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Abstract The biggest challenge in using CRISPR technologies, which limits their widespread application in medicine, is off-target effects. These effects could, in principle, be minimized by ensuring that CRISPR is activated primarily in the targeted cells, thereby reducing the likelihood of unintended genetic modifications in non-target tissues. Therefore, the development of a light activatable CRISPR approach to dynamically control gene activation in both space and time would be highly beneficial. A drawback is that the overwhelming majority of recently introduced light activatable CRISPR systems require UV or blue light exposure, severely limiting the penetration depth of light in tissue at which CRISPR can be activated, and, in the case of UV light, raising safety concerns. A small number of systems that activate CRISPR using longer wavelengths are hindered by either slow light activation or issues related to toxicity and biocompatibility of the proposed techniques in humans. To address this, we developed a split-Cas9/dCas9 system in which activation is achieved through a near-infrared photocleavable dimerization complex. This photoactivation method can be safely used in humans in vivo, easily adapted to different split-Cas9/dCas9 systems, and enables rapid, spatially precise light activation across various cell types. 

It can be difficult to employ optical techniques for analyzing biological structures smaller than or comparable to the wavelength of light, such as extracellular vesicles or some types of bacteria. Biological light scattering spectroscopy (LSS), developed to address this problem, has been successfully used for characterizing tissue on cellular and subcellular scales. At the same time, calibration with a reference sample of known optical properties can complicate LSS measurements. In this Letter, we present dual-angle LSS (daLSS), which is designed for calibration-free measurements of scatterer suspensions. It employs measurements of a sample at two distinct angles, which then allows system effects to be removed entirely. Not only does daLSS simplify and speed up the measurement procedure, but it also makes spectra more reproducible, an important feature for diagnostic techniques. We validated the technique by accurately reconstructing the sizes of polystyrene microspheres with diameters less than 100 nm and then demonstrated that not only are the daLSS spectra of several common bacteria strains easily distinguishable but they are also highly reproducible.