Strains and media
The E. coli and λ prophage genomes were engineered using a strategy derived from λ red homologous recombination coupled to Cas9 targeting71. Packaged cosmids were produced using an engineered E. coli K-12 strain carrying the thermosensitive cI857 λ prophage with its cos site deleted. When needed, a constitutively expressed SrpR repressor45 was inserted in the lacZ locus to repress the expression of genes in the production strain. The chimeric stf genes (λ-P2, λ-K1F, λ-K5 and λ-KL106) were expressed on a plasmid in trans (p938, p2292, p2058 and p1806), with the stf gene deleted from the prophage. All experiments were performed with cells grown in lysogeny broth plus 5 mM CaCl2, supplemented with antibiotics when necessary (chloramphenicol 12.5–25 µg ml−1, kanamycin 25–50 µg ml−1, trimethoprim 5–10 µg ml−1, streptomycin 50–100 µg ml−1, ampicillin 50–100 µg ml−1 and carbenicillin 50–100 µg ml−1). DAPG (100 µM, Santa Cruz Biotechnology) was added to medium or plates to induce the pPhlF promoter45. The csgA-StrepTag translation levels were analysed using YESCA agar plates (1 g l−1 yeast extract, 10 g l−1 casamino acids and 20 g l−1 agar in water) + 4% DMSO (v/v).
All titration assays were carried out with an E. coli MG1655 strain (NCBI, no. NC_000913) modified to encode the EDL933 OmpC variant, if necessary (s14269), and carrying a plasmid expressing the primase gene (p1321) if needed. mCherry (GenBank, no. QQM12952) and β-lactamase (GenBank, no. ANG10794) were used as reporter proteins encoded on the E. coli MG1655 genome (MG1655-mCherry and MG1655-bla). For chimeric gpJ/receptor analysis, plasmid stability assays and delivery efficiency assays, superfolder GFP (GenBank, no. AYN72676) was used as a reporter. E. coli MG1655 strains carrying the OmpC EDL933 receptor and bla gene were used for the primase experiments (s14269 and MG1655-bla). The clinical K. pneumoniae ST258 isolate was obtained from the International Health Management Associates (no. 1445327). Strain UTI89 is an extra-intestinal pathogenic strain isolated from a patient suffering from a urinary tract infection53; strain TN03 is a human pathogenic strain isolated from a human stool sample at Hôpital Tenon (Paris, France), and the genomic sequence was obtained by shotgun sequencing (NCBI, BioSample no. SAMN12782170)48 and PacBio sequencing (Sequel II, Centre for Genomic Research Liverpool, UK).
Streptomycin-resistant mutants were selected on M9 minimal medium plates supplemented with streptomycin overnight at 37 °C, and the rpsL gene was sequenced. E. coli strains s14269 (rpsL K42R) and TN03 (rpsL K42Q) were used for in vivo experiments (s21052 and s21476). Strain genotypes are listed in Supplementary Table 3.
Cloning and plasmid construction
Chemically competent DH10B cells (Thermo Scientific) or a modified K-12 strain carrying the SrpR repressor45 were used in cloning procedures. Molecular cloning was carried out using Gibson Assembly72. Base editors ABE8e42 and evoAPOBEC1-nCas9-UGI43 were codon optimized for E. coli and synthesized (Twist Bioscience). The obtained DNA fragments were assembled and cloned downstream of the pSrpR promoter for all constructs.
For generation of gpJ variants the insertion point was defined as one of the last stretches of amino acid identity between naturally occurring gpJ proteins and λ gpJ (amino acid position 964 in the λ gpJ protein, Uniprot accession no. P03749). A8 and 1A2 gpJ were amplified from the genome of E. coli strains in a private collection (see sequence in Supplementary Information). Sequences for the λ-P2 STF chimera were amplified from phage P2 protein H (amino acid position 251 onwards, Uniprot accession no. P26700) and phage λ genomic DNA (λ STF, amino acid positions 1–393, Uniprot accession no. P03764) and cloned downstream of the inducible pPhlF promoter. The sequence for the λ-K5 chimera was obtained by fusion of λ STF (positions 1–528, Uniprot accession no. P03764) to Genbank no. YP_009787294.1 (amino acid positions 211–272), followed by GenBank no. CAA71133.2 (amino acid position 62 onwards). The latter was codon optimized to remove rare codons and synthesized (Twist Bioscience). The λ-KL106 chimera was obtained by fusion of λ STF with the STF from a prophage identified in a KL106 K. pneumoniae ST258 strain. The K1F STF sequence was codon optimized to remove rare codons and synthesized based on gene 17 of GenBank entry no. NC_007636.1. K1F, K5 and KL106 STFs were all fused at amino acid position S528 of the λ STF; the STF P2 was fused at amino acid position R393 of the λ STF. The DNA sequences of chimeric gpJ and stf genes are provided in Supplementary Information.
