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.

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We present a fast and model-free 2D and 3D single-molecule localization algorithm that allows more than 3 million localizations per second on a standard multi-core CPU with localization accuracies in line with the most accurate algorithms currently available. Our algorithm converts the region of interest around a point spread function (PSF) to two phase vectors (phasors) by calculating the first Fourier . CC-BY-NC 4.0 International license not peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/191957 doi: bioRxiv preprint first posted online 2 coefficients in both 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.

Super-resolution microscopy is frequently employed in the life sciences, but the number of 18 freely accessible and affordable microscopy frameworks, especially for single particle tracking 19 photo-activation localization microscopy (sptPALM), remains limited. To this end, we designed 20 the miCube: a versatile super-resolution capable fluorescence microscope, which combines high 21 spatiotemporal resolution, good adaptability, low price, and easy installation. By providing all 22 details, we hope to enable interested researchers to build an identical or derivative instrument. 23The capabilities of the miCube are assessed with a novel sptPALM assay relying on the 24 heterogeneous expression of target genes. Here, we elucidate mechanistic details of 25 catalytically inactive Cas9 (dead Cas9) in live Lactococcus lactis. We show that, lacking specific 26 DNA target sites, the binding and unbinding of dCas9 to DNA can be described using simplified 27 rate constants of kbound free = 30-80 s -1 and kfree bound = 15-40 s -1 . Moreover, after providing 28 specific DNA target sites via DNA plasmids, the plasmid-bound dCas9 population size decreases 29 with increasing dCas9 copy number via a mono-exponential decay, indicative of simple 30 disassociation kinetics. 31 1.31 ± 0.05 µm 2 /s). This value is consistent with simulated cytoplasmic diffusion of dCas9-PAmCherry2