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.

Cas12a (also called Cpf1) is a representative type V-A CRISPR effector RNAguided DNA endonuclease, which provides an alternative to type II CRISPR-Cas9 for genome editing. Previous studies have revealed that Cas12a has

Highlights d Reaction rates for individual steps in the DNA cleavage reaction are characterized d Kinetic pathway of divalent ions in the DNA cleavage reaction is revealed d Sequence-preferential DNA bending is induced for the cleavage site selection d It is determined by protein-DNA interactions rather than by the intrinsic property

Homotypic fusion of endoplasmic reticulum membranes is driven by atlastin GTPases; however, the underlying mechanism remains largely unknown. Here, using a FRET-based single-vesicle fusion assay with liposomes bearing the yeast atlastin Sey1p, we investigated the molecular mechanisms of atlastin-mediated membrane tethering and fusion. Although Sey1p-bearing proteoliposomes frequently underwent membrane tethering in a GTP hydrolysis-dependent manner as reported in studies using bulk assays, only a small fraction of the tethered liposomes proceeded to fusion. Strikingly, the rest of the tethered liposomes failed to fuse or dissociate. This stable tethering, however, did not require continued GTP hydrolysis because GTP omission and magnesium chelation did not disrupt tethering. Interestingly, an increased Sey1p density on the membrane markedly accelerated tethering but barely affected the fusion rate of the tethered liposomes, indicating that Sey1p requires additional factors to support efficient fusion in vivo. Finally, the assay also revealed that Sey1p-mediated liposome fusion occurs through hemifusion, suggesting the mechanistic conservation between biological membrane fusion events despite the existence of diverse fusogens.

We analyze the ordered phases of Bernal stacked multilayer graphene in the presence of interaction induced band gaps due to sublattice symmetry breaking potentials, whose solutions can be analyzed in terms of light-mass and heavy-mass pseudospin doublets which have the same Chern numbers but opposite charge polarization directions. The application of a perpendicular external electric field reveals an effective Hund's rule for the ordering of the sublattice pseudospin doublets in a tetralayer, while a similar but more complex phase diagram develops with increasing layer number. I. FLAVOR ANTIFERRO STATES IN TETRALAYER GRAPHENEFrom the Hartree energy cost considerations, the metastable states with the lowest total energy are expected to be flavor antiferro when there is no external electric field perpendicular to the graphene layers. Within the energetically more favorable flavor antiferro states, we classify different Hall phases [1, 2]: layer antiferromagnetic (LAF) phase with the spin dependent but valley independent sublattice potential, quantum spin Hall (QSH) phase with both the spin and valley dependent sublattice potential, and quantum anomalous Hall (QAH) phase with the valley dependent but spin independent sublattice potential, as schematically shown in Fig. 1.