The primase gene from the E. coli CFT073 strain (NCBI, no. AAN79964, locus AE016759_238) was amplified from the CFT073 genome and cloned into plasmid p2076 or p1321 (constitutive and DAPG-inducible promoter, respectively). The cohesive end site (cos) of the λ genome was cloned onto plasmids designed to be packed into λ cosmid particles (Supplementary Table 4). The 23 OmpC variants were amplified from bacterial genomes and cloned onto a pSC101 vector carrying a kanamycin resistance cassette. All plasmids were purified using the Plasmid DNA Miniprep Kit (Omega Bio-Tek) and sequence verified by Sanger sequencing (Eurofins Genomics). Plasmids, gRNAs and oligonucleotide sequences are listed in Supplementary Tables 4–6. A plasmid map of the non-replicative cosmid encoding the adenine base editor and a guide RNA is depicted in Supplementary Fig. 10.
Packaged cosmid production
Packaged cosmid production was performed with an engineered E. coli K-12 strain derived from CY2120 (ref. 41) carrying a modified λ prophage (CY-1A2, CY-A8 or CY-Ur-λ; Supplementary Table 3). For the packaged cosmids shown in Fig. 1b and Extended Data Figs. 1 and 8a, the production strains contained plasmids p513 and p938; for the packaged cosmids in Fig. 1c and Extended Data Fig. 4, the production strains carried plasmids p1396 and p938 or p2327 and p938; for the packaged cosmids in Fig. 2b,c, the production strains contained plasmids p1324 and p1321; for the packaged cosmids in Figs. 2d and 3, the production strains carried plasmids p2328, p938 and p2076; for the packaged cosmids in Extended Data Fig. 2, the production strains contained plasmid p513, as well as the plasmid p938 where indicated; for the packaged cosmids in Extended Data Fig. 8b,c, the production strains contained plasmids p2074 and p2058; for the packaged cosmids in Extended Data Fig. 8d, the production strains contained plasmids p2075 and p2292 or p1806. All plasmids are listed in Supplementary Table 4.
Production strains were cultured in lysogeny broth medium supplemented with 5 mM CaCl2, and appropriate antibiotics and 100 µM DAPG if necessary, to induce λ-P2 STF or λ-K5 STF expression or primase expression. Production strains were grown overnight at 30 °C in liquid medium in an orbital shaker, diluted 1:6 the following day in fresh medium supplemented with antibiotics and DAPG when needed, and grown for 30 min at 30 °C. Packaged cosmid production was heat induced at 42 °C for 45 min. Next, cell cultures were shifted to 37 °C for 3–6 h in an orbital shaker (New Brunswick Innova 44) at 180 rpm. Samples were centrifuged for 10 min at 4,500g and cell pellets were resuspended and lysed with B-PER reagent (Thermo Scientific; 1/10 of the initial volume of cosmid production for in vitro assays and 1/50 for in vivo assays) and lysozyme (100 µg ml−1, Applichem Lifescience). DENARASE (×10,000, c-LEcta) was added to the reaction to degrade residual DNA and RNA at a dilution of 1:10,000. Bio-Beads (SM-2 resin, Bio-Rad) were added to the lysis reaction and samples incubated for 1 h in a mini-shaker (PS-3D, Grant-Bio) at room temperature. Samples were centrifuged for 5 min at 16,000g and supernatants sterile filtered (0.22 µm pore size, Sartorius Minisart). For in vivo administration, packaged cosmids were concentrated and buffer exchanged against PBS by tangential flow filtration (MWCO 100 kDa, Sartorius Vivaflow 200). Packaged cosmid concentration was analysed by E. coli transduction with diluted cosmid stocks (1:10 dilution) and consecutive colony counting on chloramphenicol plates following overnight incubation at 37 °C. An initial culture volume of roughly 4.8 ml was needed to obtain an intermediate dose of 1010 particles used per mouse, as shown in Fig. 5 (see calculation in Supplementary Table 1).
