Single-molecule techniques allow the visualization of the molecular dynamics of nucleic acids and proteins with high spatiotemporal resolution. Valuable kinetic information of biomolecules can be obtained when the discrete states within single-molecule time trajectories are determined. Here, we present a fast, automated, and bias-free step detection method, AutoStepfinder, that determines steps in large datasets without requiring prior knowledge on the noise contributions and location of steps. The analysis is based on a series of partition events that minimize the difference between the data and the fit. A dual-pass strategy determines the optimal fit and allows AutoStepfinder to detect steps of a wide variety of sizes. We demonstrate step detection for a broad variety of experimental traces. The user-friendly interface and the automated detection of AutoStepfinder provides a robust analysis procedure that enables anyone without programming knowledge to generate step fits and informative plots in less than an hour.

CRISPR–Cas immunity protects prokaryotes against invading genetic elements¹. It uses the highly conserved Cas1–Cas2 complex to establish inheritable memory (spacers)^2–5. How Cas1–Cas2 acquires spacers from foreign DNA fragments (prespacers) and integrates them into the CRISPR locus in the correct orientation is unclear^6,7. Here, using the high spatiotemporal resolution of single-molecule fluorescence, we show that Cas1–Cas2 selects precursors of prespacers from DNA in various forms—including single-stranded DNA and partial duplexes—in a manner that depends on both the length of the DNA strand and the presence of a protospacer adjacent motif (PAM) sequence. We also identify DnaQ exonucleases as enzymes that process the Cas1–Cas2-loaded prespacer precursors into mature prespacers of a suitable size for integration. Cas1–Cas2 protects the PAM sequence from maturation, which results in the production of asymmetrically trimmed prespacers and the subsequent integration of spacers in the correct orientation. Our results demonstrate the kinetic coordination of prespacer precursor selection and PAM trimming, providing insight into the mechanisms that underlie the integration of functional spacers in the CRISPR loci.

CRISPR-Cas provides RNA-guided adaptive immunity against invading genetic elements. Interference in type I systems relies on the RNA-guided Cascade complex for target DNA recognition and the Cas3 helicase/nuclease protein for target degradation. Even though the biochemistry of CRISPR interference has been largely covered, the biophysics of DNA unwinding and coupling of the helicase and nuclease domains of Cas3 remains elusive. Here, we employed single-molecule Förster resonance energy transfer (FRET) to probe the helicase activity with high spatiotemporal resolution. We show that Cas3 remains tightly associated with the target-bound Cascade complex while reeling the DNA using a spring-loaded mechanism. This spring-loaded reeling occurs in distinct bursts of 3 bp, which underlie three successive 1-nt unwinding events. Reeling is highly repetitive, allowing Cas3 to repeatedly present its inefficient nuclease domain with single-strand DNA (ssDNA) substrate. Our study reveals that the discontinuous helicase properties of Cas3 and its tight interaction with Cascade ensure controlled degradation of target DNA only. Loeff et al. report on a single-molecule fluorescence analysis of the E. coli CRISPR-Cas3 protein. The Cas3 protein uses a spring-loaded unwinding mechanism, reeling the target DNA 3 bp at a time. Facilitated by slipping, Cas3 repeatedly presents its intrinsically inefficient nuclease domain with DNA substrate, which may contribute to promoting a robust immune response.

The genome and transcriptome are constantly modified by proteins in the cell. Recent advances in single-molecule techniques allow for high spatial and temporal observations of these interactions between proteins and nucleic acids. However, due to the difficulty of obtaining functional protein complexes, it remains challenging to study the interactions between macromolecular protein complexes and nucleic acids. Here, we combined single-molecule fluorescence with various protein complex pull-down techniques to determine the function and stoichiometry of ribonucleoprotein complexes. Through the use of three examples of protein complexes from eukaryotic cells (Drosha, Dicer, and TUT4 protein complexes), we provide step-by-step guidance for using novel single-molecule techniques. Our single-molecule methods provide sub-second and nanometer resolution and can be applied to other nucleoprotein complexes that are essential for cellular processes.

Small RNA-guided protein complexes play an essential role in CRISPR-mediated immunity in prokaryotes. While these complexes initiate interference byflagging cognate invader DNA for destruction, recent evidence has implicated their involvement innew CRISPR memory formation, called priming, against mutated invader sequences. The mechanism by which the target recognition complex mediates these disparate responses-interference and priming-remains poorly understood. Using single-molecule FRET, we visualize how bona fide and mutated targets are differentially probed by E.coli Cascade. We observe that the recognition of bona fide targets is an ordered process that is tightly controlled forhigh fidelity. Mutated targets are recognized with low fidelity, which is featured by short-lived and PAM- and seed-independent binding by any segment of the crRNA. These dual roles of Cascade in immunity with distinct fidelities underpin CRISPR-Cas robustness, allowing for efficient degradation ofbona fide targets and priming of mutated DNA targets.