Fluorescent CRISPR Adaptation Reporter for rapid quantification of spacer acquisition

CRISPR-Cas systems are adaptive prokaryotic immune systems protecting against horizontally transferred DNA or RNA such as viruses and other mobile genetic elements. Memory of past invaders is stored as spacers in CRISPR loci in a process called adaptation. Here we developed a novel assay where spacer integration results in fluorescence, enabling detection of memory formation in single cells and quantification of as few as 0.05% cells with expanded CRISPR arrays in a bacterial population. Using this fluorescent CRISPR Adaptation Reporter (f-CAR), we quantified adaptation of the two CRISPR arrays of the type I-E CRISPR-Cas system in Escherichia coli, and confirmed that more integration events are targeted to CRISPR-II than to CRISPR-I. The f-CAR conveniently analyzes and compares many samples, allowing new insights into adaptation. For instance, we show that in an E. coli culture the majority of acquisition events occur in late exponential phase.

negative results 11 . PCR products or genomic DNA has also been analyzed by high-throughput DNA sequencing to quantify spacer integration 17,22,23 . PCR and sequencing methods assay populations of bacteria and require substantial sample processing before obtaining the results. Other assays are based on functionality, where new spacers mediate plasmid curing 10,13 or survival of phage infection 10,20,21,24 . However, such methods do not detect non-functional or self-targeting adaptation events, as they may result in lack of phage protection or cytotoxicity 25 .
The f-CAR method allows rapid, easy-to-use, low-cost, sensitive, and quantifiable detection of adaptation. The method also enables detection of spacer acquisition in real-time and in single cells, which previously described methods do not. Our f-CAR system is based on a previously published system where spacer integration results in expression of a chloramphenicol resistance gene 26 , which allows for selection of cells that have acquired a spacer. We developed this system further by instead using a fluorescent read-out, enabling in vivo detection and quantification of adaptation. Here, the reporter is Yfp, referred to as Yfp-CAR, but f-CAR could be developed for use with other fluorescent reporter proteins. We characterize the developed reporter system and use it to investigate biological questions on type I-E CRISPR-Cas adaptation.

Results and Discussion
Design and construction of Yfp-CAR. To construct Yfp-CAR, the synthetic constitutive promoter pJ23101 27 , a Shine-Dalgarno sequence and a translational start codon (ATG) were placed upstream of a partial CRISPR array originating from either of the two CRISPR arrays in E. coli MG1655, CRISPR-I and CRISPR-II. These two constructs are referred to as Yfp-CAR CR-I and Yfp-CAR CR-II . The arrays consist of 69 bp of the respective leader, including the sequence elements essential for adaptation 9,28 , and one spacer flanked by two repeats. The yfp sequence lacking translational start signals was inserted downstream of the CRISPR array, such that upstream initiation results in out-of-frame translation (Fig. 1). In addition, translational stop codons (TAA) in the leader sequence are in frame with the ATG. For details and sequences of Yfp-CAR constructs see Supplementary  Table S1. The reporter is designed for constitutive transcription but translation terminates within the leader sequence of the RNA. As adaptation lengthens the array by one 61 bp spacer-repeat unit, yfp is moved into frame and the stop codons in the leader out of frame, resulting in Yfp production and fluorescence (Fig. 1). Additional insertion events would move yfp out of frame again. The entire construct was inserted into the galK locus of the E. coli chromosome in a strain deleted for cas genes and both native CRISPR arrays. The latter was done to direct all integration events to Yfp-CAR and allow accurate quantification of spacer acquisition. The constructed strain cannot perform interference based on acquired spacers, ensuring that cells are not lost from the population due to self-targeting. Absence of Cascade and Cas3 prevents primed adaptation, hence all spacer acquisitions are naïve. Adaptation was enabled by plasmid-based expression of Cas1 and Cas2 from an IPTG-inducible T7 promoter 9 . Spacer acquisition assays were done in liquid culture with (or without) induction of Cas1 and Cas2 expression, followed by a period of regrowth with repression of cas1 and cas2 before measuring fluorescence (for details see Materials and Methods).

Spacers can be inserted into Yfp-CAR.
