Materials and Methods

Phage-delivered CRISPR-Cas9 for strain-specific depletion and genomic deletions in the gut microbiome

RESOURCE AVAILABILITY

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Peter Turnbaugh (ude.fscu@hguabnrut.retep).

Materials availability

Plasmids or strains generated in this study will be made available upon request.

Data and code availability

  • Sequence data have been deposited at the NCBI Sequence Read Archive under NCBI BioProject: PRJNA642411 and are publicly available as of the date of publication. Accession numbers are listed in the Key Resources Table. Additional supporting data is publicly available at https://github.com/turnbaughlab/2021_Lam_M13_CRISPRCas9. KEY RESOURCES TABLEREAGENT or RESOURCESOURCEIDENTIFIERBacterial and virus strainsMG1655 (Derivative of K-12)ATCCATCC 700926DH5α (Routine cloning; phage propagation with helper HP4M13)Thermo FisherThermo Fisher 18265017NEB 5-alpha (Routine cloning)NEBNEB C2987HXL1-Blue MRF’ (Phage propagation with helper M13KO7; TcR)AgilentAgilent 212208MG1655 _rpsL-SmR (Spontaneous rpsL-SmR (Lys42Arg) derivative of MG1655)This studyKL52W1655 F− (Derivative of K-12; M13R)ATCCATCC 23737W1655 F+ (Derivative of K-12; M13S)ATCCATCC 23590; KL68W1655 F− rpsL-SmR (Recombineered rpsL-SmR (Lys42Arg) derivative of W1655 F−)This studyKL89W1655 F+ rpsL-SmR (Recombineered rpsL-SmR (Lys42Arg) derivative of W1655 F+)This studyKL90AV01::pAV01 (MG1655 with constitutive sfgfp clonetegrated at HK022 att site; KmR)Vigouroux et al., 2018AV01::pAV01; KL106AV01::pAV02 (MG1655 with constitutive mcherry clonetegrated at lambda att site; KmR)Vigouroux et al., 2018AV01::pAV02; KL107W1655 F+ rpsL-SmR sfgfp (W1655 F+ rpsL-SmR with sfgfp transduced from AV01::pAV01; KmS)This studyKL114W1655 F+ rpsL-SmR mcherry (W1655 F+ rpsL-SmR with mcherry transduced from AV01::pAV02; KmS)This studyKL115W1655 F+ rpsL-SmR sfgfp mcherry (W1655 F+ rpsL-SmR sfgfp with mcherry transduced from AV01::pAV02; KmS)This studyKL204Bacteriophage: M13KO7 (helper phage, KmR)NEBNEB N0315SBacteriophage: VCSM13 (helper phage, KmR)AgilentAgilent 200251Bacteriophage: P1 (transducing phage)ATCCATCC 25404-B1Chemicals, peptides, and recombinant proteinsMacConkey AgarThermo FisherCat#212123USP-grade streptomycin sulfateVWRCat#0382USP-grade ampicillin sodium saltTeknovaCat#A9510USP-grade carbenicillin disodium saltTeknovaCat#C2110Critical commercial assaysZymoBIOMICS 96 MagBead DNA KitZymoCat#D4302SequelPrep Normalization Plate KitLife TechCat#A10510-01KAPA Library Quantification KitKAPACat#KK4824Ligation Sequencing KitOxford NanoporeCat#SQK-LSK109Native Barcoding KitOxford NanoporeCat#EXP-NBD114Deposited dataSequencing data repositoryThis paperNCBI BioProject: PRJNA64241116S rRNA gene sequencing dataThis paperNCBI SRA: SRR12118792-SRR12118959Isolates from mouse fecal samples after delivery of pBluescript IIThis paperNCBI SRA: SRR14278062-SRR14278073Illumina and Nanopore data for the hybrid assembly of reference strain KL68 (W1655 F+ or ATCC 23590) as well as SmR fluorescent derivatives KL114 (sfgfp) and KL204 (sfgfp mcherry)This paperNCBI SRA: SRR14296642-SRR14296644; SRR14297452-SRR14297454Sequencing of isolates after targeting with phage-delivered CRISPR-Cas9This paperNCBI SRA: SRR14289086-SRR14289109Experimental models: Organisms/strainsBALB/c miceTaconicTaconic BALB-F MPFOligonucleotidesSee Table S1See Table S1See Table S1Recombinant DNAPlasmid: pBluescript II KS(−) (Commercial phagemid; CarbR)Alting-Mees and Short, 1989Agilent 212208Plasmid: pSIJ8 (Temperature-sensitive; lambda Red recombineering; CarbR)Jensen et al., 2015RRID: Addgene68122Plasmid: pE-FLP (Temperature sensitive; constitutive flippase expression; CarbR)St-Pierre et al., 2013RRID: Addgene_45978Plasmid: pCas9 (Low-copy vector carrying _cas9, tracrRNA, and CRISPR array; CmR)Jiang et al., 2013RRID: Addgene42876Plasmid: HP4_M13 (helper plasmid, KmR)Praetorius et al., 2017RRID: Addgene_120340Plasmid: pCas9-GFPT-f1A (pCas9 with GFP-targeting spacer; f1-_bla in orientation A; CmR CarbR)This studypCas9-GFPT-f1A; pKL100Plasmid: pCas9-GFPT-f1B (pCas9 with GFP-targeting spacer; f1-bla in orientation B; CmR CarbR)This studypCas9-GFPT-f1B; pKL101Plasmid: pCas9-NT-f1A (pCas9 with Non-targeting spacer; f1-bla in orientation A; CmR CarbR)This studypCas9-NT-f1A; pKL102Plasmid: pCas9-NT-f1B (pCas9 with Non-targeting spacer; f1-bla in orientation B; CmR CarbR)This studypCas9-NT-f1B; pKL103Software and algorithmsQIIME2Bolyen et al., 2019https://qiime2.org/DADA2Callahan et al., 2016https://benjjneb.github.io/dada2/phyloseqMcMurdie and Holmes, 2013https://github.com/joey711/phyloseqphylosmithSmith, 2019https://github.com/schuyler-smith/phylosmithflowCoreHahne et al., 2009https://github.com/RGLab/flowCorePhenoflowProps et al., 2016https://github.com/rprops/PhenoFlowggcytoVan et al., 2018https://github.com/RGLab/ggcytoGuppyOxford Nanopore Technologieshttps://nanoporetech.com/communityqcatOxford Nanopore Technologieshttps://github.com/nanoporetech/qcatporechopWick et al., 2017ahttps://github.com/rrwick/PorechopNanoFiltDe Coster et al., 2018https://github.com/wdecoster/nanofiltfastpChen et al., 2018https://github.com/OpenGene/fastpUnicyclerWick et al., 2017bhttps://github.com/rrwick/Unicyclerbowtie2Langmead and Salzberg, 2012https://github.com/BenLangmead/bowtie2breseqDeatherage and Barrick, 2014https://github.com/barricklab/breseqsamtoolsLi et al., 2009https://github.com/samtools/samtoolsOtherResource website for supporting dataThis paperhttps://dx.doi.org/10.5281/zenodo.5517961Open in a separate window
  • This paper does not report original code.
  • Any additional information required to reanalyze the data reported in this paper is available from the Lead Contact upon request.

