Single-Cell Biophysics: Measurement, Modulation, and Modeling

37 / 155

Single-Cell Biophysics: Measurement, Modulation, and Modeling Monday Speaker Abstracts
32
Single-Molecule Studies of Cas9 Search Kinetics in Living Cells
Daniel Jones, Prune Leroy, Cecilia Unosson, David Fange, Johan Elf.
Uppsala University, Uppsala, Sweden.
Cas9-based DNA targeting is radically different from that of transcription factors. Cas9 can be
programmed by a guide RNA to target any sequence. We have studied how much time it takes to
located a singel target sequence in a living cell using single molecule fluorescence microscopy.
We also measure how long time the bound dCas9 stays at a target sequences and what
implications this has for repression of a targeted gene.
Decoding Chromatin Organization with Superresolution Microscopy
Melike Lakadamyali1,2.
1ICFO-Institute of Photonic Sciences, Barcelona, Spain, 2University of Pennsylvania,
Philadelphia, PA, USA.
Nucleosomes help structure chromosomes by compacting DNA into fibers. Chromatin
organizationin the kilo-to-megabase length scales plays an important role for regulating gene
expression. However, these small length scales have been impossible to visualize using
conventional microscopy methods, hence making it challenging to decipher the nanoscale
organization of chromatin inside the nucleus. To gain insight into how nucleosomes are arranged
in vivo, we have been using quantitative super-resolution nanoscopy to visualize and determine
the organization of the chromatin fiber in single nuclei. We have been able to correlate the
nanoscale organization of chromatin to cell state, enabling us to identify the level of pluripotency
as well as the differentiation state of different cell types.