Specific gene repression by CRISPRi system transferred through bacterial conjugation

Abstract

In microbial communities, bacterial populations are commonly controlled using indiscriminate, broad range antibiotics. There are few ways to target specific strains effectively without disrupting the entire microbiome and local environment. Here, we use conjugation, a natural DNA horizontal transfer process among bacterial species, to deliver an engineered CRISPR interference (CRISPRi) system for targeting specific genes in recipient Escherichia coli cells. We show that delivery of the CRISPRi system is successful and can specifically repress a reporter gene in recipient cells, thereby establishing a new tool for gene regulation across bacterial cells and potentially for bacterial population control.

Keywords: CRISPR/Cas9; conjugation; horizontal gene transfer; synthetic biology; synthetic gene regulation.

Figure 1

Design of CRISPRi Conjugative System. (A) Design of CRISPRi conjugation system. The conjugative donor strain S17–1 contains chromosomal copies of genes necessary for conjugation from natural conjugative plasmid RP4, and the recipient strain contains chromosomal insertions of mRFP and sfGFP. The conjugative plasmid encodes a CRISPRi system specifically targeting mRFP. Once the CRISPRi plasmid is conjugated from the donor into the recipient and induced to produce dCas9, sgRNA and dCas9 form a complex and block the transcription of mRFP. (B) Design of CRISPRi conjugative plasmid. The CRISPRi system was cloned into the pARO190 plasmid, which is competent for conjugative transfer by the presence of an origin of transfer (oriT).S. pyogenes dCas9 was placed under an aTc-inducible promoter (P_LtetO-1)^, while the sgRNA to mRFP was placed under a medium-level constitutive promoter (P_ON, iGEM Parts Registry BBa_J23119). Plasmid contains ampicillin/carbenicillin resistance and is approximately 10.5 kb.

Figure 2

Conjugated CRISPRi Causes Specific mRFP repression. (A) Specific repression of mRFP is seen only in the presence of the sgRNA complementary to mRFP, but sfGFP is not affected. Fluorescence results represent geometric mean ± s.t.d. of three biological replicates after induction by aTc. Control (−) is reporter strain without a conjugated plasmid. Flow cytometry data were analyzed by FlowJo 7.6.1. (B) Microscopic images of mRFP and sfGFP expression in target strains. Top panels are mRFP and lower panels are sfGFP. mRFP expression is selectively reduced with the presence of the sgRNA, as almost no fluorescence is observed. sfGFP expression remains high for all cells. Control shows cells with no fluorescent reporters. Scale bar, 10 μm.