Cas12a (also called Cpf1) is a representative type V-A CRISPR effector RNA-guided DNA endonuclease, which provides an alternative to type II CRISPR–Cas9 for genome editing. Previous studies have revealed that Cas12a has unique features distinct from Cas9, but the detailed mechanisms of target searching and DNA cleavage by Cas12a are still unclear. Here, we directly observe this entire process by using single-molecule fluorescence assays to study Cas12a from Acidaminococcus sp. (AsCas12a). We determine that AsCas12a ribonucleoproteins search for their on-target site by a one-dimensional diffusion along elongated DNA molecules and induce cleavage in the two DNA strands in a well-defined order, beginning with the non-target strand. Furthermore, the protospacer-adjacent motif (PAM) for AsCas12a makes only a limited contribution of DNA unwinding during R-loop formation and shows a negligible role in the process of DNA cleavage, in contrast to the Cas9 PAM.

Full understanding of complex biological interactions frequently requires multi-color detection capability in doing single-molecule fluorescence resonance energy transfer (FRET) experiments. Existing single-molecule three-color FRET techniques, however, suffer from severe photobleaching of Alexa 488, or its alternative dyes, and have been limitedly used for kinetics studies. In this work, we developed a single-molecule three-color FRET technique based on the Cy3-Cy5-Cy7 dye trio, thus providing enhanced observation time and improved data quality. Because the absorption spectra of three fluorophores are well separated, real-time monitoring of three FRET efficiencies was possible by incorporating the alternating laser excitation (ALEX) technique both in confocal microscopy and in total-internal-reflection fluorescence (TIRF) microscopy.

Since its first demonstration about twenty years ago, single-molecule fluorescence resonance energy transfer (FRET) has undergone remarkable technical advances. In this tutorial review, we will discuss two technical advances that increase the information content of the single-molecule FRET measurements: single-molecule multi-color FRET and single-molecule FRET combined with force or torque. Our expectations for future developments will be briefly discussed at the end.

Topoisomerase II resolves intrinsic topological problems of double-stranded DNA. As part of its essential cellular functions, the enzyme generates DNA breaks, but the regulation of this potentially dangerous process is not well understood. Here we report single-molecule fluorescence experiments that reveal a previously uncharacterized sequence of events during DNA cleavage by topoisomerase II: nonspecific DNA binding, sequence-specific DNA bending, and stochastic cleavage of DNA. We have identified unexpected structural roles of Mg ²⁺ ions coordinated in the TOPRIM (topoisomerase-primase) domain in inducing cleavage-competent DNA bending. A break at one scissile bond dramatically stabilized DNA bending, explaining how two scission events in opposing strands can be coordinated to achieve a high probability of double-stranded cleavage. Clamping of the protein N -gate greatly enhanced the rate and degree of DNA bending, resulting in a significant stimulation of the DNA cleavage and opening reactions. Our data strongly suggest that the accurate cleavage of DNA by topoisomerase II is regulated through a tight coordination with DNA bending.