Analysis of OmpC variants in E. coli
Seventy-six unique OmpC protein sequences were extracted from 525 E. coli genome assemblies using TBLASTN73 v.2.11.0 with a curated set of OmpC sequences as query. The sequences were aligned using MAFFT74 v.7.520 with default parameters and with the option –treeout to export the guide tree. The multiple sequence alignment and corresponding guide tree are provided in Supplementary Data 10 and Supplementary Fig. 1.
Packaged cosmid delivery efficiency
Delivery efficiency was analysed using cosmid particles equipped with λ-P2 STF, λ-K1F STF, λ-K5 STF or λ-KL106 STF chimeras and gpJ 1A2 or A8 into strain s14269, UTI89, TN03 or K. pneumoniae ST258, respectively. Cosmids carrying λ-P2 STF and gpJ A8 or 1A2 were also used for analysis of delivery efficiency into a series of MG1655-ΔLamB-ΔompC strains carrying different OmpC variants on a plasmid. The cosmid encodes a sfGFP, mCherry or venus gene under a constitutive promoter. Cells were grown in lysogeny broth supplemented with 5 mM CaCl2 to an optical density (OD600) of 0.2–0.6. Cell density was adjusted to OD600 = 0.025 in fresh lysogeny broth supplemented with 5 mM CaCl2, and 90 µl of cell culture was mixed with 10 µl of each cosmid serially diluted in lysogeny broth plus 5 mM CaCl2 (1:3 dilution) to reach different MOIs. The samples were incubated for 45 min at 37 °C, and 8 µl was added to 250 µl of ice-cold PBS plus 1 mg ml−1 kanamycin before analysis by flow cytometry (sfGFP and venus: excitation, 488 nm; emission, 530/30 BP; mCherry: excitation, 561 nm; emission, 620/15 BP; Attune NxT, Thermo Scientific). The gating strategy is described in Supplementary Fig. 11.
Plasmid stability assay
Plasmid stability was investigated in vitro with a time-course assay. E. coli MG1655 carrying a DAPG-inducible primase plasmid (p1321) with or without 100 µM DAPG was grown to an OD600 of 0.2–0.6 in lysogeny broth plus 5 mM CaCl2 and 50 µg ml−1 kanamycin. Samples were then diluted to an OD600 of 0.01 in fresh lysogeny broth plus 5 mM CaCl2 and 50 µg ml−1 kanamycin plus/minus 100 µM DAPG, treated with a packaged cosmid harbouring the sfGFP gene and the conditional primase origin of replication (p1324) at an approximate MOI of 40, and subsequently incubated in an orbital shaker at 37 °C. Samples (1–5 µl) were taken at different time points, mixed with 250 µl of ice-cold PBS supplemented with 1 mg ml−1 kanamycin and analysed in a flow cytometer (excitation, 488 nm; emission, 530/30 BP; Attune NxT, Thermo Scientific). To maintain cells in the exponential growth phase, samples were diluted 1:5 into fresh lysogeny broth medium supplemented with 50 µg ml−1 kanamycin plus/minus 100 µM DAPG every 2 h.
Base editing in vitro
The E. coli MG1655-mCherry strain was transformed with base editor payload p2316 or p2326, grown for 2 h in SOC medium (30 °C, 180 rpm) and selected on chloramphenicol plates overnight at 30 °C. Forty-eight individual colonies were resuspended in 250 µl of PBS supplemented with 1 mg ml−1 kanamycin in a 96-well plate, and mCherry fluorescence was measured by flow cytometry (excitation, 561 nm; emission, 620/15 BP; Attune NxT, Thermo Scientific). As a control, base editors were transformed with a non-targeting gRNA (SapI spacer) and the mCherry fluorescence of two colonies was analysed. The target region of mCherry was amplified from a minimum of three individual colonies per experiment, and PCR products were sequenced to confirm base editing.