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Animals

Animal procedures were approved by the University of California, San Francisco (UCSF) Institutional Animal Care and Use Committee (IACUC), and animal experiments performed were in compliance with ethical regulations. Specific-pathogen-free 6–10 week old female BALB/c mice from the vendor Taconic were used for all mouse experiments. Mice that arrived co-housed were distributed equally across experimental groups. Mice were orally gavaged with known doses of phage and provided food and antibiotic water ad libitum. All phage experiments were carried out using singly-housed mice to reduce the risk of cross-contamination of modified bacterial strains.

Microorganisms

E. coli strains, plasmids, and phage used in this study, including descriptions of relevant characteristics, are provided in the Key Resources Table (TcR, tetracycline-resistant; SmR, streptomycin-resistant; KmR, kanamycin-resistant; KmS, kanamycin-sensitive; M13R, M13-resistant; M13S, M13-sensitive; CarbR, carbenicillin-resistant; CmR, chloramphenicol-resistant). E. coli strains were routinely cultured in liquid using lysogeny broth (LB) or terrific broth (TB) and on solid media using LB or MacConkey agar at 37°C; strains harboring a temperature-sensitive plasmid were cultured at 30°C. M13 phage carrying phagemid DNA were generated using helper phage M13KO7 or VCSM13, or helper plasmid HP4_M13, and were stored in PBS at −80°C with added glycerol as cryoprotectant. P1 phage lysates for transduction were generated using plate lysates and stored at 4°C.

METHOD DETAILS

Oligonucleotides

Oligonucleotides used in this study are listed in Table S1.

Minimum inhibitory concentration (MIC) assay

Cells were prepared by standardizing an overnight culture to an OD600 of 0.1 using saline (0.85% NaCl), and further diluted ten-fold in saline then ten-fold in LB. The drug was prepared by dissolving the antibiotic in vehicle (sterile distilled water) and filter-sterilizing, then serially diluting two-fold in vehicle to prepare 100 × stock solutions, and finally diluting ten-fold in LB for 10 × stock. To wells of a 96-well plate, 60 μl of LB, 15 μl of drug, and 75 μl of cells were added and mixed well. Final drug concentrations ranged between 0.002 μg/ml to 1000 μg/ml for ampicillin and 0.24 μg/ml to 2000 μg/ml for carbenicillin. The plate was incubated overnight at 37°C without shaking and OD600 was measured the following morning after agitation.

