Chaubet, L. | bioRxiv Science

bioRxiv2021

Cas12a is a dynamic and precise RNA-guided nuclease without off-target activity on {lambda}-DNA

Cas12a is an RNA-guided endonuclease that is emerging as a powerful genome-editing tool. Here we combined optical tweezers with fluorescence to monitor Cas12a binding onto {lambda}-DNA, providing insight into its DNA binding and cleavage mechanisms. At low forces Cas12a binds DNA specifically with two off-target sites, while at higher forces numerous binding events appear driven by the mechanical distortion of the DNA and partial matches to the crRNA. Despite the multiple binding events, cleavage is only observed on the target site at low forces, when the DNA is flexible. Activity assays show that the preferential off-target sites are not cleaved, and the {lambda}-DNA is severed at the target site. This precision is also observed in Cas12a variants where the specific dsDNA and the unspecific ssDNA cleavage are dissociated or nick the target DNA. We propose that Cas12a and its variants are precise endonucleases that efficiently scan the DNA for its target but only cleave the selected site in the {lambda}-DNA.

biophysics↗

bioRxiv2019

Dynamic crosslinking of the actin cytoskeleton governs cell mechanics

Cells precisely control their mechanical properties to organize and differentiate into tissues. The architecture and connectivity of cytoskeletal filaments changes in response to mechanical and biochemical cues, allowing the cell to rapidly tune its mechanics from highly-crosslinked, elastic networks to weakly-crosslinked viscous networks. While the role of actin crosslinking in controlling actin network mechanics is well-characterized in purified actin networks, its mechanical role in the cytoplasm of living cells remains unknown. Here, we probe the frequency-dependent intracellular viscoelastic properties of living cells using multifrequency excitation and in situ optical trap calibration. At long timescales in the intracellular environment, we observe that the cytoskeleton becomes fluid-like. The mechanics are well-captured by a model in which actin filaments are dynamically connected by a single dominant crosslinker. A disease-causing point mutation (K255E) of the actin crosslinker -actinin 4 (ACTN4) causes its binding kinetics to be insensitive to tension. Under normal conditions, the viscoelastic properties of wild type (WT) and K255E+/- cells are similar. However, when tension is reduced through myosin II inhibition, WT cells relax 3x faster to the fluid-like regime while K255E+/- cells are not affected. These results indicate that dynamic actin crosslinking enables the cytoplasm to flow at long timescales.

biophysics↗