Rewiring capsule production by CRISPRi-based genetic oscillators demonstrates a functional role of phenotypic variation in pneumococcal-host interactions

Phenotypic variation is the phenomenon in which clonal cells display different traits even under identical environmental conditions. This plasticity is thought to be important for processes including bacterial virulence1–8, but direct evidence for its relevance is often lacking. For instance, variation in capsule production in the human pathogen Streptococcus pneumoniae has been linked to different clinical outcomes9–14, but the exact relationship between variation and pathogenesis is not well understood due to complex natural regulation15–20. In this study, we used synthetic oscillatory gene regulatory networks (GRNs) based on CRISPR interference together with live cell microscopy and cell tracking within microfluidics devices to mimic and test the biological function of bacterial phenotypic variation. We provide a universally applicable approach for engineering intricate GRNs using only two components: dCas9 and extended sgRNAs (ext-sgRNAs). Our findings demonstrate that variation in capsule production is beneficial for pneumococcal fitness in traits associated with pathogenesis providing conclusive evidence for this longstanding question.

strain VL3251. First part is the amplification by PCR of pVL1305 with primers OVL3387 (with BsmBI restriction site) and OVL2625. Second part is the amplification by PCR of pVL1305 with primers OVL3386 (with BsmBI restriction site) and OVL726.
Strain VL3308 (D39V, Δprs1::PF6-lacI/tetR (gen), bgaA::Plac-dCas9sp (tet)) was constructed by amplification by PCR of the upstream homologous region to integrate in the S. p. bgaA locus, the tet marker, Plac-dCas9sp and the downstream homologous region using chromosomal DNA (gDNA) of strain VL1998 as template with primers OVL173 and OVL174. After purification, the PCR product was used to transform strain VL333 with tetracycline selection. The bgaA locus of the resulting strain, VL3308, was confirmed by Sanger sequencing.
Strain VL3436 (D39V, Δprs1::PF6-lacI/tetR (gen), bgaA::Plac-dCas9sp (tet), cep::P3-BS3-sgRNA2 (spc)) was constructed by Golden Gate assembly of two parts. First part is the amplification by PCR of the upstream homologous region to integrate in the S. p. cep locus, the spc marker, the promoter P3 and the BS3 from the gDNA of strain VL3253 with primers OVL3504 (with BsmBI restriction site) and OVL2625. Second part is the amplification by PCR of the sgRNA2, the dCas9 handle, the sgRNA terminator and the downstream homologous region to integrate in S. p. cep locus, from the gDNA of strain VL3253 with OVL3205 (with BsmBI restriction site) and OVL2628. After purification, the two parts were digested with BsmBI (NEB) at 55°C for 2h. After purification, ligation was realized at room temperature with T4 DNA ligase (Vazyme) for 1h and directly used to transform strain VL3308 with spectinomycin selection. The cep locus of the resulting strain, VL3436, was confirmed by Sanger sequencing.
Strain VL3437 (D39V, Δprs1::PF6-lacI/tetR (gen), bgaA::Plac-dCas9sp (tet), cep::P3-BS6-sgRNA3 (spc)) was constructed by Golden Gate assembly of two parts. First part is the amplification by PCR of the upstream homologous region to integrate in S. p. cep locus, the spc marker, the promoter P3 and the BS3 from the gDNA of strain VL3256 with the primers OVL3506 (with BsmBI restriction site) and OVL2625. Second part is the amplification by PCR of the sgRNA3, the dCas9 handle, the sgRNA terminator and the downstream homologous region to integrate in S. p. cep locus, from the gDNA of strain VL3256 with primers OVL3207 (with BsmBI restriction site) and OVL2628. After purification, the two parts were digested with BsmBI (NEB) at 55°C for 2h. After purification, ligation was realized at room temperature with T4 DNA ligase (Vazyme) for 1h and directly used to transform strain VL3308 with spectinomycin selection. The cep locus of the resulting strain, VL3437 was confirmed by Sanger sequencing.
