Intein-based thermoregulated meganucleases for containment of genetic material

Abstract

Limiting the spread of synthetic genetic information outside of the intended use is essential for applications where biocontainment is critical. In particular, biocontainment of engineered probiotics and plasmids that are excreted from the mammalian gastrointestinal tract is needed to prevent escape and acquisition of genetic material that could confer a selective advantage to microbial communities. Here, we built a simple and lightweight biocontainment system that post-translationally activates a site-specific DNA endonuclease to degrade DNA at 18°C and not at higher temperatures. We constructed an orthogonal set of temperature-sensitive meganucleases (TSMs) by inserting the yeast VMA1 L212P temperature-sensitive intein into the coding regions of LAGLIDADG homing endonucleases. We showed that the TSMs eliminated plasmids carrying the cognate TSM target site from laboratory strains of Escherichia coli at the permissive 18°C but not at higher restrictive temperatures. Plasmid elimination is dependent on both TSM endonuclease activity and intein splicing. TSMs eliminated plasmids from E. coli Nissle 1917 after passage through the mouse gut when fecal resuspensions were incubated at 18°C but not at 37°C. Collectively, our data demonstrates the potential of thermoregulated meganucleases as a means of restricting engineered plasmids and probiotics to the mammalian gut.

Graphical Abstract

Figure 1.

Strategy for the development of a thermosensitive biocontainment endonuclease. (A) Schematic of the thermosensitive biocontainment system shown in the context of plasmid containment in the mammalian gut. (B) Linear product formation over time in vitro for wild-type I-OnuI incubated at different temperatures with pTarget-Onu. The line of best fit through the mean value is plotted for each temperature condition. Each point represents an independent replicate (n = 3). (C) Thermoregulation of LAGLIDADG homing endonucleases (LHEs) using the L212P VMA1 intein. (top) The VMA1/LHE open reading frame (not to scale). (bottom) A functional reconstituted meganuclease is created by intein splicing at temperatures below 20°C but not at higher temperatures. (D) Multiple sequence alignment of 10 LHEs with the two conserved glycine-cysteine sites for the insertion of the VMA1 intein labelled as GC1 and GC2 in purple and red, respectively. (E) Crystal structure alignment of the LHEs I-OnuI, I-GpeMI and I-PanMI modified in PyMol. I-OnuI is shown in blue (PDB:6BDA), I-GpeMI in green (PDB:4YHX) and I-PanMI in silver (PDB:5ESP). GC1 and GC2 are highlighted in purple and red, respectively.

Figure 2.

Thermoregulation of endonuclease activity using an intein-based approach. (A) Schematic of pEndo and pTarget used in the two-plasmid selection experiments. oriV, plasmid origin of replication; pBAD, arabinose-inducible promoter; TSM, temperature-sensitive meganuclease; Amp^R, ampicillin resistance gene; Kan^R, kanamycin resistance gene. The LHE target site is cleaved by the spliced TSM at the permissive temperature and the extent of pTarget loss can be determined by the ratio of colonies on solid media containing (+Kan) or lacking (-Kan) kanamycin. (B) I-OnuI TSM activity in E. coli NEB5a on M9 minimal media incubated at either 37°C or 18°C and spot plated on solid media containing (+Kan) or lacking (-Kan) kanamycin. (C) Two-plasmid assay in E. coli NEB5a with TSMs developed from three LHEs. Plasmid retention was calculated as the ratio of colonies grown on M9 minimal media with ampicillin and kanamycin, over media with only ampicillin. Each data point represents individual replicates (n=3).

Figure 3.