Yfp-CAR was first tested by performing a spacer acquisition assay followed by conventional detection of integration by PCR (Fig. 2a). When the Cas1-Cas2 complex was expressed, PCR products corresponding to unexpanded and expanded arrays were detected for both Yfp-CAR CRISPR arrays (Fig. 2b). This demonstrated that the reporter arrays are targeted for integration. As expected, no expansion of the arrays was detected with non-functional Cas1 (Fig. 2b).

Spacer insertion into Yfp-CAR generates fluorescence. When cells from a spacer acquisition assay
were transferred to solid media, fluorescent colonies could be observed after overnight incubation. As expected, the majority of colonies were non-fluorescent, but fluorescent colonies that maintained fluorescence after restreaking were readily detected (Figs 2c, S1 and S2). To confirm spacer integration in fluorescent colonies, we performed colony PCR on Yfp positive (Yfp+) and negative (Yfp−) colonies. All 27 tested fluorescent colonies Figure 1. Construction of fluorescent CRISPR adaptation reporter. An unexpanded CRISPR array, consisting of two repeats (diamonds), one spacer (rectangle) and partial leader (grey) with in-frame stop codon, is inserted after a constitutive promoter and RBS (top). The reporter gene, here yfp, is in the +2 frame of the ATG and not translated with unexpanded array (top). Spacer acquisition adds 61 bp to the array (bottom), moving yfp into frame and stop codons out of frame, resulting in fluorescence.
SCientifiC REPORTS | 7: 10392 | DOI:10.1038/s41598-017-10876-z had expanded CRISPR arrays whereas the 24 tested non-fluorescent colonies were unexpanded (Fig. 2d, supplementary Fig. S2, data not shown). We conclude that spacer integration leads to Yfp expression. Sequencing of PCR products from four selected Yfp+ colonies identified acquired spacers and their origin ( Supplementary  Fig. S3), demonstrating another useful application for the f-CAR. Three spacers were unambiguously derived from plasmid. The fourth spacer was from lacI, which is present on both plasmid and chromosome. However, the source is likely plasmid lacI as it is present in more copies than chromosomal lacI. The sequenced spacers were all different and corresponded to individual spacer integration events that likely occurred in the liquid culture before plating.
To further characterize adaptation of the Yfp-CAR CRISPR arrays, a spacer acquisition assay was performed and PCR-amplified arrays from the experiment were analyzed by high throughput sequencing. Out of a total of 28,305 and 28,139 array sequences, 34 and 197 demonstrated expanded arrays for Yfp-CAR CR-I and Yfp-CAR CR-II , respectively (Table 1). Previous analyses indicated that approximately 95% of integrated spacers should be 32 bp 19 . Spacers with different length would not move the reporter gene into frame, but we detect only one spacer of aberrant length (33 bp) that, incidentally, also contained a 1 bp deletion in a repeat so the array was still extended by a total of 61 bp. In addition, newly incorporated spacers can contain in-frame stop codons resulting in translation termination and absence of fluorescence. We found that 53% of spacers inserted into Yfp-CAR CR-I and 68% into Yfp-CAR CR-II had no in-frame stop codons in any of the ten possible positions (Table 1), and would therefore permit Yfp expression after adaptation. These numbers correlate well with the calculation that 59% of spacer integration events should allow f-CAR fluorescence, on average 26 . It should be noted that the frequency of fluorescent cells (here Yfp+) is thus not equal to the absolute adaptation frequency. Relative differences are, however, still accurately measured. In line with previously observed bias in spacer acquisition from plasmids rather than the chromosome in E. coli 9 , the majority of acquired spacers were derived from the plasmid expressing Cas1 and Cas2, and only 6 and 18 spacers originating from the chromosome for Yfp-CAR CR-I and Yfp-CAR CR-II , respectively ( Table 1, Supplementary Table S2.) As mentioned above lacI spacers likely originated from the plasmid and are listed as such. Three spacers did not map to the plasmid or the chromosome, and could be derived from sequences present in the organism but not represented in the available plasmid and genome sequences. The spacers origins were distributed over the entire plasmid and indicated no strand bias ( Supplementary Fig. S4). Most sequenced spacers were unique, corresponding to individual acquisition events, and not amplifications of spacers due to e.g. growth advantage or PCR bias.