16S rRNA gene sequencing

Mouse fecal pellets were stored at −80°C. DNA was extracted from single pellets using a ZymoBIOMICS 96 MagBead DNA Kit (Zymo D4302) and 16S rRNA gene sequencing was performed using a dual indexing strategy (Gohl et al., 2016). Briefly, a 22-cycle primary PCR was performed using KAPA HiFi Hot Start DNA polymerase (KAPA KK2502) and V4 515F/806R Nextera primers. The reaction was diluted in UltraPure DNase/RNase-free water (Life Tech 0977–023) and used as template for a 10-cycle secondary (indexing) PCR using sample-specific dual indexing primers. The reactions were normalized using a SequelPrep Normalization plate (Life Tech A10510–01) and the DNA was eluted and pooled. To purify and concentrate the DNA, 5 volumes of PB Buffer (QIAGEN 28004) were added, mixed, and purified using a QIAquick PCR Purification Kit (QIAGEN 28106). The DNA was gel extracted using a MinElute Gel Extraction Kit (QIAGEN 28604), quantified by qPCR using a KAPA Library Quantification Kit for Illumina Platforms (KAPA KK4824), and paired-end sequenced on the Illumina MiSeq platform. Data were processed using a 16S rRNA gene analysis pipeline (https://github.com/turnbaughlab/AmpliconSeq) based on QIIME2 (Bolyen et al., 2019) incorporating DADA2 (Callahan et al., 2016), and analyzed using R packages qiime2R (v0.99.23; https://github.com/jbisanz/qiime2R), phyloseq (v1.33.0) (McMurdie and Holmes, 2013), and phylosmith (v1.0.4) (Smith, 2019).

Construction of streptomycin-resistant E. coli strains

Strains resistant to the antibiotic streptomycin were generated by either selection for spontaneous resistance or by lambda Red recombineering (Datsenko and Wanner, 2000; Jensen et al., 2015). Spontaneous resistant mutants were selected by plating overnight cultures on LB supplemented with 500 μg/ml streptomycin. Lambda Red recombineering was later used to introduce a specific allele for genetic consistency between strains as different mutations in the rpsL gene can confer resistance to streptomycin (Timms et al., 1992). Briefly, cells were transformed with the CarbR temperature-sensitive plasmid pSIJ8 (Jensen et al., 2015), and electrocompe-tent cells were prepared from cells grown in LB carbenicillin at 30°C to early exponential phase and lambda Red recombinase genes were induced by addition of L-arabinose to 7.5 mM. Cells were electroporated with an rpsL-SmR PCR product amplified from a spontaneous streptomycin-resistant mutant of MG1655 using primers PS-rpsL1 and PS-rpsL2, and recombinants were selected on LB supplemented with 500 μg/ml streptomycin. The pSIJ8 plasmid was cured by culturing in liquid at 37°C in the absence of carbenicillin, plating for single colonies, and confirming CarbS. The rpsL gene of SmR strains was confirmed by Sanger sequencing.

Construction of fluorescently marked E. coli strains

P1 lysates were generated of AV01∷pAV01 and AV01∷pAV02 carrying “clonetegrated” sfgfp and mcherry, respectively (Vigouroux et al., 2018). Briefly, 150 μl of overnight culture in LB supplemented with 12.5 μg/ml kanamycin was mixed with 1 μl to 25 μl P1 phage (initially propagated from ATCC on MG1655). The mixture was incubated for 10 min at 30°C to aid adsorption, added to 4 mL LB 0.7% agar, and overlaid on pre-warmed LB agar supplemented with 25 μg/ml kanamycin 10 mM MgSO4. Plates were incubated overnight at 30°C, and phage were harvested by adding 5 mL SM buffer, incubating at room temperature for 10 min, and breaking and scraping off the top agar into a conical tube. Phage suspensions were centrifuged to pellet agar; the supernatant was passed through a 100 μm cell strainer, then through a 0.45 μm syringe filter, and lysates were stored at 4°C. For transduction, 1–2 mL of recipient overnight culture was pelleted and resuspended in 1/3 volume LB 10 mM MgSO4 5 mM CaCl2. 100 μl of cells was mixed with 1 μl to 10 μl P1 lysate and incubated at 30°C for 60 minutes. To minimize secondary infections, 200 μl 1 M sodium citrate was added, followed by 1 mL of LB. The mixture was incubated at 30°C for 2 h, then plated on LB 10 mM sodium citrate 25 μg/ml kanamycin to select for transductants. For excision of the vector backbone including the kanamycin resistance gene and heat-inducible integrase, cells were electroporated with pE-FLP (St-Pierre et al., 2013); transformants were selected on carbenicillin and confirmed for KmS. pE-FLP was cured by culturing in liquid at 37°C in the absence of carbenicillin, plating for single colonies, and confirming CarbS. Strains were subsequently grown routinely at 37°C. For imaging fluorescent strains on agar, plates were typically incubated at 37°C overnight, transferred to room temperature to allow fluorescence intensity to increase, and then imaged.

