Specialized iNANO Lecture by Associate Professor Johannes Hohlbein, Wageningen University & Research

Associate Professor Johannes Hohlbein, Laboratory of Biophysics, Wageningen University & Research, the Netherlands

Exploring life at the single-molecule level

Single-molecule localisation microscopy (SMLM) allows monitoring molecular interactions in live cells and other complex samples. In the first part of my presentation, I will introduce SMLM and describe the miCube microscopy framework that we developed to increase the accessibility and affordability of SMLM. Developmental milestones include new algorithms for ultrafast data analysis and the addition of adaptive optics for localising proteins in turbid media.

The second part of my presentation will focus on the biology of CRISPR-Cas. Even though widely applied for genomic editing, the kinetics by which Cas proteins find target DNA have remained elusive. Cas proteins continuously screen the cell for protospacer adjacent motifs (PAM) even in absence of targets. We quantified the rapid switching between DNA-bound and freely-diffusing states revealing that dCas9 averages around 20 ms per PAM binding event in Gram-positive Lactoccous lactis. In presence of targets, we derived the copy number dependent rate of target binding. Taken together, our quantitative data and analysis facilitates further optimisation of the CRISPR-Cas toolbox.