Yfp-CAR accurately and sensitively quantifies cells with expanded arrays.
To determine the detection limit and accuracy of Yfp-CAR, cells from a designed pre-expanded control strain (Yfp+) were mixed at different ratios with cells carrying the unexpanded array (Yfp−) and analyzed by PCR and flow cytometry (Fig. 3). For all flow cytometry analysis, gates were set so that less than 0.01% positive events were detected in the strain with an unexpanded array and a non-functional Cas1 (Fig. 3a). By PCR, spacer integration could be detected in a sample with 0.5% cells with expanded arrays, but not with 0.25% (Fig. 3b). This correlates well with   Table 1. Summary of analysis of expanded arrays by SMRT sequencing. Spacer acquisition assay using Yfp-CAR CR-I and Yfp-CAR CR-II was analyzed by SMRT sequencing of the CRISPR arrays in the two populations. the previously reported PCR detection limit of 0.4% expanded arrays 9 . In contrast, flow cytometry reproducibly detected expanded arrays when present in as few as 0.05% cells (Fig. 3c). Furthermore, samples with 0.5, 0.75 and 1% expanded arrays generated PCR bands of similar intensity, obscuring quantitative differences between these samples, whereas the expected percentages could be reproducibly measured by flow cytometry (Fig. 3b,c). We conclude that Yfp-CAR is at least 10-fold more sensitive than PCR-based assays, and that reliable quantification of adaptation events can be obtained even at low frequencies.

Quantification and detection of adaptation using Yfp-CAR. Spacer integration in Yfp-CAR CR-I and
Yfp-CAR CR-II after spacer acquisition assays was quantified by flow cytometry. With Yfp-CAR CR-I , 0.6% of the cells were fluorescent (Fig. 4b) whereas 3.0% of the cells with Yfp-CAR CR-II were fluorescent (Fig. 4b), suggesting that spacer integration is about five times more frequent in Yfp-CAR CR-II than in Yfp-CAR CR-I . Congruent with this, a 5.8-fold preference for adaptation into Yfp-CAR CR-II was observed by comparing the number of expanded sequences obtained by high throughput sequencing ( Table 1). The qualitative difference is in line with a previous report where more integration events were detected in CRISPR-II than in CRISPR-I 10 . Adaptation could also be detected in Yfp-CAR CR-II in a strain with the two wildtype CRISPR arrays and the endogenous chromosomal cas genes (data not shown), demonstrating that integration occurs into Yfp-CAR even in the presence of native arrays. It should be noted that the endogenous E. coli cas genes are silenced by H-NS 29 , so potentially lethal targeting of chromosome, or plasmid with selection marker, by CRISPR-Cas (which would preclude detection of spacer acquisition) does not occur under these conditions. Without induction of Cas1 and Cas2 proteins, the percentage of fluorescent cells was dramatically reduced, but still above that in the negative controls (Fig. 4b). This is probably due to low-level leaky expression of Cas1 and Cas2.
Cells from assayed strains were also examined by microscopy. As expected, fluorescent cells were detected in a fraction of the population (Fig. 4c). Spacer integration in cells carrying Yfp-CAR CR-II was also investigated by manual time-lapse microscopy. Analysis after 24 h of Cas1 and Cas2 protein expression detected the presence of fluorescent cells. These cells continued to produce fluorescent offspring ( Supplementary Fig. S5), suggesting that no new spacers are integrated during imaging. Some cells were non-fluorescent at the beginning of regrowth but later became fluorescent ( Supplementary Fig. S5). They likely represent integration events that occurred late in stationary phase, without enough time for detectable Yfp expression before initial imaging, but could also correspond to real-time observations of spacer integration.