Strain VL3438 (D39V, Δprs1::PF6-lacI/tetR (gen), bgaA::Plac-dCas9sp (tet), cep::P3-BS2-sgRNA6 (spc)) was constructed by Golden Gate assembly of two parts. First part is the amplification by PCR of the upstream homologous region to integrate in S. p. cep locus, the spc marker, the promoter P3 and the BS2 from the gDNA of strain VL3252 with the primers OVL3502 (with BsmBI restriction site) and OVL2625. Second part is the amplification by PCR of the sgRNA6, the dCas9 handle, the sgRNA terminator and the downstream homologous region to integrate in the S. p. cep locus, from the gDNA of strain VL3252 with OVL3203 (with BsmBI restriction site) and OVL2628. After purification, the two parts were digested with BsmBI (NEB) at 55°C for 2h. After purification, ligation was realized at room temperature with T4 DNA ligase (Vazyme) for 1h and directly used to transform strain VL3308 with spectinomycin selection. The cep locus of the resulting strain, VL3438 was confirmed by Sanger sequencing.
Strain VL3439 (D39V, Δprs1::PF6-lacI/tetR (gen), bgaA::Plac-dCas9sp (tet), cep::P3-BS3-sgRNA2 (spc), zip::P3-BS2-mNeonGreen-opt (ery)) was constructed by Golden Gate assembly of three parts. First part is the amplification by PCR of the upstream homologous region to integrate in the S. p. zip locus and the ery marker from the plasmid pASR103 with primers OVL3531 and OVL3851 (with BsmBI restriction site). Second part is the amplification by PCR of T_rpsI, T_tuf, T_B1002, T_B0015, the promoter P3 and the BS2 from the gDNA of strain VL3252 with primers OVL3533 and OVL3534 (both with BsmBI restriction site). Third part is the amplification by PCR of mNeonGreen-optimized and the downstream homologous region to integrate in S. p. zip locus from the plasmid pASR110 with primers OVL3535 (with BsmBI restriction site) and OVL3536. After DpnI treatment for parts 1 and 3 and purification, the three parts were digested with BsmBI (NEB) at 55°C for 3h. After purification, ligation was realized at room temperature with T4 DNA ligase (Vazyme) for 1h and directly used to transform strain VL3436 with erythromycin selection. The zip locus of the resulting strain, VL3439 was confirmed by Sanger sequencing.
Strain VL3440 (D39V, Δprs1::PF6-lacI/tetR (gen), bgaA::Plac-dCas9sp (tet), cep::P3-BS3-sgRNA2 (spc), zip::P3-BS2-mNeonGreen-opt (ery)) was constructed by Golden Gate assembly of three parts. First part is the amplification by PCR of the upstream homologous region to integrate in S. p. zip locus and the ery marker from plasmid pASR103 with primers OVL3531 and OVL3851 (with BsmBI restriction site). Second part is the amplification by PCR of T_rpsI, T_tuf, T_B1002, T_B0015, the promoter P3 and the BS6 from gDNA of strain VL3253 with primers OVL3533 and OVL3537 (both with BsmBI restriction site). Third part is the amplification by PCR of mTurquoise2-optimized and the downstream homologous region to integrate in S. p. zip locus from gDNA of template strain VL3312 (Veening lab collection) with primers OVL3538 (with BsmBI restriction site) and OVL3536. After DpnI treatment of part 1 and purification, the three parts were digested with BsmBI (NEB) at 55C for 3h. After purification, ligation was realized at room temperature with T4 DNA ligase (Vazyme) for 1h and directly used to transform strain VL3437 with erythromycin selection. The zip locus of the resulting strain, VL3440, was confirmed by Sanger sequencing.
Strain VL3441 (D39V, Δprs1::PF6-lacI/tetR (gen), bgaA::Plac-dCas9sp (tet), cep::P3-BS2-sgRNA6 (spc), zip::P3-BS6-mScarletI-opt (ery)) was constructed by Golden Gate assembly of three parts. First part is the amplification by PCR of the upstream homologous region to integrate in S. p. zip locus and the ery marker from plasmid pASR103 with primers OVL3531 and OVL3851 (with BsmBI restriction site). Second part is the amplification by PCR of T_rpsI, T_tuf, T_B1002, T_B0015, the promoter P3 and the BS2 from gDNA of strain VL3256 with primers OVL3533 and OVL3539 (both with BsmBI restriction site). Third part is the amplification by PCR of mScarletI-optimized and the downstream homologous region to integrate in S. p. zip locus from the template strain VL3309 (Veening lab collection) with primers OVL3540 (with BsmBI restriction site) and OVL3536. After DpnI treatment of part 1 and purification, the three parts were digested with BsmBI (NEB) at 55°C for 3h. After purification, ligation was realized at room temperature with T4 DNA ligase (Vazyme) for 1h and directly used to transform strain VL3438 with erythromycin selection. The zip locus of the resulting strain, VL3441, was confirmed by Sanger sequencing.
