The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA–DNA base-pairing to target foreign DNA in bacteria. Cas9–guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9–RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9–RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9–RNA. Competition assays provide evidence that DNA strand separation and RNA–DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate scission of double-stranded DNA.

Deep learning for protein interactions: The use of deep learning has revolutionized the field of protein modeling. Humphreys et al . combined this approach with proteome-wide, coevolution-guided protein interaction identification to conduct a large-scale screen of protein-protein interactions in yeast (see the Perspective by Pereira and Schwede). The authors generated predicted interactions and accurate structures for complexes spanning key biological processes in Saccharomyces cerevisiae . The complexes include larger protein assemblies such as trimers, tetramers, and pentamers and provide insights into biological function. —VV

Condensin is a highly processive DNA motor: Condensin is thought to regulate genome architecture by creating DNA loops. Terakawa et al. used single-molecule imaging to show that yeast condensin is a highly processive mechanochemical motor capable of translocating along DNA (see the Perspective by Nasmyth). Their findings elucidate how the rapid ATP hydrolysis–dependent motor activity of condensin provides the driving forces necessary to support three-dimensional chromosome organization and compaction through a loop extrusion mechanism. Science , this issue p. 672 ; see also p. 589

Cohesin is essential for the hierarchical organization of the eukaryotic genome and plays key roles in many aspects of chromosome biology. The conformation of cohesin bound to DNA remains poorly defined, leaving crucial gaps in our understanding of how cohesin fulfills its biological functions. Here, we use single-molecule microscopy to directly observe the dynamic and functional characteristics of cohesin bound to DNA. We show that cohesin can undergo rapid one-dimensional (1D) diffusion along DNA, but individual nucleosomes, nucleosome arrays, and other protein obstacles significantly restrict its mobility. Furthermore, we demonstrate that DNA motor proteins can readily push cohesin along DNA, but they cannot pass through the interior of the cohesin ring. Together, our results reveal that DNA-bound cohesin has a central pore that is substantially smaller than anticipated. These findings have direct implications for understanding how cohesin and other SMC proteins interact with and distribute along chromatin.