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Real-time observation of CRISPR spacer acquisition by Cas1–Cas2 integrase

Abstract

Cas1 integrase associates with Cas2 to insert short DNA fragments into a CRISPR array, establishing nucleic acid memory in prokaryotes. Here we applied single-molecule FRET methods to the Enterococcus faecalis (Efa) Cas1–Cas2 system to establish a kinetic framework describing target-searching, integration, and post-synapsis events. EfaCas1–Cas2 on its own is not able to find the CRISPR repeat in the CRISPR array; it only does so after prespacer loading. The leader sequence adjacent to the repeat further stabilizes EfaCas1–Cas2 contacts, enabling leader-side integration and subsequent spacer-side integration. The resulting post-synaptic complex (PSC) has a surprisingly short mean lifetime. Remarkably, transcription effectively resolves the PSC, and we predict that this is a conserved mechanism that ensures efficient and directional spacer integration in many CRISPR systems. Overall, our study provides a complete model of spacer acquisition, which can be harnessed for DNA-based information storage and cell lineage tracing technologies.

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Fig. 1: Reconstitution of the integration reaction at the single-molecule level.
Fig. 2: Mechanism of target searching by EfaCas1–Cas2.
Fig. 3: Stability of half- and full-integration complexes.
Fig. 4: Prespacer processing and unidirectional integration.
Fig. 5: Resolution of the post-synaptic complex.
Fig. 6: In vivo evidence that transcription from the CRISPR locus promotes new spacer incorporation.
Fig. 7: Model explaining how transcription-coupled repair resolves PSC and allows the final spacer incorporation into a CRISPR array.

Data Availability

Source data for Figs. 2d,f, 3b,d and 6b are available with the paper online. Information extracted from single-molecule movies is presented in Figs. 16 and Extended Data Figs. 110 in the manuscript. Raw data for the movies are available upon reasonable request.

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Acknowledgements

This work is supported by National Institutes of Health (NIH) grant GM118174 to A.K.

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Authors

Contributions

J.B.B., Y.X., G.S. and A.K. designed the research. J.B.B. was the main contributor to the single-molecule data collection and analysis and the biochemical experiments. Y.X. made a significant contribution to protein preparation and mutagenesis. G.S. designed and carried out the in vivo spacer acquisition experiments. A.C. contributed significantly to the bulk and single-molecule data production. C.H. and F.D. contributed to material preparation. A.K., G.S. and J.B.B wrote the manuscript.

Corresponding author

Correspondence to Ailong Ke.

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The authors declare no competing interests.

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Peer review information Anke Sparmann was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Bulk biochemistry showing that the structure-guided fluorescent labeling scheme did not alter the integration activity of EfaCas1–Cas2.

a, Location of the Cy3 (green) and Cy5 (red) fluorophores and the six possible integration schemes (leader-half, spacer-half, and full-integration in two prespacer orientations); b, The expected length of the fluorophore-containing products from each integration scheme on a. c, Product of EfaCas1–Cas2 catalyzed integration over time, resolved on Urea-PAGE. Green band: Cy3-containing products; red band: Cy5-containing products; yellow: products containing both Cy3 and Cy5 fluorophores; leftmost lane: 5’-Cy3-labeled ssDNA size ladder. Uncropped gel images for panel c are shown in the Source Data. Source data

Extended Data Fig. 2 Efficient target capture by EfaCas1–Cas2–PS(4,4) and interpretation of denaturing FRET states after SDS wash.

a, Histogram (native condition) collected from 25 short movies each having 325 FRET pairs on average after 10 min of 10 nM Cas1–Cas2–PS(4,4) incubation. Only two peaks were observed in steady state representing two orientation of prespacer, but it is not clear whether prespacer is integrated into the leader-side/spacer side, or, in half or full integration state. EFRET= center ± s.d. b-d, Representative smFRET traces showing potential binding, half-integration or full-integration events. Within five minutes of recording, more than 90% of traces recorded Cas1–Cas2 activities in the form of binding-unbinding or integration-disintegration. e, f, Oligonucleotide annealing scheme to mimic the leader-side half integration in two prespacer orientations. g, h, FRET histogram from single-molecule constructs depicted in e and f, respectively. i, smFRET histogram (denatured condition) after EfaCas1–Cas2 catalyzed integration from half-integration-only prespacers [that is PS(4, 4ddC)]. Integration only took place from the non-dideoxy end of the prespacer. Leader-side integration was strongly preferred. Spacer-side integration peak was only present after extended incubation.

Extended Data Fig. 3 Spacer-side labeling scheme revealed DNA bending and four native FRET levels for half and full integration.

a, The Crystal structure of half and full integration shown in both prespacer orientations; half to full conversion bends the target DNA and changes the FRET states, positions of donor and acceptor fluorophores are as indicated on DNA. b, Schematic of half and full integration in native states; six integration possibilities are shown. c, Steady-state FRET efficiency histogram showing binding-integration of Cas1–Cas2–PS(4,4) in the native state. Only four peaks were observed, two for half integration (unbent target) and two for the bent state in each orientation. Mostly, bent state corresponded to full integration (as detected by SDS wash), but a small fraction of bent population also showed half integration (both leader and spacer side) due to integration-disintegration phenomenon (see TDP); EFRET= center ± s.d. d, EFRET transition from the native to denatured state tabulated. e, Schematics of six integration configurations in the protein-denatured state.

