas12a (FndCas12a+) or the pSEVA221 control plasmid (FndCas12a-). Data are presented as the mean ± standard deviation (SD) from three biological replicates

Fig. 5

Metabolic genetic switch targeting the endogenous gapA gene. (A) Schematic representation of an aTC-inducible gfp gene linked to a crRNA cassette targeting the gapA gene (crRNA(GapA)) with a T244 terminator filter. (B) Schematic representation of an aTC-inducible gapC gene linked to a crRNA cassette targeting the gapA gene (crRNA(GapA)) with a T244 terminator filter. FndCas12a was constitutively expressed under the control of the P_J23114 promoter. (C) Cell growth (OD₆₀₀) of E. coli DH5α strains harboring the P_TET-gfp-T244-crRNA(GapA) circuit co-transformed with either P_J23114-FndCas12a (red circles) or the pSEVA221 control plasmid (open circles). (D) Cell growth (OD₆₀₀) of E. coli DH5α strains harboring the P_TET-gapC-T244-crRNA(GapA) circuit co-transformed with either P_J23114-FndCas12a (red circles) or the pSEVA221 control plasmid (open circles). Data are presented as the mean ± standard deviation (SD) from three biological replicates