Methods

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

Strains, plasmids, phage, and oligonucleotides

Bacterial strains, plasmids, and phage used in this study, including descriptions and sources, are provided in Supplementary Table 1. Oligonucleotides used in this study are provided in Supplementary Table 2.

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 and 16S rRNA gene sequencing was performed using a dual indexing strategy32. 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/jbisanz/AmpliconSeq) based on QIIME233 incorporating DADA234, and analyzed using R packages qiime2R (v0.99.23; https://github.com/jbisanz/qiime2R), phyloseq (v1.33.0)35, and phylosmith (v1.0.4)36. See Data Availability for more information.

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 recombineering37, 38. 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 streptomycin39. Briefly, cells were transformed with the CarbR temperature-sensitive plasmid pSIJ838, and electrocompetent 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, respectively40. 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-FLP41; 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

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 female BALB/c mice from the vendor Taconic were used for all mouse experiments. 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 ampicillin42. 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 15,000–21,000g 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 100g, ten-fold serial dilutions were made in PBS, and 5 µl of each dilution was spotted on Difco MacConkey agar (BD 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 pCas922 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 NT40) 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.

Preparation of M13 carrying pBluescript II

This protocol was adapted from those to generate phage display libraries43. 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 10,000g 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 15,000g 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)44 was transformed with the GFPT phagemid (pCas9-GFPT-f1A or pCas9-GFPT-f1B) or the NT phagemid (pCas9-GFPT-f1A or pCas9-GFPT-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 250 ml of the same media, and incubated overnight. Cells were pelleted at 10,000g 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 20,000g 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 21,000g 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 6×10 13 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 timepoint were processed i mmediately. 5 00 μl P BS w as added, samples were i ncubated for 5 min at room temperature, then suspended by manual mixing and vortexing. Samples were centrifuged at 21,000g for 1 min, the supernatant was transferred to a new tube, and phage titer was determined against i ndicator strain XL1-Blue MRF’ by diluting samples i n PBS, i ncubating with cells, and plating on LB supplemented with carbenicillin. For all dilutions and the undiluted suspension, 10 μl was used to i nfect 90 μl cells; additionally, for the undiluted suspension, 100 μl was used to i nfect 900 μl cells to maximize the l imit of detection.

Assay for acid survival. Phage M13(pBluescript II) stored i n PBS was diluted 1-in-100 i n saline. Solutions varying i n 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 1×10 9 M13(pBluescript II). Phage were i ncubated i n the solution, and 10 μl was sampled at 5, 15, and 60 min. Samples were diluted 1-in-100 i n PBS to make acidic samples neutral and phage titer was determined against i ndicator 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 i ndicator strain i n 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 21,000g 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 with sfgfp and mcherry-marked 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 timepoint and then incubated for 24 h with further sampling every 4 h. At each timepoint, 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 21,000g 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 21,000g for 1 min, and the supernatant was transferred to a new tube and frozen at −20°C until all timepoints 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 (Supplementary Fig. 18). Samples were vortexed to mix, large particulate matter was pelleted by centrifuging at 100g for 30 seconds, 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)45, Phenoflow (v1.1.2)46, and ggcyto (v1.14.0)47. 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 timepoints. 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 timepoints, and GFP-mCherry+ events from Day 0 (pre-phage) were used to subtract background of red fluorescence at subsequent timepoints. For exclusion of timepoints due to lack of colonization, the background threshold was calculated as the maximum background observed for that population across all timepoints 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 21,000g 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 gene48.

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 6,000g 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 20,000g 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 2,000g 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 2,000g 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 2,000g 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 (ThermoFisher). Genomic DNA was normalized to 0.18 ng/ μl f or l ibrary preparation. Nextera XT libraries were constructed i n 384-well plates using a custom, miniaturized version of the standard Nextera XT protocol. Small volume l iquid 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 l ibrary. 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 i ndices were used to avoid i ndex 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). Basecalling 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) (https://github.com/rrwick/Porechop) using the discard middle option. Reads were filtered using NanoFilt (v2.6.0)49 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)50 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)51 for hybrid assembly; default parameters were used unless otherwise noted. See Data Availability for more information.

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

Isolates were cultured on LB or Difco MacConkey agar plates supplemented with carbenicillin or both carbenicillin and streptomycin. Isolates from in vitro GFP-targeting experiments were streak purified 4 times on agar to ensure clonality. Isolates from in vivo experiments were obtained by suspending a fecal pellet in 500 µl PBS, streaking the suspension onto agar, followed by streak purification of single colonies. For DNA extraction, single 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 well, 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 20 min to precipitate proteins. Precipitate was removed by centrifuging 21,000g for 5 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 21,000g for 2.5 min. The aqueous phase was transferred to a new tube, centrifuged at 21,000g 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)50 and reads were aligned using bowtie2 (v2.3.5.1)52 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, deletions were visualized by using samtools (v1.9)53 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; breseq (v0.35.4)54 was used to assess deletion size. See Data Availability for more information.

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 to Escherichia coli within the mouse gastrointestinal (GI) tract. Delivery of a programmable exogenous CRISPR-Cas9 system enabled 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 enabled E. coli to 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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