Both leader and repeat sequences affect adaptation frequency. While the leader sequence of CRISPR-I and CRISPR-II differ substantially, their leader-proximal repeats display only one nucleotide difference (Fig. 4a). To investigate the sequence elements affecting adaptation frequencies and causing the observed differences in integration into Yfp-CAR CR-I and Yfp-CAR CR-II , minimal Yfp-CAR reporters were constructed with CRISPRs consisting only of the leader sequence and the leader-proximal repeat (Fig. 4d). Four combinations of leader and repeat sequences were tested for integration (Fig. 4d,e). Higher integration frequencies were observed for Yfp-CAR L-II, R-II (corresponding to CRISPR-II) than for Yfp-CAR L-I, R-I (corresponding to CRISPR-I), similar to previous results. Average adaptation frequencies into the minimal reporter arrays were comparable to those of the longer arrays, 0.5% for Yfp-CAR L-I, R-I vs. 0.6% Yfp-CAR CR-I and 3.4% Yfp-CAR L-II, R-II vs. 3.0% for Yfp-CAR CR-II (cf. Fig. 4b and e), indicating that arrays with only one repeat are as functional as the longer arrays. Interestingly, the arrays with combinations of leader and repeat from different arrays exhibited intermediate adaptation frequencies (Fig. 4e), suggesting that both leader and repeat sequences contribute to adaptation efficiency. The sequence of the leader-repeat boundary has previously been shown to affect spacer integration frequencies, both in vitro and in vivo 28 , but within this region only one base pair differs between the arrays. This single base pair difference in the repeat sequence decreases adaptation frequency by almost half, possibly due to a critical position in the insertion site for the new spacer 28 . The leader sequences are less conserved (Fig. 4a) and further studies are required to elucidate the sequences within the leader that affect adaptation frequencies.
New spacers are primarily integrated during late exponential phase. As a further demonstration of the utility of f-CAR, the percentage of Yfp fluorescent cells was monitored over time during an acquisition assay. This time course assay addressed whether adaptation preferentially occurs during a specific growth phase. As integration is more frequent into CRISPR-II than into CRISPR-I, Yfp-CAR CR-II was used. Samples were taken at regular time intervals, and the percentage of Yfp+ cells was measured after a short regrowth, thus cancelling out differences in fluorescence due to e.g. different Yfp-CAR promotor activity or Yfp maturation in the different growth phases. During early exponential phase (Fig. 4f, white area), the percentage of Yfp+ cells remained unchanged, while it rapidly increased during late exponential phase (Fig. 4f, light grey area). The fraction of Yfp+ cells continued to increase in early stationary phase and plateaued during late stationary phase (Fig. 4f, dark grey area) to the same level observed in the previous acquisition assays with Yfp-CAR CR-II (Fig. 4b and f). We conclude that, under the experimental conditions used, adaptation occurs during late exponential/early stationary phase, with very little or no adaptation during exponential growth and late stationary phase. Since Cas protein expression was induced we cannot exclude that the induction kinetics affect the observed growth phase dependence. It is however tempting to speculate that chromosome topology, replication or presence/absence of other factors may also play a role. As an example the IHF protein, which is required for adaptation 28 , has been demonstrated to be most abundant in late exponential phase 30 .

Conclusion and Outlook
The fluorescent reporter for adaptation (f-CAR) described in this communication enables real-time, quantitative detection of acquisition events by the CRISPR-Cas system in E. coli. The method allow several options for SCientifiC REPORTS | 7: 10392 | DOI:10.1038/s41598-017-10876-z measuring spacer integration by fluorescence; in single cells by e.g. flow cytometry or microscopy as well as in colonies. The system could be developed for other organisms and different types of CRISPR-Cas systems as long as the repeat-spacer unit is not (always) a multiple of three base pairs. Other possible developments are addition of a full set of cas genes to the system which, together with expression of pre-crRNA, could allow investigation of interference and primed adaptation. Also, detection of multiple spacer insertions could be achieved by further shifting the frame of yfp or by inserting multiple fluorescent reporter genes in different frames.
To demonstrate this read-out system's advantageous properties to address CRISPR-Cas biology, we performed several experiments. We confirm that adaptation is more frequent for CRISPR-II than for CRISPR-I, and demonstrate that both the leader and repeat sequences of CRISPR-II contribute to it being more favored for insertion than CRISPR-I. Furthermore, we show that adaptation primarily takes place in late exponential/early stationary phase.
In conclusion, f-CAR is a fluorescent reporter system that provides new and significantly improved possibilities for detection and quantification of CRISPR-Cas adaptation. The f-CAR method will hopefully assist further investigations of both fundamental aspects and details of the adaptation process of CRISPR-Cas immune systems.

