Materials and Methods

Protospacer-Adjacent Motif Specificity duringClostridioides difficileType I-B CRISPR-Cas Interference and Adaptation

MATERIALS AND METHODSBacterial strains and growth conditions. Bacterial strains used in this study are listed in Table S1 in the supplemental material. C. difficile strains were grown in brain heart infusion (BHI) (Difco) medium at 37°C under anaerobic conditions (5% H2, 5% CO2, and 90% N2), within an anaerobic chamber (Jacomex). When needed, thiamphenicol (Tm) at the final concentration of 15 μg/ml was added to C. difficile cultures. Cefoxitin (Cfx) and d-cycloserine (Cs) were used for counterselection of E. coli donor cells during conjugation into C. difficile. E. coli strains were grown in LB medium (56), supplemented with ampicillin (Amp) (100 μg/ml) and chloramphenicol (Cm) (15 μg/ml) when it was necessary. The nonantibiotic analogue anhydrotetracycline (ATc) at a concentration of 250 ng/ml was used for induction of the inducible Ptet promoter of pRPF185 vector derivatives in C. difficile (57).Construction of plasmids and conjugation into C. difficile. Plasmids and oligonucleotides used in this work are presented in Table S1 and Table S2, respectively. To construct plasmid PAM libraries, we used the pRPF185Δgus vector. Single-stranded synthetic oligonucleotides containing four random nucleotides on the 5′ end, a selected protospacer sequence corresponding to the first spacer of CRISPR 3 (identical to CRISPR 16) or CRISPR 13 arrays for C. difficile 630 and {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291 strains, respectively, and regions overlapping the pRPF185Δgus vector (37) were synthesized. Subsequently, these single-stranded synthetic oligonucleotides were amplified by PCR using short complementary primers to generate the double-stranded fragments (Table S2). To generate the PAM libraries, the double-stranded fragments were cloned into SacI and BamHI sites of pRPF185Δgus using a Gibson assembly reaction (New England BioLabs) (58).TABLE S2Oligonucleotides used in this study. Download Table S2, PDF file, 0.2 MB.Copyright © 2021 Maikova et al.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.For CRISPR-Cas interference assays, the synthetic complementary (5′→3′ and 3′→5′) single-stranded oligonucleotides containing SacI and BamHI restriction sites and different PAM and protospacer sequences were used to construct conjugative plasmid vectors carrying PAM-protospacer sequences. The single-stranded oligonucleotides were annealed to each other, and the resulting double-stranded fragments were ligated into SacI and BamHI sites of the pRPF185Δgus vector.To create plasmids overexpressing Cas proteins for naive adaptation assays, C. difficile 630Δerm cas1-cas2 and cas4-cas1-cas2 gene regions, including ribosome-binding sites (−21 to +1252 relative to translational start site of cas2 gene and −37 to +1773 relative to translational start site of cas4 gene, respectively) were amplified by PCR and introduced into SacI and BamHI sites of pRPF185Δgus under the control of the ATc-inducible Ptet promoter, resulting in pCas1-2 and pCas1-2-4 plasmids (Table S1).DNA sequencing was conducted to confirm the plasmid construction. All resulting plasmids were transformed into E. coli strain HB101 (RP4). E. coli transformants were subsequently mated with C. difficile cells on BHI agar plates for 24 h at 37°C. C. difficile transconjugants were selected on BHI agar containing Tm (15 μg/ml), d-cycloserine (Cs) (25 μg/ml), and cefoxitin (Cfx) (8 μg/ml).Conjugation with PAM libraries and high-throughput sequencing. Plasmid PAM libraries were transformed into E. coli NEB10 beta cells (New England BioLabs). A sufficient number of Cm-resistant colonies (8,000 to 9,000) was selected and used for plasmid DNA extraction. This DNA served as a template for PCR with primers carrying Illumina adaptors, giving the control DNA sample for input libraries (named “PAM libraries before the conjugation”).For output library preparation, the plasmid PAM libraries were transformed into E. coli HB101 RP4 cells for further conjugation into C. difficile cells (approximately 4.9 × 1010 plasmid copies for the 630Δerm library and 2.8·1010 plasmid copies for the {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291 library). A sufficient number of Tm-resistant transconjugants (up to 4,000) was selected. All the transconjugants were then transferred to liquid BHI medium supplemented with antibiotics to eliminate remaining E. coli cells. Tm was used to maintain plasmids within C. difficile cells, while Cfx and Cs were used to counterselect E. coli cells sensitive to these antibiotics. Cells from the resulting liquid cultures were collected and used for the preparation of InstaGene (Bio-Rad) extracts that served as a template for PCR amplification with primers carrying Illumina adaptors, giving the DNA sample for sequencing named “PAM libraries after the conjugation.”The DNA samples “PAM libraries before the conjugation” and “PAM libraries after the conjugation” were sequenced using an Illumina NextSeq 500 system with 2-million-read coverage. Sequence reads were aligned with reference sequences using BWA software (59). All unmapped reads were discarded from the analysis. Randomized PAM regions in selected reads were extracted using a custom-written Python script (version 3.4).The numbers of each PAM counts were compared for two libraries (Table S3A and B). Significantly depleted PAM sequences were determined using Pearson’s chi-square test. P values adjusted using standard multiple testing corrections kept all possible PAM variants as depleted. Therefore, we used a P value of 10−12 to filter the highly depleted PAMs. The depleted sequences were assembled in a special data set, where the number of counts for each PAM was normalized to that of the lowest depleted PAM. The