Mouse experiments with E. coli colonization, antibiotic water, and phage treatment

Streptomycin water was prepared by dissolving USP grade streptomycin sulfate (VWR 0382) in autoclaved tap water to a final concentration of 5 mg/ml and passing through 0.45 μm filtration units. Mice were provided streptomycin water for 1 day, followed by oral gavage of 0.2 mL containing approximately 109 CFU of streptomycin-resistant E. coli. Mice were kept on streptomycin water thereafter to maintain colonization. For selection with β-lactam antibiotics, USP grade ampicillin sodium salt (Teknova A9510) or USP grade carbenicillin disodium salt (Teknova C2110) was also dissolved in the water to a final concentration of 1 mg/ml; carbenicillin was preferred for its increased stability over ampicillin (Bobrowski and Borowski, 1971). Drinking water containing streptomycin was prepared fresh weekly; with the addition of a β-lactam antibiotic, it was prepared fresh every 3–4 days. For phage treatment, filtered phage solutions stored at −80°C were thawed and used directly for oral gavage. Unfiltered phage solutions were precipitated by diluting approximately 5-fold in PBS, adding 0.2 volumes phage precipitation solution (20% PEG-8000, 2.5 M NaCl), incubating for 15 min on ice, pelleting at 15,000–21,000 g for 15 min at 4°C, resuspending in PBS, centrifuging to pellet insoluble matter, and filtering through 0.45 μm. Heat-inactivated phage were prepared by incubating 1 mL aliquots at 95°C in a water bath for 30 min. Streptomycin-treated mice colonized with SmR E. coli were orally gavaged with 0.2 mL of phage and placed on drinking water containing both streptomycin and carbenicillin.

Enumeration and culture of E. coli from mouse feces

Fecal pellets were collected from individual mice and CFU counts were performed on the same day to determine CFU per gram feces. Briefly, fecal samples (typically 10–40 mg) were weighed on an analytical balance and 250 μl to 500 μl PBS or saline was added. Samples were incubated for 5 min at room temperature and suspended by manual mixing and vortexing. Large particulate matter was pelleted by centrifuging at 100 g, ten-fold serial dilutions were made in PBS, and 5 μl of each dilution was spotted on Difco MacConkey agar (Thermo Fisher 212123) supplemented with the appropriate antibiotics, i.e., streptomycin (100 μg/ml) or carbenicillin (50 μg/ml). For qualitative assessment of the fluorescent strains in feces, samples were spotted onto LB supplemented with the appropriate antibiotics. For isolating E. coli from fecal samples for genomic or plasmid DNA analysis, the fecal suspension was streaked on agar, and single colonies were further streak-purified.

Construction of CRISPR-Cas9 phagemid vectors

Cultures were grown in LB or TB media supplemented with the appropriate antibiotics. Plasmid DNA was prepared by QIAprep Spin Miniprep Kit (QIAGEN 27106), eluted in TE buffer, and incubated at 60°C for 10 min. Samples were quantified using a NanoDrop One spectrophotometer. The vector pCas9 (Jiang et al., 2013) was digested with BsaI (NEB R0535) and gel extracted with a QIAquick Gel Extraction Kit (QIAGEN 28706). Spacers were generated by annealing and phosphorylating the two oligos (PSP116 and PSP117 for GFPT; PSP120 and PSP121 for NT (Vigouroux et al., 2018)) at 10 μM each in T4 ligation buffer (NEB B0202S) with T4 polynucleotide kinase (NEB M0201S) by incubating at 37°C for 2 h, 95°C for 5 min, and ramping down to 20°C at 5°C/min. The annealed product was diluted 1 in 200 in sterile distilled water and used for directional cloning by ligating (Thermo Scientific FEREL0011) to 60 ng of BsaI-digested, gel extracted pCas9 overnight at room temperature. Ligations were used to transform NEB 5-alpha competent cells (NEB C2987H) and the cloned spacer was verified by Sanger sequencing using primer PSP108. The trailing repeat was later confirmed to lack the starting 5′G, which did not interfere with GFP-targeting function. The 1.8-kb fragment carrying the f1 origin of replication and β-lactamase gene (f1-bla) was amplified from pBluescript II with SalI adapters using primers KL215 and KL216 and KOD Hot Start DNA polymerase (Millipore 71842–3). The PCR product was purified using a QIAquick PCR Purification Kit (QIAGEN 28104), digested with SalI (Thermo Fisher FD0644), gel extracted, and used to ligate to SalI-digested, FastAP-dephosphorylated (Thermo Fisher FEREF0651) vector. Ligations were used to transform DH5α and clones were screened by restriction digest for both possible insert orientations (A or B) using XbaI (Thermo Scientific FD0684) and one of each orientation was saved for both the GFPT and NT phagemids, generating pCas9-GFPT-f1A, pCas9-GFPT-f1B, pCas9-NT-f1A, and pCas9-NT-f1B.