The E. coli strain MG1655-bla transformed with base editor payload p1396 or p2327 was grown for 2 h in SOC medium (30 °C, 180 rpm) before spotting of 10 µl of individual cell dilutions on chloramphenicol/carbenicillin plates, as well as on chloramphenicol plates. As a control, base editors were transformed with a non-targeting gRNA (SapI spacer) on the payload. Editing efficiency was analysed by colony counting on plates following overnight incubation at 30 °C. The target site of bla was amplified from a minimum of three individual colonies, and PCR products were sequenced to confirm targeted base editing.
Base editing using packaged cosmids in E. coli strains MG1655-bla, UTI89 and TN03 or K. pneumoniae ST258 was performed similarly to transformation assays. The target strain was cultured to mid-log phase, diluted to an OD600 of either 0.025 (Figs. 1c and 4) or 0.005 (Fig. 2d) and transduced with serial dilutions (1:1 or 1:2) of the packaged cosmid yielded in a 96-well plate. Cells were grown for 2 h in lysogeny broth medium supplemented with 5 mM CaCl2 (30 or 37 °C, 180 rpm) before spotting of individual dilutions on either lysogeny broth plates or lysogeny broth + carbenicillin plates. As a control, cells were treated with lysogeny broth medium rather than packaged cosmid solution. For all experiments the target gene was amplified via PCR from the genome of individual colonies and base editing was confirmed by Sanger sequencing. For the UTI89, TN03 and K. pneumoniae ST258 experiments, the target gene was amplified via PCR from the genome of cell populations (more than 105 cells) and base-editing efficiencies from Sanger sequencing data were analysed using the tool EditR75.
In situ editing of E. coli in the mouse gut
Specific-pathogen-free 5–9-week-old female BALB/cYJ mice were supplied by Charles River Laboratories and housed in an animal facility in accordance with Institut Pasteur’s guidelines and European recommendations. Animal procedures were approved by Institut Pasteur (approval ID: 20040) and the French Research Ministry (APAFIS ID: 28717), and animal experiments were performed in compliance with applicable ethical regulations. Water and food were provided ad libitum. Animals were randomly assigned into cages upon reception. After acclimatation, cages where randomly assigned to treatment groups.
Animals were acclimated for 5 days before the addition of streptomycin sulfate (5 mg ml−1, Sigma-Aldrich, no. S9137) to autoclaved drinking water to decrease the number of facultative aerobic/anaerobic resident bacteria76. Drinking water containing streptomycin was prepared fresh weekly. Three days later (D0), mice were orally gavaged with approximately 1 × 108 CFU of strain s21052 or s21476, grown overnight in lysogeny broth and resuspended in 200 µl of sterile gavage buffer (20% sucrose, 2.6% sodium bicarbonate, pH 8.0). Starting at D5, mice were orally administered (200 µl per mouse) with either gavage buffer or packaged cosmids diluted 1:1 in buffer. The appropriate dose was achieved by diluting the packaged cosmid suspension in PBS before formulation in buffer, and checked by E. coli transduction.
Metagenomic analysis
For 16S analysis, faecal samples were collected from individual mice at D4, D5 (immediately before oral gavage with packaged cosmids), D6, D9 and D12 of colonization for all mice that received 1 × 1010 tu per animal, and samples were immediately frozen at −80 °C to preserve microbial diversity. Total microbial faecal DNA was extracted using a QiaAMP Fast DNA Stool Mini Kit (Qiagen) following the manufacturer’s instructions and sent for sequencing (Illumina NovaSeq, 250 base pair, paired-end reads, Novogene).