consensus of resulting sequence subsets was then visualized using the WebLogo tool (60). For the additional PAM sequence visualization, PAM wheels were constructed according to Leenay et al. using KronaExcelTemplate (https://github.com/marbl/Krona/releases) (61). For each individual PAM sequence, a depletion score was estimated as the ratio of the normalized read count in output PAM libraries to the normalized read count in the control. In cases where PAM happened to be enriched in the “after the conjugation” library, the depletion score was changed to zero. The depletion scores were then used as the input for the Krona plot (61).TABLE S3(A) Sequencing read counts for 630Δerm PAM libraries before and after conjugation. (B) Sequencing read counts for {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291 PAM libraries before and after conjugation. Download Table S3, PDF file, 0.09 MB.Copyright © 2021 Maikova et al.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.Plasmid conjugation efficiency assays. To evaluate the conjugation efficiency, conjugative plasmids carrying PAM-protospacer were transformed into the E. coli HB101 (RP4) strain and transferred to the C. difficile 630Δerm or C. difficile {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291 strain by conjugation. The ratio of C. difficile transconjugants to the total number of CFUs was estimated by subculturing conjugation mixtures on BHI agar supplemented with Tm, Cs, and Cfx and comparing the CFU to the number of CFUs obtained after plating serial dilutions on BHI agar plates containing Cfx only.CRISPR adaptation assay and high-throughput sequencing of newly acquired spacers. After overnight growth in BHI medium supplemented with Tm and ATc, pCas1-2-4-containing cells were twice transferred to BHI medium supplemented with ATc without Tm (I and II reseeding) (Fig. 4A). These additional steps were necessary to enrich the bacterial culture with cells that acquired new spacers. After each reseeding, two rounds of PCR were performed to detect spacer acquisition. For amplification, we used a specific set of primers for each array. Forward primers annealed to leader regions of CRISPR arrays and reverse primers annealed to the first or the second spacer (CRISPR 10 array) of native CRISPR arrays (Fig. 4A). Primers are listed in the Table S2.PCR products corresponding to expanded CRISPR arrays were extracted from the gel and used for nested PCR with primers containing Illumina adapters for further high-throughput sequencing and bioinformatic analysis. The amplicons were sequenced using the Illumina NextSeq 500 system with 2-million-read coverage. Sequence reads were analyzed in R using ShortRead and Biostrings packages (62) as described previously (63, 64). Graphical representation of results was performed using ggplot2 package (65) and the EasyVisio tool, developed by E. Rubtsova.Newly acquired spacers of 10 to 79 bp in length were mapped to the reference genomes of Clostridium difficile 630 (NCBI reference sequence {"type":"entrez-nucleotide","attrs":{"text":"NC_009089.1","term_id":"126697566","term_text":"NC_009089.1"}}NC_009089.1), pCD630 (NCBI reference sequence {"type":"entrez-nucleotide","attrs":{"text":"NC_008226.2","term_id":"1374003393","term_text":"NC_008226.2"}}NC_008226.2), and pCas1-2-4, with one mismatch allowed. Three nucleotides upstream of the first protospacer position were considered a PAM sequence. Spacers that aligned to multiple positions within the same molecule were removed from the analysis. Spacers that aligned to a single DNA molecule were referred to as “unique,” and spacers that aligned to several molecules (but to a single position within each molecule) were referred to as “nonunique” and analyzed separately (Table S4). “Shifters” and “flippers” were removed from analysis (66).In total, for CRISPR 8 and CRISPR 9, we found 5,077 spacers that mapped to 1,380 individual genomic protospacer positions (Table S4). One percent of all positions (14 protospacers) contributed most to sequenced spacers and corresponded to 27% of sequenced genomic spacers. The genomic coordinates of these seemingly “hot” protospacers were different for CRISPR 8 and CRISPR 9; therefore, it is unlikely that these positions represent true “hot” protospacers. We assumed that these seemingly “hot” protospacers could arise due to early acquisition of corresponding spacers followed by their spread in the population during prolonged cultivation. Alternatively, they could be the result of heterogeneity in amplification during two subsequent rounds of PCR. To avoid unwanted biases caused by these seemingly “hot” protospacers, we removed them from subsequent analyses of spacer lengths, protospacer distributions along the genome, and frequencies of associated PAM motifs.RNA extraction and qRT-PCR. Total RNA was isolated from the C. difficile 630Δerm strain after 4, 6, and 10 h of growth in tryptone-yeast extract (TY) medium corresponding to early exponential, late exponential, and stationary phases, respectively, as previously described (67). cDNA synthesis by reverse transcription and quantitative real-time PCR analysis was performed as previously described (68) using a Bio-Rad CFX Connect real-time system. The expression levels of CRISPR arrays were calculated relative to that of the 16S RNA gene (69).Data availability. Raw sequencing data have been deposited in the National Center for Biotechnology Information Sequence Read Archive under BioProject identifier (ID) PRJNA719030.

Article TitleProtospacer-Adjacent Motif Specificity duringClostridioides difficileType I-B CRISPR-Cas Interference and Adaptation

Abstract

Raw sequencing data have been deposited in the National Center for Biotechnology Information Sequence Read Archive under BioProject identifier (ID)PRJNA719030.


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