CRISPR-Cas9 is widely used in genomic editing, but the kinetics of target search and its relation to the cellular concentration of Cas9 have remained elusive. Effective target search requires constant screening of the protospacer adjacent motif (PAM) and a 30 ms upper limit for screening was recently found. To further quantify the rapid switching between DNA-bound and freely-diffusing states of dCas9, we developed an open-microscopy framework, the miCube, and introduce Monte-Carlo diffusion distribution analysis (MC-DDA). Our analysis reveals that dCas9 is screening PAMs 40% of the time in Gram-positive Lactoccous lactis , averaging 17 ± 4 ms per binding event. Using heterogeneous dCas9 expression, we determine the number of cellular target-containing plasmids and derive the copy number dependent Cas9 cleavage. Furthermore, we show that dCas9 is not irreversibly bound to target sites but can still interfere with plasmid replication. Taken together, our quantitative data facilitates further optimization of the CRISPR-Cas toolbox.

We present a fast and model-free 2D and 3D single-molecule localization algorithm that allows more than 3 × 10 6 localizations per second to be calculated on a standard multi-core central processing unit with localization accuracies in line with the most accurate algorithms currently available. Our algorithm converts the region of interest around a point spread function to two phase vectors (phasors) by calculating the first Fourier coefficients in both the x-and y-direction. The angles of these phasors are used to localize the center of the single fluorescent emitter, and the ratio of the magnitudes of the two phasors is a measure for astigmatism, which can be used to obtain depth information (z-direction). Our approach can be used both as a stand-alone algorithm for maximizing localization speed and as a first estimator for more time consuming iterative algorithms.

Highlights d 20 Cascade complexes are required to provide 50% protection d Cascade spends equal time probing DNA (30 ms) and diffusing to a next site d Cas8e dynamically associates with Cascade in cells d CRISPR target search and invader replication compete in a kinetic ''arms race''

150) 28CRISPR-Cas systems encode RNA-guided surveillance complexes to find and cleave 29 invading DNA elements. While it is thought that invaders are neutralized minutes after cell 30 entry, the mechanism and kinetics of target search and its impact on CRISPR protection levels 31 have remained unknown. Here we visualized individual Cascade complexes in a native type I 32 CRISPR-Cas system. We uncovered an exponential relationship between Cascade copy 33 number and CRISPR interference levels, pointing to a time-driven arms race between invader 34 replication and target search, in which 20 Cascade complexes provide 50% protection. Driven 35 by PAM-interacting subunit Cas8e, Cascade spends half its search time rapidly probing DNA 36 (~30 ms) in the nucleoid. We further demonstrate that target DNA transcription and CRISPR 37 arrays affect the integrity of Cascade and impact CRISPR interference. Our work establishes 38 the mechanism of cellular DNA surveillance by Cascade that allows the timely detection of 39 invading DNA in a crowded, DNA-packed environment. 40 41 42

We study the diffraction of Damon-Eshbach-type spin waves incident on a one-dimensional grating realized by microslits in a thin Permalloy film. By means of time-resolved scanning Kerr microscopy, we observe unique diffraction patterns behind the grating which exhibit replications of the spin wave field at the slits. We show that these spin wave images, with details finer than the wavelength of the incident Damon-Eshbach spin wavelength, arise from the strongly anisotropic spin wave dispersion.