The obtained paired-end reads were filtered by length (cutadapt v.3.3 (ref. 73); –minimum-length parameter set to 20), merged (FLASH77 v.1.2.11, parameters –min-overlap 10 –max-mismatch-density 0.2) and filtered by quality (fastp78 v.0.23.1; parameters -q 19 -u 15). Subsequently chimeric sequences were removed using vsearch79 v.2.16.0 with the Silva reference database80. At this point, one sample (D5 M36) had to be excluded from subsequent analysis due to low sequencing depth (11,499 non-chimeric reads). Sequences were clustered into operational taxonomic units at a 97% similarity threshold with Uparse81 v.7.0.1001 and annotated with taxonomy information using QIIME82 v.1.9.1 and the Silva database. The QIIME toolkit was also used for both taxonomic profiling of samples and obtaining beta-diversity values. The phylogenetic tree supplied to UniFrac distance computation83 was generated using MUSCLE84 3.8.1551 (-maxiters parameter set to 2). Microbiome composition at different phylogenetic levels is provided in Supplementary Data 3–9.
Evaluation of base-editing efficiency from mouse faeces by direct plating
Fresh faecal samples were collected at D0 and subsequent relevant time points as a proxy for assessment of intestinal colonization levels of s21052. In brief, samples were weighed on an analytical balance and 1 ml of PBS added. Samples were incubated for 2 min at room temperature and suspended by manual mixing and vortexing. Serial dilutions were performed in PBS, 5 μl of each dilution was spotted onto Drigalski agar plates (Bio-Rad) supplemented with 100 µg ml−1 streptomycin and plates were incubated overnight at 37 °C. Estimation of editing efficacy was performed the following day by repatching individual colonies (up to 12 colonies per mouse and per time point) onto agar plates, with or without 50 µg ml−1 carbenicillin, to investigate loss of resistance to β-lactams subsequent to editing of the bla gene (Extended Data Fig. 5). In addition, separate faecal samples were collected and frozen at −80 °C within 1 h of collection to assess editing efficacy by ddPCR.
Evaluation of base-editing efficiency by ddPCR
Primers F3 (5′-GGATCTCAACAGCGGTAAG-3′) and R3 (5′-GGCATCAACACGGGATAATA-3′), both with a melting temperature of 61 °C, were designed to amplify a 112-base-pair region of the bla gene in E. coli s21052 spanning the target site for base editing. Two Taqman probes were designed to bind this amplicon with the target site towards the middle of the probes, before or after successful editing (A to G): P1 (5′-FAM-CT+TT+T+A+AA+GTT+C+T+GC-3′) and P2 (5′-HEX-CT+TT+T+G+AAGTT+CT+GC-3′). Each probe contained a different fluorophore (FAM or HEX), as well as carefully positioned locked nucleic acid bases (symbolized by base A, T, C or G preceded by a + sign in the sequences above). Locked nucleic acid nucleotides allow for a greater melting temperature (Tm) difference between matching and mismatched probes while retaining small probe size, further improving discrimination82. Tm for either probe matching its specific sequence was predicted to be 66 °C, compared with 55 °C in the case of binding to the non-matching sequence (OligoAnalyzer Tool, IDT).
Reactions were conducted in a final volume of 8 µl with PerfeCTa Multiplex qPCR Toughmix, 100 nM fluorescein, 250 nM of each primer and 250 nM of each probe using a Naica ddPCR system (Stilla Technologies). The following two-step cycling programme was applied: initial denaturation for 3 min at 95 °C, followed by 50 cycles at 95 °C for 10 s and 57 °C for 30 s. This Taqman assay was validated for specificity using purified gDNA from overnight bacterial cultures of either wild-type s21052 or in vitro-edited s21052 (Supplementary Fig. 4), and fluorescence spillover compensation was carried out using the appropriate control reactions following the manufacturer’s recommendations.
A similar strategy was used to quantify the editing of the csgA gene in TN03, with primers F5 (5′-GCGTGACACAACGTTAATTTCCATTC-3′) and R5 (5′-AGAGCGCTACCGGAGAATACG-3′) and probes P3 (5′-FAM-AC+A+T+GAA+A+CT+T+TTAAAA+G+T+A+GC-3′) and P4 (5′-HEX-AC+A+C+GAA+A+CT+TT+TAAAA+GT+A+GC-3′).
Stool samples collected from mice were weighed, resuspended at 100 mg ml−1 in ultrapure water, homogenized and heat treated at 98 °C for 10 min. Following brief vortexing and 1 min cooling at room temperature, supernatant was pipetted from the top of the suspension to avoid major debris, diluted at least ten times in ultrapure water and analysed immediately by ddPCR without further processing.
