The prokaryotic CRISPR (clustered regularly interspaced palindromic repeats)-associated protein, Cas9, has been widely adopted as a tool for editing, imaging, and regulating eukaryotic genomes. However, our understanding of how to select single-guide RNAs (sgRNAs) that mediate efficient Cas9 activity is incomplete, as we lack insight into how chromatin impacts Cas9 targeting. To address this gap, we analyzed large-scale genetic screens performed in human cell lines using either nuclease-active or nuclease-dead Cas9 (dCas9). We observed that highly active sgRNAs for Cas9 and dCas9 were found almost exclusively in regions of low nucleosome occupancy. In vitro experiments demonstrated that nucleosomes in fact directly impede Cas9 binding and cleavage, while chromatin remodeling can restore Cas9 access. Our results reveal a critical role of eukaryotic chromatin in dictating the targeting specificity of this transplanted bacterial enzyme, and provide rules for selecting Cas9 target sites distinct from and complementary to those based on sequence properties.DOI: http://dx.doi.org/10.7554/eLife.12677.001

The RNA-guided CRISPR-associated protein Cas9 is used for genome editing, transcriptional modulation, and live-cell imaging. Cas9-guide RNA complexes recognize and cleave double-stranded DNA sequences on the basis of 20-nucleotide RNA-DNA complementarity, but the mechanism of target searching in mammalian cells is unknown. Here, we use single-particle tracking to visualize diffusion and chromatin binding of Cas9 in living cells. We show that three-dimensional diffusion dominates Cas9 searching in vivo, and off-target binding events are, on average, short-lived (<1 second). Searching is dependent on the local chromatin environment, with less sampling and slower movement within heterochromatin. These results reveal how the bacterial Cas9 protein interrogates mammalian genomes and navigates eukaryotic chromatin structure.

Ring-shaped hexameric helicases and translocases support essential DNA-, RNA-, and protein-dependent transactions in all cells and many viruses. How such systems coordinate ATPase activity between multiple subunits to power conformational changes that drive the engagement and movement of client substrates is a fundamental question. Using the Escherichia coli Rho transcription termination factor as a model system, we have used solution and crystallographic structural methods to delineate the range of conformational changes that accompany distinct substrate and nucleotide cofactor binding events. Small-angle X-ray scattering data show that Rho preferentially adopts an open-ring state in solution and that RNA and ATP are both required to cooperatively promote ring closure. Multiple closed-ring structures with different RNA substrates and nucleotide occupancies capture distinct catalytic intermediates accessed during translocation. Our data reveal how RNA-induced ring closure templates a sequential ATP-hydrolysis mechanism, provide a molecular rationale for how the Rho ATPase domains distinguishes between distinct RNA sequences, and establish structural snapshots of substepping events in a hexameric helicase/translocase.ATPase | helicase | motor protein | transcription | translocase H exameric helicases and translocases are motor proteins that play a central role in cellular transactions ranging from replication and repair to transcriptional regulation, chromosome packaging, and proteolytic homeostasis (1-4). Used to drive the processive and, at times, highly rapid movement of extended nucleic acid or protein chains through a central pore, ring-shaped motors face several challenges to their operation. One is that certain enzymes must transition through controlled ring opening and/or subunit assembly events to allow long, polymeric substrates that lack freely accessible ends to access interior motor elements (5-7). Another is that, once loaded, the molecular plasticity inherent to these assemblies must be harnessed to precisely coordinate ATP binding and hydrolysis between multiple subunits with the powering of substrate translocation, while at the same time alternating between tight and loose grips on the substrate to allow for processive movement. The substrate-dependent molecular rearrangements that underpin ring dynamics during these events remain poorly understood, not only for hexameric helicases and translocases, but for related ring-shaped switches as well.The Escherichia coli Rho transcription termination factor is a well-established model system for understanding hexameric translocase and helicase function (8, 9). During termination, Rho uses a cytosine-specific RNA-binding domain appended to the N terminus of a RecA-type ATPase fold (10, 11) to bind nascent RNA transcripts at cytosine-rich sequences [known as Rho utilization (rut) sites] (12, 13). Once loaded, Rho consumes ATP to translocate 5′→3′ toward a paused RNA polymerase, eventually promoting transcription bubble collapse and RNA release (14-19).Str...