Extended Data Fig. 4 Kinetic measurement of Kd by counting Cy5 spots.

a, A schematic of target and Cas1–Cas2–PS(4,4) used in experiments. b, Plot of bound or integrated single molecule population (measured via Cy5 signal on target) after the introduction of EfaCas1–Cas2–PS(4,4) at different concentration into the flow cell. Fitting the data with single-exponential equation yields rate constant kobs for each concentration, which when plotted against concentration (c) gives equilibrium constant Kd and a reaction rate constant, k2.

Extended Data Fig. 5 Measurement of leader side reaction rate khalf.

a, A schematic of target and Cas1–Cas2–PS(4,4) used in experiments b, FRET histogram collected following SDS denaturation at varied reaction times, showing the changing free and integrated population (leader side) at different reaction times. c, Plot of leader-side half integrated population vs reaction time. Data fitting gives the rate of formation (khalf) of leader-side half integration. The khalf is comparable to k2 (Extended Data Fig. 3) and represents a lower limit of reaction rate because the integration reaction was difficult to perform reliably by hand for a reaction time of 1s or less. d, A representative trace showing binding and unbinding events obtained for unlabeled prespacer loaded, Cy-5 labeled Cas1-Cas2. e, A representative trace showing occasional binding and unbinding events in the absence of a prespacer.

Extended Data Fig. 6 Capturing binding-unbinding and integration-disintegration events using one-ended ddC prespacer.

a, A representative smFRET trace from a 15-min long movie with prespacer PS(4, 4ddC). O1 has longer dwell time than O2 because of integration from 3’-OH. The trace captures binding and unbinding, integration and disintegration, and FRET transition from native to the denatured state upon SDS treatment on a single shot. Two orientations of prespacer are shown by dashed lines. b, Plot of transitions from two native peaks, O1 and O2, to corresponding denatured peaks representing leader and spacer side integration, respectively. The plot was generated for PS(4,4ddC). c, Two smFRET traces for prespacer PS(4ddC, 4) after swapping -OH group and -ddC from PS(4, 4ddC). The dwell time for O1 and O2 is reversed due to swap.

Extended Data Fig. 7 Long movie trace showing finite stability of half and full integration.

a, Schematics of construct used in photo-stability test with 532 nm excitation, Cy3 emission, and FRET-induced Cy5 emissions. b, Representative smFRET traces showing eventual photobleaching of Cy3 (top) and Cy5 (bottom) after long-time excitation. c, Percentage of live molecules vs survival time for Cy3 (green) and Cy5 (red). d, A schematic of target and Cas1–Cas2–PS(4,4) used in experiments. e, f, Representative smFRET traces from 20-min long recording. As the one Cas1–Cas2–PS(4,4) molecule integrates and later disintegrates, another comes and interacts with the target as the excess molecules were not washed out. After 18 minutes of recording, SDS solution flowed through the channel to identify the fate of PS(4,4) prespacer if it was integrated at the time of flow. The last part of trace was used to create TDP as it contains both native and denatured state FRET levels.

Extended Data Fig. 8 Kinetic measurement of full-integration reaction.

a, A schematic of target and Cas1–Cas2–PS(4,4) used in experiments. b-e, Denaturing FRET histogram of the integration reaction quenched at different time point using SDS wash. Prespacer PS(4,4) was used in the measurement to allow full integration. Histogram for each time point was constructed from 25 short movies, each with about 300 FRET pairs. f, Histograms in b-e were quantified and the percentage of half- (black) and full-integration (red) products were plotted against reaction time, which shows the depletion of half-integration and the compensatory accumulation of full-integration population. The rate of formation full-integration (kfull) was derived from fitting the single-exponential equation, \(y = y0 + A \ast {\it{exp}}\left( { - k_{full} \ast x} \right)\) where y is population, x is reaction time.

Extended Data Fig. 9 Integration of various prespacer precursors.

a, Both overhangs 4 nt for positive control; both native and SDS histogram are shown. b, c, One side overhang 4 nt, another side overhang 5 and 6 nt, respectively. Histograms under native condition show integration from only one orientation, i.e. O1, and histograms under SDS treatment indicates that only one side of prespacer is attached as half integration. d, e, EfaCas1–Cas2 can spontaneously unwind 4 bp duplex blunt end. As a result, both orientations O1 and O2 appear under the native condition, and SDS wash resolves native peaks into several new peaks as seen before (Fig. 1d). f, With duplex length extended to 30 bp from its optimal 22 bp length, only one side with 4 nt overhang is integrated. Data were collected only for the denaturing condition.

Extended Data Fig. 10 Probe does not bind to repeat without transcription.

a, Cy3 spots from the 200bp target duplex five minutes after the flow of Cas1-Cas2-PS(4,4). The integration was detected by the appearance of Cy5 spots in the acceptor channel (but Cy5 spots not shown). b, The Cy3 spots were photobleached quickly by introducing imaging solution without gloxy under regular illumination of green laser (~25 mW). c, Image collected after the flow of Cy3-IR1 probe. The lack of Cy3 spots suggests that repeat is not exposed where the probe is expected to bind. A slight increase in spot number compared to ‘b’ (second image) may be due to non-specific binding of probe on surface-adsorbed Cas1–Cas2 that did not have prespacer or reappearance of some dark Cy3 (which appeared photobleached in ‘b’). d, Gel image showing multiple integrated spacers (bands) after 80 minutes under the different condition of replication and transcription.

Supplementary information

Supplementary Information

Supplementary text and Supplementary Table 1 for oligos used in the study.

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Original gel image

Source Data Extended Data Fig. 1

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Budhathoki, J.B., Xiao, Y., Schuler, G. et al. Real-time observation of CRISPR spacer acquisition by Cas1–Cas2 integrase. Nat Struct Mol Biol 27, 489–499 (2020). https://doi.org/10.1038/s41594-020-0415-7

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