Base-editing off-target analysis
Target strain MG1655-bla or TN03 was cultured to mid-log phase, diluted to an OD600 of 0.005 and mixed with serial dilutions (1:2) of the yielded packaged cosmid in a 96-well plate. Cells were incubated for 2 h (30 °C, 180 rpm), serially diluted and 10 µl spotted on lysogeny broth plates followed by incubation overnight at 30 °C. As a control, cells were treated with lysogeny broth medium supplemented with 5 mM CaCl2 rather than packaged cosmid solution. The following day, cells were recovered from different spots (over 105 cells per spot) corresponding to different MOIs and the csgA gene was amplified by PCR from the genome. Base editing of csgA was analysed by Sanger sequencing; meanwhile, plates and samples were stored at 4 °C. For downstream next-generation sequencing analysis we selected samples based on our Sanger sequencing results. We selected samples that were treated with the lowest MOI (roughly 227 (S1) and roughly 195 (S2) for the MG1655-bla experiment, and with MOI of about 51 (S1 and S2) for the TN03-csgA experiment) in which no wild-type peak at the target site was detectable on the chromatogram. Both treated and control samples were grown for 2–3 h from these two independent experiments (S1 and S2) and gDNA was extracted (Wizard Genomic DNA Purification Kit, Promega). Sample concentrations were measured and dilutions sent for next-generation sequencing (Illumina PE150, Eurofins). Samples were sequenced at an average genome-wide coverage greater than 6,000.
The program fastp v.0.23.2 (ref. 75) was used for quality control of raw reads. Because multiple libraries were sequenced to obtain the desired depth, all reads passing quality control from the same sample were merged into a single file. The E. coli MG1655 complete reference genome was downloaded from NCBI (nuccore accession no. NC_000913.3), manually modified to reflect genetic modifications to the wild-type strain and further corrected using breseq85 v.0.37.1 with reads from control sample S1. For TN03 (NCBI assembly accession no. GCA_015186165.1), the complete reference genome obtained from a previous PacBio sequencing run of the same strain was used. The versions of both reference genomes used in the following analyses are available at https://github.com/Eligo-Bioscience/in-situ-targeted-base-editing-of-gut-bacteria-in-mice. The four readsets (two repeats for base-edited samples ABE S1 and ABE S2 and two for controls S1 and S2) corresponding to each of the two strains were aligned against their respective reference genome using bwa86 v.0.7.17-r1188 (mem algorithm, default parameters). For each position of the reference genome, the frequency of each nucleotide was computed with a custom Python script using the pile-up function of pysam v.0.20.0 (ref. 87). The ‘read mismatch frequency’ was calculated as the ratio between the number of aligned reads differing from the reference and total alignment coverage at each position.
Figures 2e and 4d and Supplementary Figs. 2, 3, 8 and 9 were generated using the libraries Matplotlib and Seaborn. The merged readsets passing quality control for each sample are available on NCBI SRA (Bioproject, no. PRJNA944658). The predicted off-target sites (positions in the reference genomes with up to seven mismatches in the target sequence, including up to two in the ten PAM-proximal nucleotides) corresponding to individual off-target IDs for MG1655-bla or TN03 are listed in Supplementary Data 1 and 2. The code used for the analysis and generation of the figures is available at https://github.com/Eligo-Bioscience/in-situ-targeted-base-editing-of-gut-bacteria-in-mice.
In vivo competition assay
To ensure that both edited and unedited clones used in the competition assay were as close as possible to each other, we performed an in vitro base-editing experiment using E. coli strains s21052 and s21476 and selected sequence-confirmed edited and unedited clones treated in the same manner. For each E. coli strain, five groups of three mice were conditioned with streptomycin and orally gavaged with independent pairs of clones (wild type and edited) at an approximate ratio of 1:1. Stool samples were collected regularly for up to 29 days, and bacterial colonization levels were measured by plating resuspended stool samples onto selective agar plates. Duplicate stool samples were also collected and frozen, and the ratio of edited to wild-type clones was later estimated by ddPCR using the assays described above.