Strain VL3746 (D39V, Δprs1::PF6-lacI/tetR (gen), bgaA::Plac-dCas9sp (tet), zip::P3-BS6-sgRNA3-P3-BS2-mNeonGreen-opt (ery)) was constructed by Golden Gate assembly of three parts. First part is the amplification by PCR of the upstream homologous region to integrate in S. p. zip locus, the ery marker and the terminators T_rpsI, T_tuf from the template strain VL3441 with primers OVL3252 and OVL4250 (with BsmBI restriction site). Second part is the amplification by PCR of P3, BS6, sgRNA3, the dCas9 handle and the sgRNA terminator using gDNA of strain VL3437 as template with OVL4251 and OVL4252 (both with BsmBI restriction site). Third part is the amplification by PCR of terminators T_B1002, T_B0015, P3, BS2, mNeonGreen-optimized, T_B1006 and the downstream homologous region to integrate in S. p. zip locus from the template strain VL3439 with OVL4253 (with BsmBI restriction site) and OVL3255. After purification, the three parts were digested and ligated together during an assembly reaction with T4 DNA ligase buffer (Vazyme), T4 DNA ligase (Vazyme) and Eps3i (NEB; isoschizomer of BsmBI). The assembly mixture was incubated in a thermocycler (PCR Max) for step 1: 1.5 min at 37°C, step 2: 3 min at 16°C, steps 1 and 2 repeated 25x, step 3: 5 min at 37°C and step 4: 10 min at 80°C. Transformation of VL3308 with the assembly mixture and selection with erythromycin. The zip locus of the resulting strain, VL3746, was confirmed by Sanger sequencing.
Strain VL3747 (D39V, Δprs1::PF6-lacI/tetR (gen), bgaA::Plac-dCas9sp (tet), cep::P3-BS6-mScarletI-opt-P3-BS3-sgRNA2 (spc)) was constructed by Golden Gate assembly of three parts. First part is the amplification by PCR of the upstream homologous region to integrate in S. p. cep locus, the spc marker and the terminators T_rpsI, T_tuf from the template strain VL3436 with the primers OVL725 and OVL4254 (with BsmBI restriction site). Second part is the amplification by PCR of P3, BS6 and mScarletI-optimized from the template strain VL3441 with primers OVL4255 and OVL4256 (both with BsmBI restriction site). Third part is the amplification by PCR of terminators T_B1002, T_B0015, P3, BS3, sgRNA2, the dCas9 handle, the terminator S. pyogenes, T_B1006 and the downstream homologous region to integrate in S. p. cep locus from the template strain VL3436 with primers OVL4257 (with BsmBI restriction site) and OVL726. After purification, the three parts were digested and ligated together during an assembly reaction with T4 DNA ligase buffer (Vazyme), T4 DNA ligase (Vazyme) and Eps3i (NEB). The assembly mixture was incubated in a thermocycler (PCR Max) for step 1: 1.5 min at 37°C, step 2: 3 min at 16°C, steps 1 and 2 repeated 25x, step 3: 5 min at 37°C and step 4: 10 min at 80°C. Transformation of VL3308 with the assembly mixture and selection with erythromycin. The cep locus of the resulting strain, VL3747, was confirmed by Sanger sequencing.