Methods
Reagents and growth conditions. Routine growth of E. coli was done in LB (5 g/L yeast extract, 10 g/L NaCl, 10 g/L tryptone), supplemented with the appropriate antibiotics (50 mg/L kanamycin, 100 mg/L ampicillin, 50 mg/L streptomycin, 15 mg/L chloramphenicol or 15 mg/L tetracycline). Media components were from Oxoid. All other chemicals, and all oligonucleotides, were from Sigma-Aldrich.  Table S3). The kanamycin cassette was subsequently removed using the Flp recombinase encoded by pCP20 as previously described 32 , creating a marker-less deletion of cas3-CRISPR-I. CRISPR-II was deleted by scar-less mutagenesis using the kan-sacB cassette 33 . Briefly, the CRISPR-II array was replaced by a kan-sacB cassette by Lambda Red recombination and subsequent kanamycin selection. Next, a single-stranded DNA oligonucleotide, LA157, was used to remove the kan-sacB cassette in a second recombination step. Sucrose toxicity in cells expressing SacB was used to select for cells that had successfully replaced the kan-sacB cassette with the provided oligonucleotide.
Cas1 and Cas2 were expressed from pCas1 + 2 9 (Supplementary Table S4), where Cas1 and Cas2 are cloned under an IPTG-inducible T7-promoter. Cas1 with a point-mutation inactivating its DNase activity was used as a negative control; pCas1 D221A + 2 (Supplementary Table S4) 9 . The gene encoding the T7 polymerase was inserted in the araBAD-operon by P1 mediated transduction from MLS640. MLS640 is a BW25113 derivative, constructed by insertion of the T7 polymerase and tetracycline resistance gene in the araBAD-locus by Lambda Red recombination. The T7 polymerase and tetR were amplified from BL21AI using LA120 and LA121 primers. Primers and overhangs were chosen so that the intergenic region between araC and the araBAD promoter was reconstituted in the constructed BW25113-strain, but the araBAD operon was not. All relevant primers and DNA oligonucleotides can be found in Supplementary Table S3. All relevant strains and plasmids can be found in Supplementary Table S4.

Construction of Yfp-CAR.
A yfp gene (sYFP2, Genbank KM018300 34 ) was cloned after the CRISPR-array from pCSIR-T 26 , which corresponds to parts of CRISPR-I. The yfp was cloned out of frame (+2) of the start codon (ATG) with the CRISPR-I array between the ATG and the yfp. This also places a stop codon (located in the leader) in frame with the start codon, before the yfp. The partial array includes 69 bp of the leader, two repeats, one spacer and one partial spacer. The sequence is identical to CRISPR-I in MG1655 except for a point mutation in the leader (−42 from the first repeat) to prevent an in-frame stop codon after spacer integration 26 . To improve the fluorescence signal, an unstructured linker was placed between the CRISPR array and the yfp. This construct was PCR amplified with primers LA151 and LA152 and integrated in the galK locus after the constitutive, synthetic promoter J23101 27 replacing a previously integrated rfp in such a way that the ATG of the pCSIR-T construct was substituted with that of the rfp gene, creating strain MLS904 (Fig. 1). The CRISPR array was placed in the opposite direction of transcription to prevent crRNA production which could interfere with the translation of Yfp, e.g. if the construct is combined with Cascade which would bind the repeat of the crRNA. An Yfp-positive control for Yfp-CAR CR-I was constructed by inserting a 61 bp random sequence without stop codons in the first spacer (strain MLS902). To create Yfp-CAR CR-II with the CRISPR-II array, a kan-sacB cassette was inserted between the translational start codon and yfp in strain MLS904 replacing the CRISPR-I array (strain MLS988). The kan-sacB cassette was subsequently replaced with 69 bp of the leader and a repeat-spacer-repeat unit from CRISPR-II, PCR amplified from MG1655 (primers LA164 and LA166) as described above, creating a scar-less replacement of CRISPR-I by CRISPR-II in Yfp-CAR (strain MLS989). A positive control for Yfp-CAR CR-II was created by adding one base pair to the inserted sequence thereby moving the yfp into frame (primers LA165 and LA166, creating strain MLS990). Sequences of the Yfp-CAR constructs can be found in Supplementary Table S1.