Preparation of M13 carrying pBluescript II

This protocol was adapted from those to generate phage display libraries(Tonikian et al., 2007). XL1-Blue MRF’ was transformed with pBluescript II (Agilent 212208). An overnight culture of this strain was prepared in 5 mL LB supplemented with tetracycline (5 μg/ml) and carbenicillin (50 μg/ml) and subcultured the following day 1-in-100 into 5 mL 2YT supplemented with the same antibiotics. At an OD600 of 0.8, cells were infected with helper phage M13KO7 (NEB N0315S) or VCSM13 (Agilent 200251) at a multiplicity of infection of approximately 10-to-1 for 1 h at 37°C The infected cells were used to seed 2YT supplemented with carbenicillin (100 μg/ml) and kanamycin (25 μg/ml) at 1-in-100, and the culture was grown overnight to produce phage. Cells were pelleted at 10,000 g for 15 min, and the supernatant containing phage was transferred. Phage were precipitated by adding 0.2 volumes phage precipitation solution, inverting to mix well, and incubating for 30 min on ice. Phage were pelleted at 15,000 g for 15 min at 4°C and the supernatant was discarded. The phage pellet was resuspended in PBS at 1%–4% of the culture volume. The resuspension was centrifuged to pellet insoluble material and transferred to a new tube. Glycerol was added to a final concentration of 10%–15%. Phage preparations were aliquoted into cryovials and stored at −80°C.

Preparation of M13 carrying CRISPR-Cas9 phagemids

DH5α(HP4_M13) (Praetorius et al., 2017) was transformed with the GFPT phagemid (pCas9-GFPT-f1A or pCas9-GFPT-f1B) or the NT phagemid (pCas9-NT-f1A or pCas9-NT-f1B) and plated on LB media containing carbenicillin and kanamycin. Transformants were inoculated into 5 mL 2YT supplemented with 100 μg/ml carbenicillin and 25 μg/ml kanamycin, incubated overnight, used 1-in-100 to seed a large volume of the same media, and incubated overnight. Cells were pelleted at 10,000 g for 15 min, and the supernatant containing phage was transferred. Phage were precipitated by adding 0.2 volumes phage precipitation solution, inverting to mix well, and incubating for 30 min on ice. Phage were pelleted at 20,000 g for 20 min at 4°C with slow deceleration. The supernatant was completely removed, phage were resuspended in PBS at 1% of the culture volume, and glycerol was added to a final concentration of 10%–15%. The phage solution was centrifuged at 21,000 g to pellet insoluble matter, filtered through 0.45 μm, and stored at −80°C.

Titration of M13 phage carrying phagemid DNA

Phage titer was determined using indicator strain XL1-Blue MRF’ or SmR W1655 F+. An overnight culture of the indicator strain in LB supplemented with the appropriate antibiotics was subcultured 1-in-100 or 1-in-200 into fresh media and grown to an OD600 of 0.8. To estimate titer, serial ten-fold dilutions of the phage preparation were made in PBS, and 10 μl of each dilution was used to infect 90 μl of cells. After incubating at 37°C for 30 min with shaking, 10 μl of the infection mix was spotted onto LB supplemented with carbenicillin. For more accurate titration, 100 μl of phage dilutions were mixed with 900 μl cells in culture tubes, incubated at 37°C for 30 min with shaking, and 100 μl was plated on LB carbenicillin.

Enumeration of viable M13 from mouse feces

Mice were orally gavaged with 6 × 1013 M13(pBluescript II) or as negative controls, heat-inactivated phage or PBS. Approximately 100 mg of feces were collected at 0, 3, 6, 9, and 24 h post-gavage, and samples at each time point were processed immediately. 500 μl PBS was added, samples were incubated for 5 min at room temperature, then suspended by manual mixing and vortexing. Samples were centrifuged at 21,000 g for 1 min, the supernatant was transferred to a new tube, and phage titer was determined against indicator strain XL1-Blue MRF’ by diluting samples in PBS, incubating with cells, and plating on LB supplemented with carbenicillin. For all dilutions and the undiluted suspension, 10 μl was used to infect 90 μl cells; additionally, for the undiluted suspension, 100 μl was used to infect 900 μl cells to maximize the limit of detection.

Assay for acid survival

Phage M13(pBluescript II) stored in PBS was diluted 1-in-100 in saline. Solutions varying in pH (1.2, 2, 3, 4, 5, 6, and 7) were prepared by mixing different ratios of 0.2 M sodium phosphate dibasic and 0.1 M citric acid and adjusting with concentrated HCl. 200 μl of each pH solution was transferred to the wells of a microtiter plate, and 10 μl of phage was added containing 1 × 109 M13(pBluescript II). Phage were incubated in the solution, and 10 μl was sampled at 5, 15, and 60 min. Samples were diluted 1-in-100 in PBS to make acidic samples neutral and phage titer was determined against indicator strain XL1-Blue MRF’ by plating on LB supplemented with carbenicillin. Solution-only controls were assayed simultaneously and cells were plated on LB to confirm viability of the indicator strain in the presence of samples originating from an acidic pH.

Targeting experiments in vitro with M13 CRISPR-Cas9

Overnight cultures of fluorescently marked SmR W1655 F+ sfgfp and mcherry were prepared in LB supplemented with streptomycin, subcultured 1 in 200 into fresh media, and grown to an OD600 of 0.8. 900 μl cells (approximately 1 × 109) was transferred to a culture tube, 100 μl phage (approximately 1 × 1010 for f1A vectors and approximately 5 × 1010 for f1B vectors) was added, and the tube was incubated at 37°C for 30 min. The infection culture was transferred to a microfuge tube, cells were pelleted at 21,000 g for 1 min, and the supernatant was removed. Cells were washed twice by adding 1 mL PBS, vortexing, pelleting cells, and removing supernatant. Cells were resuspended in 1 mL PBS, and ten-fold serially diluted in PBS. 10 μl of each dilution was spotted onto LB supplemented with carbenicillin and 100 μl was plated on larger plates for isolating single colonies for analysis. Colonies were picked and streak-purified four times to ensure phenotypic homogeneity and clonality.