Strain VL3748 (D39V, Δprs1::PF6-lacI/tetR (gen), bgaA::Plac-dCas9sp (tet), cil::P3-BS3-mTurquoise2opt-Ptet-BS2-sgRNA6 (kan)) was constructed by Golden Gate assembly of five parts. First part is the amplification by PCR of the upstream homologous region to integrate in S. p. cil locus, the kan marker and MCS from the template strain VL2969 with the primers OVL3318 and OVL4258 (with BsmBI restriction site). Second part is the amplification by PCR of P3, BS3 and mTurquoise2-opt from the template strain VL3440 with primers OVL4259 and OVL4260 (both with BsmBI restriction site). Third part is the amplification by PCR of terminators T_rrnB and T_rpsI from the template strain VL2969 with primers OVL4261 and OVL4262 (both with BsmBI restriction site). The fourth part is the amplification by PCR of BssgRNA2, sgRNA-6 the dCas9 handle, the sgRNA terminator from template strain VL3438 with primers OVL4263 and OVL4264 (both with BsmBI restriction site). Fifth part is the amplification by PCR of the terminator T_tufA and the downstream homologous region to integrate in S. p. cil locus from the template strain VL2969 with the primers OVL4265 (with BsmBI restriction site) and OVL4266. After purification, the five parts were digested and ligated together during an assembly reaction with T4 DNA ligase buffer (Vazyme), T4 DNA ligase (Vazyme) and Eps3i. The assembly mixture was incubated in a thermocycler (PCR Max) for step 1: 1.5 min at 37°C, step 2: 3 min at 16°C, steps 1 and 2 repeated 25x, step 3: 5 min at 37°C and step 4: 10 min at 80°C. The assembly mixture was amplified by PCR with OVL3318 and OVL4266. After purification by gel extraction, the product was used to transform strain VL3308 and selection with kanamycin. The cil locus of the resulting strain, VL3748, was confirmed by Sanger sequencing. This construct has a point mutation in the mTurquoise2-opt gene (leading to a F72I amino acid change) as well as an insertion of a second MCS sequence between the T_tufA and the downstream homologous region to integrate in S. p. cil locus.
Strain VL3870 (D39V, bgaA::Plac-dcas9sp (gen)) was constructed by Golden Gate assembly to replace the tet marker from VL3753 to gen marker. The flanking regions were amplified by PCR from strain VL3753 with primers OVL5139 and OVL5140 (with SapI restriction site) for the upstream and OVL5143 (with SapI restriction site) and OVL5144 for the downstream. The gen marker was amplified from pASR102 with OVL5141 and OVL5142 (both with SapI restriction site). After purification, the three parts were digested and ligated together during an assembly reaction with T4 DNA ligase buffer (Vazyme), T4 DNA ligase (Vazyme) and SapI. The assembly mixture was incubated in a thermocycler (PCR Max) for step 1: 1.5 min at 37°C, step 2: 3 min at 16°C, steps 1 and 2 repeated 25x, step 3: 5 min at 37°C and step 4: 10 min at 80°C. Strain VL1 was directly transformed with the assembly product with gentamycin selection. The bgaA locus of the resulting strain, VL3870, was confirmed by Sanger sequencing.
Strain VL3872 (D39V, Pcps::P3-BS6-mScarletI-opt-cps (tet)) was constructed by Golden Gate assembly. The flanking regions were amplified by PCR from the strain VL3703 with the primers OVL5145 and OVL5146 (with SapI restriction site) for the upstream and OVL5149 (with SapI restriction site) and OVL5150 for the downstream. P3-BS6-mScarletI-opt was amplified from strain VL3747 with primers OVL5147 and OVL5148 (both with SapI restriction site). The assembly mixture was incubated in a thermocycler (PCR Max) as described for VL3871. Strain VL1 was directly transformed with the assembly product with tetracycline selection. The cps locus of the resulting strain, VL3872, was confirmed by Sanger sequencing.
Strain VL4315 (D39V, zip::P3-BS6-sgRNA3-P3-BS2-mNeonGreen-opt (ery), Pcps::P3-BS6-mScarletIopt-cps (tet), cep:: P3-BS3-sgRNA2 (spc), bgaA::Plac-dCas9-P3-BS3-mTurquoise2-opt-P3-BS2-sgRNA6 (gen)) was constructed by Golden Gate assembly of three parts. First part is the amplification by PCR of the upstream homologous region to integrate in S. p. bgaA locus, the gen marker and Plac-dCas9 with OVL174 and OVL5899 (with BsaI restriction site) using genomic DNA of strain VL3870 as template. The second part is the amplification by PCR of BssgRNA3-mTurquoise2-opt-P3-BS2-sgRNA6 from strain VL3879 with primers OVL5900 and OVL5901 (both with BsaI restriction site). The third part is the amplification by PCR of the downstream homologous region to integrate in S. p. bgaA locus from the strain VL3879 with primers OVL5902 (with BsaI restriction site) and OVL173. After purification, the three parts were digested and ligated. The ligation product was used to transform strain VL3875 with gentamycin selection. The resulting strain VL4315 was confirmed by whole genome sequencing (illumina) and also known as the CAPSUlator.