Thermosensitivity of the I-OnuI TSM in E. coli Nissle 1917 (EcN). (A) Two-plasmid assay with the I-OnuI TSM to determine the temperature at which plasmid cleavage is no longer observed. Each data point represents individual replicates (n=3). The asterisk (*) indicates small colony morphology observed on solid LB media incubated at 20°C. (B) Elimination of pTarget-Onu in vivo in liquid culture over time. Aliquots of the liquid culture were taken at the specified time points and plated on solid LB media, Plasmid retention was calculated at the indicated time points as described in Figure 2C for the indicated variants. Each data point represents individual replicates (n = 3). (C) Change in the quantity of pTarget-Onu relative to the cspA gene on the EcN chromosome number after 12 h of incubation at 18°C determined by quantitative PCR. Three biological and five technical replicates were performed for each sample, and each data point represents one of the replicates.

Figure 4.

Characterizing escape and inactivation of the I-OnuI TSM. (A) Escape frequency from a single- (pEndo) and dual-endonuclease (pDual) setup. (left) The frequency of TSM inactivation in pEndo and pDual. The escape frequency was determined by the number of colonies grown on solid LB media with kanamycin relative to media without kanamycin. Each data point represents individual replicates (n = 3). (right) Schematic of the dual-endonuclease plasmid, pDual. oriV, plasmid origin of replication; Amp^R, ampicillin resistance gene; pAnderson, constitutively active Anderson promoter; TSM, temperature-sensitive meganuclease. Two variations of the Anderson promoter were used, with either high- (1.0) or mid- (0.5) expression relative to the cognate Anderson promoter. Each Anderson promoter controls the expression of a separate TSM. (B) Identification of inactivating mutations in the TSM coding region from escape mutants of pEndo in E. coli Nissle 1917. Data is shown as a bar plot, with the percentage of each of the type of mutations shown relative to the total number of escape plasmids. (C) Mapping the locations of IS911 insertions and the 12 bp VMA1 intein deletion on the I-OnuI TSM. Black arrows represent IS911 insertion sites on the I-OnuI TSM open reading frame, and red arrows represent the location of the 12 bp VMA1 intein deletion.

Figure 5.

Containment of a biotherapeutic plasmid using the I-OnuI TSM. (A) Schematic of the pTarget-gReg plasmid. oriV, plasmid origin of replication; Kan^R, kanamycin resistance gene; gus^R, glucuronide-responsive transcription factor; pGusA, promoter from the gusA regulatory region; dCas9, catalytically-inactive Cas9; pTet, constitutively-active tetracycline resistance gene promoter; sgRNA, single guide RNA. (B) Schematic outlining the β-glucuronidase activity assay. Cleavage and subsequent degradation of pTarget-gReg relieves suppression of the endogenous gus operon, restoring GusA activity to wild-type levels. GusA activity is reported by the formation of the chromogenic 4-nitrophenol product by cleavage of pNPG (4-nitrophenyl-β-D-glucuronide). (C) β-Glucuronidase activity assay in E. coli Nissle 1917 with the I-OnuI TSM. Shown is GusA activity in strains with active nuclease (I-OnuI TSM), no target site (pTarget), I-OnuI TSM with an intein-splicing mutations (pEndo N454Q), or backbone vector (pEndo). Each data point represents an individual replicates (n = 3).

Figure 6.

Containment of plasmids with single or multiplexed TSMs in the mouse gut. (A) Schematic outlining the mouse model experiments. Gut microbiome knockdown was performed with 1 g/l ampicillin and 2.5% sucrose. Mice were gavaged with 10⁸ CFUs of pEndo or pDual. Fecal samples were resuspended in PBS (150μl/mg) and plated on solid media containing (+Kan) or lacking (–Kan) kanamycin to determine pTarget loss. (B) Depletion of pTarget by pEndo and pDual. Plasmid retention was calculated as the ratio of colonies grown on MacConkey agar as depicted in panel (A). Each data point represents fecal samples collected from the 3 mice used in each experimental group (n = 3). (C) Escape frequency of pEndo and pDual in a mouse model. The escape frequency was determined by the ratio of colonies recovered from plates incubated at 18°C relative to colonies recovered at 37°C. Each data point represents an individual replicate (n = 3).