Co-culture experiments of strains infected with M13 CRISPR-Cas9

Overnight cultures of fluorescently marked SmR W1655 F+ sfgfp and mcherry were prepared in LB supplemented with streptomycin. For each culture, three serial ten-fold dilutions were made in PBS, followed by a fourth ten-fold dilution into LB. Equal volumes of each were combined and 5 mL aliquots were transferred to culture tubes. Using a CFU assay, the input was determined to be 6 × 106 CFU of each strain or 1 × 107 CFU total. 10 μl (5 × 109) M13 carrying CRISPR-Cas9 was added, the co-culture was incubated at 37°C for 30 min, and carbenicillin was added to a final concentration of 100 μg/ml. The co-culture was sampled for the t = 0 time point and then incubated for 24 h with further sampling every 4 h. At each time point, 200 μl was taken; 100 μl was used to assay carbenicillin in the media (see section: Carbenicillin bioassay) and the remaining 100 μl was used for plating as follows. To the 100 μl sample of culture, 900 μl was added and cells were washed by vortexing. Cells were pelleted by centrifuging at 21,000 g for 1 min, and 900 μl of the supernatant was removed. To remove residual phage and antibiotic, the wash was repeated once more by adding 900 μl PBS, vortexing, pelleting cells, and removing 900 μl. Cells were resuspended in the remaining 100 μl. Serial ten-fold dilutions were made in PBS and 10 μl of each dilution was spotted onto LB or LB carbenicillin.

Carbenicillin bioassay

Cultures were sampled over time, cells were pelleted at 21,000 g for 1 min, and the supernatant was transferred to a new tube and frozen at −20°C until all time points were collected. The supernatants were thawed and assayed using a Kirby-Bauer disk diffusion test. An overnight culture of the indicator organism (Bacillus subtilis 168) was diluted in saline to an OD600 of 0.1. A cotton swab was dipped into this dilution and spread across LB agar, antibiotic sensitivity disks (Fisher Scientific S70150A) were overlaid using tweezers, and 20 μl of the supernatant was applied to the disk. At the same time, carbenicillin standards were prepared from 1 μg/ml to 100 μg/ml and also applied to discs. Plates were incubated overnight at 37°C and imaged the following morning.

Flow cytometry

For turbid in vitro cultures, samples were diluted 1-in-10,000 in PBS. For mouse fecal pellets, samples were used fresh or thawed from −80°C, and suspended in 500 μl PBS by manual mixing and vortexing. Fecal suspensions were incubated aerobically at 4°C overnight to improve fluorescence signal. Samples were vortexed to mix, large particulate matter was pelleted by centrifuging at 100 g for 30 s, and the sample was diluted 1-in-100 in PBS. Samples were run on a BD LSRFortessa flow cytometer using a 530/30 nm filter for GFP fluorescence and 610/20 nm for mCherry fluorescence, with the following voltages: 750 V for FSC, 400 V for SSC, 700 V for mCherry, and 700–800 V (in vivo) or 650 V (in vitro) for GFP. Flow cytometry data were analyzed in R using packages flowCore (v1.52.1) (Hahne et al., 2009), Phenoflow (v1.1.2) (Props et al., 2016), and ggcyto (v1.14.0) (Van et al., 2018). Typically, between 10,000 and 100,000 events were collected per sample, and data were rarefied after gating on FSC and SSC. Background events were accounted for on a per-mouse basis. For co-colonization with the sfgfp-marked and mcherry-marked strains, GFP+ and mCherry+ events from Day −3 (pre-E. coli) were used to subtract background at subsequent time points. For colonization with the double-marked strain, GFP+ mCherry+ events from Day −5 (pre-E. coli) were used to subtract background of double fluorescence at subsequent time points, and GFP− mCherry+ events from Day 0 (pre-phage) were used to subtract background of red fluorescence at subsequent time points. For exclusion of time points due to lack of colonization, the background threshold was calculated as the maximum background observed for that population across all time points multiplied by a factor of three. See Code Availability for more information.

Quick extraction and PCR analysis of genomic DNA from in vitro or in vivo isolates

Genomic DNA was extracted crudely to use as template for PCR. Briefly, 1.5 mL to 3 mL of culture was transferred to a microfuge tube, cells were pelleted by centrifuging, and the supernatant was discarded. The pellet was frozen, allowed to thaw on ice, resuspended in 100 μl TE, and incubated at 100°C for 15 min in an Eppendorf ThermoMixer. Samples were cooled on ice, cell debris was pelleted by centrifuging at 21,000 g for 1 min, the supernatant was transferred to a new tube, and diluted 1-in-100 in TE to use as template DNA. PCR was performed using KOD Hot Start DNA polymerase (Millipore 71842–3) using primers KL207/KL200 for the sfgfp gene and primers BAC338F/BAC805R for the 16S rRNA gene (Yu et al., 2005).