To construct the minimal CRISPR arrays corresponding to leader and a single repeat, with all combinations of the sequences, the leader and repeat was amplified from MLS904 with pCas1 D221A + 2 and MLS989 with pCas1 D221A + 2 using primers LA168 and LA171 or LA172, with LA171 corresponding to a CRISPR-I repeat, and LA172 corresponding to a CRISPR-II repeat. The resulting PCR products were used as template in a second PCR with LA168 and LA173 to add the overhangs needed for insertion by Lambda Red recombination. The minimal CRISPR arrays were inserted between the ATG and the yfp in MLS988 by Lambda Red recombination as described for CRISPR-II above. Minimal CRISPR constructs were defined by leader-repeat combinations, Yfp-CAR L-I, R-I (MLS1000), Yfp-CAR L-II, R-I (MLS1001), Yfp-CAR L-I, R-II (MLS1002), and Yfp-CAR L-II, R-II (MLS1003). Sequences of the minimal Yfp-CAR array constructs can be found in Supplementary Table S1.
Spacer acquisition assay. For spacer acquisition assays, overnight cultures, made in LB with 50 mg/L streptomycin, were diluted 1:600 in LB with 50 mg/L streptomycin and 0.1 mM IPTG and 0.2% arabinose to induce Cas protein expression. Assay was performed at 37 °C for 24 h. To allow for expression and maturation of Yfp from late integration events, the cultures were subsequently diluted 1:100 in LB with 0.2% glucose, to repress further Cas protein expression, and grown for 5 h at 37 °C. After 5 h, samples were taken for flow cytometry for single-cell detection of integration as well as for PCR for detection of integration in the whole population (see below).
For time course experiment, overnight cultures were made in LB with 50 mg/L streptomycin, 1% glucose and 0.1 M potassium phosphate buffer in order to prevent leaky expression of Cas1 and Cas2 which may cause premature spacer integration. Overnight cultures were diluted as before and, at indicated time points, samples were taken and OD 600 was measured. The samples were centrifuged for 2 minutes to pellet cells and cells were resuspended in LB with 0.2% glucose to approximately the same OD 600 (0.025) for all time points. Regrowth was done for 1.5 h at 37 °C. After regrowth, cells were pelleted by centrifugation for 2 min, and resuspended in ice cold, sterile filtered phosphate-buffered saline (PBS). Samples were kept on ice for the duration of the time course and integration was detected as Yfp + cells by flow cytometry as described below.

Spacer integration detection by PCR.
For PCR, 500 µl of culture was pelleted and cells were resuspended in 100 µl sterile water. The sampled volume was adjusted according to OD 600 to sample approximately the same number of cells for all assayed cultures. For spacer integration assays, OD 600 were 3.5-4.5 after 24 h of growth. Cells were lysed by boiling 10 min at 95 °C. Cell debris was pelleted and 5 µl of culture supernatant was used for a 50 µl PCR reaction using DreamTaq Green DNA polymerase (Thermo Fisher Scientific) and primers amplifying the CRISPR array (for Yfp-CAR CR-I : LA112 and LA007, for Yfp-CAR CR-II : LA112 and LA168; Supplementary  Table S3). 10 µl PCR product was analyzed on a 1.5% agarose Tris-borate-EDTA (TBE) gel with SYBR safe (Thermo Fisher Scientific) visualized in ChemiDoc TM MP with Image lab v. 4.0 software (Bio-Rad) using the pre-set SYBR Safe application (Excitation: UV trans illumination, Emission: Standard filter).

Spacer integration detection in colonies.
Cells were plated on LA (5 g/L yeast extract, 10 g/L tryptone, 10 g/L NaCl, 15 g/L agar) with 0.2% glucose directly after the spacer acquisition assay and grown overnight at 37 °C. Fluorescent colonies were visualized in ChemiDoc TM MP with Image lab v. 4.0 software (Bio-Rad) using Alexa 546 settings (excitation: blue epi illumination, emission: 605/50 filter). For selected colonies, the CRISPR array was investigated by colony PCR, using the same primers as above, and Illustra PuRe Taq Ready-to-Go PCR beads (GE Healthcare). The same colonies were re-streaked to confirm the fluorescent phenotype. 5 µl PCR product was analyzed on 1.5% agarose TBE gel with SYBR safe (Thermo Fisher Scientific) and visualized in ChemiDoc TM MP (Bio-Rad) as described above.