Protein translation analysis of the base-edited csgA gene
A StrepTag (NWSHPQFEK) was fused to the C-terminal site of the csgA gene (roughly 16 kDa) in strain TN03 and the genomic insertion was confirmed by sequencing. Base editing of this strain was performed as described in ‘Base editing in vitro’. Twelve single colonies from the highest MOI were picked, resuspended in 40 µl of PBS and serial dilutions were spotted on lysogeny broth plates. The following day, the target gene was amplified from single colonies by PCR from the genome of individual colonies to confirm targeted base editing by Sanger sequencing. Strain BE 1bp carried a base edit at the target site 6A (start codon) in the editing window, whereas strain BE 2bp carried the target mutation 6A as well as a bystander mutation at position 10A located upstream of the csgA start codon in the editing window. Base-edited strains carrying one or two base pair mutations at the target region were grown in lysogeny broth and stored at −80 °C. The two base-edited strains, as well as the unedited strain (TN03-csgA-StrepTag), were plated on YESCA agar plates + 4% DMSO (v/v) and incubated at 26 °C for 72 h (curli-inducing conditions88).
Translation of the cell-associated curli protein CsgA was investigated by dot-blot analysis. The lawn from different strains was scooped and resuspended in water and OD600 was adjusted to 2.0. Cells were pelleted and the supernatant removed. Each pellet was treated with 100 µl of hexafluoroisopropanol and incubated for 1 h at room temperature to dissociate curli subunits. Hexafluoroisopropanol was removed by overnight incubation of open Eppendorf tubes under a fume hood at room temperature. Samples were resuspended in 75 µl of 1× Bolt LDS Sample buffer (Thermo Scientific) supplemented with 2% beta-mercaptoethanol. Samples were incubated for 10 min at 95 °C, and 5 µl of each sample was blotted on a nitrocellulose membrane (0.2 µm, Thermo Scientific). Dots were allowed to dry under a laminar flow hood for 20 min at room temperature. The blot was blocked by incubation with 5% skimmed milk in 0.1% PBS-Tween 20. Either mouse anti-StrepTag (BioLegend) at 0.5 µg ml−1 or mouse anti-GAPDH (Thermo Scientific) at 1 µg ml−1 was used as primary antibody, with incubation overnight at 4 °C. As a secondary antibody, horseradish peroxidase-conjugated anti-mouse antibody (Invitrogen) was used at 0.2 µg ml−1 and the blot was incubated for 1.5 h at room temperature. SuperSignal West PICO PLUS Chemiluminescent Substrate (Thermo Scientific) was used for detection.
Payload delivery into faecal samples
To investigate the ability of our vectors to deliver to bacteria in the mouse gut, faeces were collected from mice that had either (1) undergone streptomycin conditioning only, (2) undergone streptomycin conditioning followed by colonization with a streptomycin-resistant MG1655 strain or (3) been left untreated. Faeces were resuspended in PBS at 1 mg ml−1, and 180 µl aliquots were treated with 20 µl of four different λ-derived vectors carrying a payload with a chloramphenicol resistance marker and harbouring different gpJ and STF chimeras (A8-P2, A8-K5, 1A2-P2 or 1A2-K5). Following a 3 h incubation at 37 °C, the samples were diluted in series and 150 µl (undiluted, 1:100 or 1:10,000) plated on either BHI medium or BHI medium supplemented with 24 µg ml−1 chloramphenicol. Plates were then incubated in an anaerobic chamber for 24 h at 37 °C (Supplementary Fig. 6).
Delivery of DNA payloads to other bacterial species
Eight λ-derived vectors harbouring different versions of gpJ and STFs (combinations of gpJ A8 or 1A2 with STF chimeras P2, K1, K5 or KL106) were used to transduce ten different Enterobacteria species and strains and six non-enterobacterial species (Supplementary Table 3). Bacteria were grown to an OD600 of 0.1 and 90 µl of cells treated with 10 µl of various λ-derived vectors carrying an mCherry payload. Following 2 h of incubation, 2 µl of these reactions was resuspended in 250 µl of PBS supplemented with 1 mg ml−1 kanamycin in a 96-well plate, and mCherry fluorescence measured by flow cytometry (excitation, 561 nm; emission, 620/15 BP; Attune NxT, Thermo Scientific). To test for background fluorescence events, untreated bacterial samples were processed and measured as for treated samples.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.