Extraction of DNA for hybrid assembly

E. coli strains KL68 (W1655 F+ or ATCC 23590), KL114 (W1655 F+ rpsL-SmR sfgfp), and KL204 (W1655 F+ rpsL-_SmR _sfgfp mcherry) were cultured in 50 mL LB supplemented with streptomycin. Cells were collected by centrifuging at 6,000 g for 10 min at room temperature, washed in 10 mL 10 mM Tris 25 mM EDTA (pH 8.0), and resuspended in 4 mL of the same buffer. 12.5 mg lysozyme (Sigma-Aldrich L6876), 100 μl 5 M NaCl, and 50 μl 10 mg/ml RNase A (Thermo-Fisher EN0531) were added and the mixture was incubated at 37°C for 15 min. To lyse cells, 350 μl 5 M NaCl, 20 μl 20 mg/ml Proteinase K (Ambion AM2546), and 500 μl 10% SDS were added, and the mixture was incubated at 60°C for 1 h with gentle inversions. 2.75 mL of 7.5 M ammonium acetate was added, and the mixture was incubated on ice 20 min to precipitate proteins. Debris was removed by centrifuging 20,000 g for 10 min and the supernatant was transferred to a new tube. To extract, an equal volume of chloroform was added and mixed; phases were separated by centrifuging at 2,000 g for 10 min, and the aqueous phase was transferred to a new tube. To precipitate the DNA, 1 volume of isopropanol was added, and the tube was inverted until a white precipitate formed. The DNA was pelleted by centrifuging at 2,000 g for 10 min and the supernatant was removed. The pellet was washed with 500 μl ice-cold 70% ethanol, allowed to dry, 1 mL TE was added, and the pellet allowed to dissolve overnight at 4°C. To further remove RNA, 250 μl of the genomic prep was transferred to a new tube, 12.5 μl 10 mg/ml RNase A was added, and the mixture was incubated at 37°C for 2 h with mixing every 30 min. To precipitate the DNA, 0.1 volume of 3 M sodium acetate was added followed by 3 volumes of 100% ethanol, and the mixture was inverted until a white precipitate formed. DNA was pelleted by centrifuging at 2,000 g for 10 min, the supernatant was removed, the pellet washed with 100 μl 70% ethanol, allowed to dry, and resuspended in 100 μl TE. Samples were quantified by Qubit dsDNA BR Assay and DNA integrity was confirmed by 0.4% agarose gel electrophoresis using GeneRuler High Range DNA Ladder (Thermo-Fisher FERSM1353). DNA was used for both Oxford Nanopore sequencing and Illumina sequencing.

Illumina whole genome sequencing

DNA concentration was quantified using PicoGreen (Thermo Fisher). Genomic DNA was normalized to 0.18 ng/μl for library preparation. Nextera XT libraries were constructed in 384-well plates using a custom, miniaturized version of the standard Nextera XT protocol. Small volume liquid handlers such as the Mosquito HTS (TTP LabTech) and Mantis (Formulatrix) were used to aliquot precise reagent volumes of < 1.2 μl to generate a total of 4 μl per library. Libraries were normalized and 1.2 μl of each normalized library was pooled and sequenced on the Illumina NextSeq or MiSeq platform using 2×146 bp configurations. 12 bp unique dual indices were used to avoid index hopping, a phenomenon known to occur on ExAmp based Illumina technologies. See Data Availability for more information.

Oxford Nanopore sequencing and hybrid Nanopore/Illumina assembly

PCR-free long read libraries were prepared using the Ligation Sequencing Kit (SQK-LSK109), multiplexed using the Native Barcoding Kit (EXP-NBD114), and sequenced on the MinION platform using flow cell version MIN106 (Oxford Nanopore Technologies). Base-calling of MinION raw signals was done using Guppy (v2.2.2, Oxford Nanopore Technologies). Reads were demultiplexed with qcat (v1.1.0, Oxford Nanopore Technologies). Quality control was achieved using porechop (v0.2.3 seqan2.1.1) (Wick et al., 2017a) using the discard middle option. Reads were filtered using NanoFilt (v2.6.0) (De Coster et al., 2018) with the following parameters: minimum average read quality score of 10 (−q 10) and minimum read length of 100 (−l 100). Illumina reads were quality filtered using fastp (v0.20.1) (Chen et al., 2018) with the following parameters: cut front, cut tail, cut window size 4, cut mean quality 20, length required 60. Filtered MinION and Illumina reads were then provided to Unicycler (v0.4.8) (Wick et al., 2017b) for hybrid assembly; default parameters were used unless otherwise noted.