Strain VL4316 (D39V, zip:: P3-BS6-sgRNA3-P3-BS2-mNeonGreen-opt (ery), Pcps::P3-BS6-mScarletIopt-∆cps (tet) (NO cps)) was constructed by Golden Gate assembly of two parts. First part is the amplification by PCR of the upstream homologous region to integrate in S. p. cps locus, tetM, tetR and P3-BS6-mScarletI-opt from strain VL3872 with primers OVL3689 and OVL5883 (with Esp3I restriction site). The second part is the downstream homologous region to integrate in S. p. cps locus from strain VL3872 with primers OVL5156 and OVL5884 (with Esp3I restriction site). After purification, the two parts were digested and ligated together during an assembly reaction with T4 DNA ligase buffer (Vazyme), T4 DNA ligase (Vazyme) and Esp3I (NEB). The assembly mixture was incubated in a thermocycler (PCR Max) for step 1: 1.5 min at 37°C, step 2: 3 min at 16°C, steps 1 and 2 repeated 25x, step 3: 5 min at 37°C and step 4: 10 min at 80°C. Strain VL4313 was directly transformed with the assembly product with tetracycline selection. The cps locus of the resulting strain, VL4316, was confirmed by Sanger sequencing.
Strain VL4319 (D39V, zip:: P3-BS6-sgRNA3-P3-BS2-mNeonGreen-opt (ery), Pcps::P3-BS6-mScarletIopt-cps (tet), cep:: P3-BS3 (spc) (NO sgRNA2)) was constructed by Golden Gate assembly of two parts. First part is the amplification by PCR of the upstream homologous region to integrate in the S. p. cep locus, the spc marker and P3 -BS3 from strain VL3873 with primers OVL873 and OVL5885 (with Esp3I restriction site). Second part is the amplification by PCR of the downstream homologous region to integrate in the S. p. cep locus from strain VL3873 with primers OVL5886 (with Esp3I restriction site) and OVL1259. After purification, the two parts were digested and ligated together during an assembly reaction with T4 DNA ligase buffer (Vazyme), T4 DNA ligase (Vazyme) and Esp3I (NEB). The assembly mixture was incubated in a thermocycler (PCR Max) for step 1: 1.5 min at 37°C, step 2: 3 min at 16°C, steps 1 and 2 repeated 25x, step 3: 5 min at 37°C and step 4: 10 min at 80°C. Strain VL4318 was directly transformed with the assembly product with spectinomycin selection. The cep locus of the resulting strain, VL4319, was confirmed by Sanger sequencing.
Strain VL4323 (D39V, zip:: P3-BS6-sgRNA3-P3-BS2-mNeonGreen-opt (ery), Pcps::P3-BS1-mScarletIopt-cps (tet) (NO BS6 but BS1), cep:: P3-BS3-sgRNA2 (spc)) was constructed by Golden Gate assembly of two parts. First part is the amplification by PCR of the upstream homologous region to integrate in S. p. cps locus, tetM, tetR and P3 from strain VL3872 with primers OVL5145 and OVL6409 (with SapI restriction site). Second part is the amplification by PCR the downstream homologous region to integrate in S. p. cps locus from strain VL3872 with primers OVL6410 (with SapI restriction site) and OVL3692. After purification, the two parts were digested and ligated together during an assembly reaction with T4 DNA ligase buffer (Vazyme), T4 DNA ligase (Vazyme) and SapI (NEB). The assembly mixture was incubated in a thermocycler (PCR Max) for step 1: 1.5 min at 37°C, step 2: 3 min at 16°C, steps 1 and 2 repeated 25x, step 3: 5 min at 37°C and step 4: 10 min at 80°C. Strain VL4320 was directly transformed with the assembly product with tetracycline selection. The cps locus of the resulting strain, VL4323, was confirmed by Sanger sequencing.