Rostamian, H. | Search bioRxiv

bioRxiv2026

Rapid CRISPR-Cas9 Genome Editing in S. cerevisiae

This protocol enables rapid CRISPR-Cas9 genome editing in Saccharomyces cerevisiae by replacing restriction/ligation guide cloning with PCR-based protospacer installation and seamless plasmid recircularization. It describes in silico HDR donor and SgRNA design, install guide sequences into cas9 plasmid by PCR and seamless assembly, plasmid cloning and sequence verification in E. coli, and LiAc/PEG co-transformation of yeast with Cas9-sgRNA plasmid plus HDR donor. The workflow selects yeast colonies on G418 and confirms edits by PCR and sequencing.

cell biology↗

bioRxiv2023

Correlative single molecule lattice light sheet imaging reveals the dynamic relationship between nucleosomes and the local chromatin environment

In the nucleus, biological processes are driven by proteins that diffuse through and bind to a meshwork of nucleic acid polymers. To better understand this interplay, we developed an imaging platform to simultaneously visualize single protein dynamics together with the local chromatin environment in live cells. Together with super-resolution imaging, new fluorescent probes, and biophysical modeling, we demonstrated that nucleosomes display differential diffusion and packing arrangements as chromatin density increases whereas the viscoelastic properties and accessibility of the interchromatin space remain constant. Perturbing nuclear functions impacted nucleosome diffusive properties in a manner that was dependent on local chromatin density and supportive of a model wherein transcription locally stabilizes nucleosomes while simultaneously allowing for the free exchange of nuclear proteins. Our results reveal that nuclear heterogeneity arises from both active and passive process and highlights the need to account for different organizational principals when modeling different chromatin environments.

biophysics↗