Analysis of isolates after in vitro or in vivo M13-mediated delivery of phagemid

Isolates from in vitro GFP-targeting experiments were streak purified 4 times to ensure clonality. Isolates from in vivo experiments were obtained by streaking fecal suspensions followed by streak purification of single colonies. For DNA extraction, colonies were inoculated into LB or TB supplemented with the appropriate antibiotics. For analysis of the phagemid, plasmid DNA was extracted using a QIAprep Spin Miniprep Kit (QIAGEN 27106), eluted in TE buffer, and incubated at 60°C for 10 min. DNA was quantified using a NanoDrop One spectrophotometer and 200–600 ng was digested with FastDigest restriction enzymes (KpnI, Thermo Scientific FD0524; XbaI, Thermo Scientific FD0684) for 10 min at 37°C followed by gel electrophoresis. Spacer sequences on phagemids were confirmed by Sanger sequencing using primer PSP108. For genome sequencing, genomic DNA was either extracted using a DNeasy Blood & Tissue Kit (QIAGEN 69506) or an in-house protocol. Briefly, isolates were cultured in 3 mL TB supplemented with streptomycin and carbenicillin. Cells were pelleted and resuspended in 460 ul of freshly prepared buffer per sample: 400 ul 10 mM Tris (pH 8.0) 25 mM ETDA, 50 μl 5 M NaCl, and 10 μl 10 mg/ml RNase A (Thermo-Fisher EN0531). 50 μl 10% SDS was added, mixed, and samples were incubated at 60°C for 1 h with periodic inversions. 260 μl of 7.5 M ammonium acetate was added, and the mixture was incubated on ice for 20 min to precipitate proteins. Precipitate was removed by centrifuging 21,000 g for 5 min and the supernatant was transferred to a new tube. An equal volume of chloroform was added and mixed; phases were separated by centrifuging at 21,000 g for 2.5 min. The aqueous phase was transferred to a new tube, centrifuged at 21,000 g for 2.5 min, and 500 ul was transferred to a new tube. To precipitate the DNA, 500 μl isopropanol was added, and the tube was inverted until a white precipitate formed. Using a pipette tip, the clump was transferred to a new tube, washed with 100 μl cold 70% ethanol, and allowed to dry. 50 μl TE was added and the pellet was allowed to dissolve at 4°C overnight. DNA integrity was confirmed by gel electrophoresis and used for Illumina whole genome sequencing (see section: Illumina whole genome sequencing). Sequence reads were quality filtered using fastp (v0.20.1) (Chen et al., 2018) and reads were aligned using bowtie2 (v2.3.5.1) (Langmead and Salzberg, 2012) to reference genomes and phagemid sequences; complete reference genomes were generated using hybrid assembly (see section: Oxford Nanopore sequencing and hybrid Nanopore/Illumina assembly). For CarbR isolates obtained after delivery of the pBluescript II phagemid, reads were simultaneously aligned to the genome of strain KL68 (W1655 F+) and the pBluescript II sequence (NCBI accession X52329.1). For CarbR isolates obtained after delivery of CRISPR-Cas9 phagemids, reads were simultaneously aligned to the genome of the strain used for in vitro (KL114; sfgfp) or in vivo (KL204; sfgfp mcherry) experiments and the sequence of the delivered phagemid. For isolates from GFP-targeting experiments, breseq (v0.35.4) (Deatherage and Barrick, 2014) was used to assess deletion size; additionally, deletions were visualized by using samtools (v1.9) (Li et al., 2009) to filter multi-mapping and low-quality read alignments with MAPQ < 2 (view −q 2), and depth was calculated using a sliding window of 20.

QUANTIFICATION AND STATISTICAL ANALYSIS

The number of biological and/or technical replicates is specified in each figure legend, along with the definition of center and variance used. Statistical significance was evaluated using R (version 3.6.2) unpaired Mann-Whitney tests (Figure 5E, wilcox.test, alternative = “less”; Figure S2, wilcox.test, alternative = “two.sided”) and a logistic regression (Figure 1D, glm, family = “binomial”).

Highlights

  • Filamentous phage M13 can deliver DNA to E. coli cells colonizing the mouse gut
  • Engineered M13 carrying CRISPR-Cas9 can specifically target a strain in the gut
  • M13-delivered CRISPR-Cas9 can induce chromosomal deletions in vitro and in vivo

Article TitlePhage-delivered CRISPR-Cas9 for strain-specific depletion and genomic deletions in the gut microbiome

Abstract

Mechanistic insights into the role of the human microbiome in the predisposition to and treatment of disease are limited by the lack of methods to precisely add or remove microbial strains or genes from complex communities. Here, we demonstrate that engineered bacteriophage M13 can be used to deliver DNA toEscherichia coliwithin the mouse gastrointestinal (GI) tract. Delivery of a programmable exogenous CRISPR-Cas9 system enables the strain-specific depletion of fluorescently marked isogenic strains during competitive colonization and genomic deletions that encompass the target gene in mice colonized with a single strain. Multiple mechanisms allowE. colito escape targeting, including loss of the CRISPR array or even the entire CRISPR-Cas9 system. These results provide a robust and experimentally tractable platform for microbiome editing, a foundation for the refinement of this approach to increase targeting efficiency, and a proof of concept for the extension to other phage-bacterial pairs of interest.


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