Single cell detection of spacer integration by flow cytometry. For single cell analysis of spacer integration events, flow cytometry was performed using a MACSQuant VYB (Miltenyi Biotec). Samples were appropriately diluted in sterile filtered PBS and loaded in a 96-well polystyrene plate for analysis. SYFP2 was excited with a blue laser (488 nm; bandpass filter 525/50 channel B1). 100,000 events were recorded for each sample. Data were acquired with the MACSQuantify TM Software (Miltenyi Biotec) and processed with FlowJo Software (FlowJo, LLC).
Events were gated for bacterial cells using side scatter measurements; these were subsequently classified as Yfp positive or negative by bifurcating the Yfp channel. The Yfp signal was always gated in such a way that there were <0.01% positive events in the negative control (strain MLS904 or MLS989 with pCas1 D221A + 2). The number of Yfp positive events for three (Yfp-CAR CR-II ) or six (Yfp-CAR CR-I ) biological replicates were averaged, error bars indicate standard deviation.
High-throughput sequencing of acquired spacers. Yfp-CARs were sequenced after a spacer acquisition assay using Single Molecule Real Time (SMRT) sequencing. To create the PCR products used for SMRT sequencing, cell lysate from a representative spacer integration assay of Yfp-CAR CR-I , Yfp-CAR CR-II was used as template. Cell lysate preparation was done as described above. PCR reactions were performed using Phusion Hot Start Flex DNA polymerase (New England Biolabs) for 35 rounds of amplification. The same primer pair, LA112 and LA168, was used for both Yfp-CARs to minimize potential amplification bias caused by primer differences. PCR product was purified using AMPure PB beads (Pacific BioSciences). Templates were prepared using maximum recommended amount of adapters and sequenced on a PacBio RS II sequencer (Pacific BioSciences) using one SMRT cell per sample. Circular consensus sequencing (CCS) algorithm was used to generate sequences, and information about newly acquired spacers was extracted.
Test of detection limit. To examine the detection limit of spacer integration by PCR and flow cytometry, we performed a dilution test using the Yfp-CAR CR-I pre-expanded Yfp positive control (MLS902) and the strain with unexpanded array (MLS904), both with the inactive Cas1 to prevent integration of new spacers. Overnight cultures were diluted 1:100 in LB with 0.2% glucose and grown for 5 h at 37 °C. OD 600 was adjusted to 4 for both cultures before serial dilution of the pre-expanded control in the strain with unexpanded CRISPR array. Samples were taken for analysis by flow cytometry and 500 µl was used for spacer integration PCR as described above. In the same way, three more dilution series were made using the Yfp-CAR CR-II positive control (MLS990) and unexpanded strain (MLS989), again with pCas1 D221A + 2. Overnight cultures were back diluted as above and the fresh cultures were diluted to an OD 600 of 4. The Yfp positive control strain (MLS990) was serially diluted in the unexpanded strain (MLS989) in three independent series and analyzed by flow cytometry.
Gating of cells after flow cytometry was done as described above. The numbers of Yfp positive events from the four dilution series (one with Yfp-CAR CR-I and three with Yfp-CAR CR-II ) were averaged and the standard deviation was calculated.
Fluorescence microscopy. After spacer acquisition assay and subsequent regrowth, samples were taken for microscopy. Cells were imaged using a Nikon Ti-Eclipse inverted microscope for phase contrast and Yfp fluorescence using a 20 ms capture time. Images were captured using a sCMOS camera (Andor, UK) with a Nikon CFI Plan Apo Lambda 60X Oil objective and all images were processed the same way using Adobe Photoshop CS6 software for overlays.
For real-time imaging (Supplementary Fig. S5) cells were placed on a 1% agarose pad supplemented with 1x LB directly after spacer acquisition assay, and images were acquired at three time points (0 h, 1 h, and 3 h) as described above.