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.

No abstract

We assessed whether exposure to solar ultraviolet-B radiation (UV-B) affects the mass loss of Larrea tridentata (creosotebush) litter in the Sonoran Desert of central Arizona. We placed three types of litter (leaves, twigs, or a natural mixture of leaves, twigs, and seeds) in bags constructed of UV-B-transmitting or UV-Babsorbing filter material that allowed either 85% (near-ambient UV-B treatment) or 15% (reduced UV-B treatment) of the biologically effective solar UV-B to reach litter inside the bags. Bags were placed outdoors for 4-5 months during the winter at two sites: a balcony or on the soil surface of the desert. Mass loss of leaf litter was greater under near-ambient UV-B than reduced UV-B at both sites: 21 (near-ambient) vs. 18% (reduced) on the balcony, and 18 vs. 14% at the desert site. Mass loss of twig litter was also greater under near-ambient UV-B at the desert site. Mass loss of the natural mixture of litter was also greater when exposed to near-ambient UV-B on the balcony, and tended to be greater at the desert site. We estimate that about 14-22% of the total mass loss of leaf litter during our 4-5 month experiments was attributable to solar UV-B exposure. Leaf litter exposed to near-ambient UV-B had lower concentrations of lignin, and fats and lipids, and slightly higher concentrations of holocellulose. The greater mass loss of litter under near-ambient UV-B appeared mainly attributable to loss of lignin, although losses of fats and lipids were also appreciable. A primary reason for greater mass loss of litter under solar UV-B appeared to be photodegradation, particularly of lignin.