Dual CRISPR-Cas3 system for inducing multi-exon skipping in DMD patient-derived iPSCs.

To restore dystrophin protein in various mutation patterns of Duchenne muscular dystrophy (DMD), the multi-exon skipping (MES) approach has been investigated. However, only limited techniques are available to induce a large deletion to cover the target exons spread over several hundred kilobases. Here, we utilized the CRISPR-Cas3 system for MES induction and showed that dual crRNAs could induce a large deletion at the dystrophin exon 45-55 region (∼340 kb), which can be applied to various types of DMD patients. We developed a two-color SSA-based reporter system for Cas3 to enrich the genome-edited cell population and demonstrated that MES induction restored dystrophin protein in DMD-iPSCs with three distinct mutations. Whole-genome sequencing and distance analysis detected no significant off-target deletion near the putative crRNA binding sites. Altogether, dual CRISPR-Cas3 is a promising tool to induce a gigantic genomic deletion and restore dystrophin protein via MES induction.

Nano-size dependent protein corona formation by SARS-CoV-2 Omicron spike protein over gold nano-colloid and reversible aggregation.

The adsorption process of SARS-CoV-2 Omicron spike protein to the nano-gold colloid surfaces was examined by monitoring the surface plasmon resonance (SPR) band shift of gold-nano particles ranging between diameters of d = 10-100 nm. The externally changed pH between 3 and 11 at 24.5 ± 0.4 °C initiated a reversible formation of the gold colloid aggregates, where formation/deformation of the aggregates were monitored by red/blue shift of the peak of the SPR band. There was no sign of reversible aggregation for d = 10, 15, and 20 nm gold colloids. A clear undulation of the peak shift corresponding to pH hopping between pH ~3 and ~11 was confirmed for colloidal d > 30 nm. This degree of the reversibility was compared to previously reported SARS-CoV-2 Alpha spike protein coated gold colloids. It was concluded that Omicron spike protein possesses a similar low affinity for gold nano particle d < 20 nm and possesses the higher affinity to the gold nanoparticles of d > 30 nm. However, the Omicron spike protein conformation was presumed to be more denatured compared to the SARS-CoV-2 Alpha spike protein. Our finding suggested Omicron spike protein was more acid labile/flexible.

Spectroscopic investigation on the affinity of SARS-CoV-2 spike protein to gold nano-particles.

The affinity of the SARS-CoV-2 spike protein (S protein) to gold nano-particles was examined through spectral shifts of SPR (Surface Plasmon Resonance) band. Gold nano-colloidal particles are sensitive to the conformational change of the protein adsorbed over the particles' surface. As the pH value was gradually lowered from approximately neutral pH to an acidic pH (ca. pH 2), all mixtures of S protein with the gold colloids ≥30 nm in diameter exhibited a drastic red-shift of the average SPR band peak at one pH value more than that observed for bare gold colloids. The surface coverage fraction (Θ) of S protein over the nano-particle's surface was extracted and all showed relatively small coverage values (i.e., Θ ~ 0.30). The SPR band peak shift was also examined as the pH values were hopped between pH ~ 3 and pH ~ 10 (pH hopping). As the pH values hopped, an alternation of the average SPR band peaks were observed. A significant amplitude of an alternation was especially observed for the mixture of S protein with gold ≥30 nm of gold size implying the reproduction of pH induced reversible protein folding. We hypothesize that the pH hopping scheme captured a reversible transition between folded or Down conformation (pH ≥ ~7) and unfolded or Up (pH ~ 3) conformation of RBD (receptor binding domain). The acidic condition may also dimerize the S protein through RBD. The Up conformation or dimerization of S protein are considered to be connected to the other gold nano particles forming gold nano-particle aggregates.

Nano-size dependence in the adsorption by the SARS-CoV-2 spike protein over gold colloid.

Gold nano-particles were coated with the spike protein (S protein) of SARS-CoV-2 and exposed to increasingly acidic conditions. Their responses were investigated by monitoring the surface plasmon resonance (SPR) band shift. As the external pH was gradually changed from neutral pH to pH ∼2 the peak of the SPR band showed a significant red-shift, with a sigmoidal feature implying the formation of the gold-protein aggregates. The coating of S protein changed the surface property of the gold enough to extract the coverage fraction of protein over nano particles, Θ, which did not exhibit clear nano-size dependence. The geometrical simulation to explain Θ showed the average axial length to be a = 7. 25 nm and b =8.00 nm when the S-protein was hypothesized as a prolate shape with spiking-out orientation. As the pH value externally hopped between pH∼3 and pH∼10, a behavior of reversible protein folding was observed for particles with diameters >30 nm. It was concluded that S protein adsorption conformation was impacted by the size (diameter, d) of a core nano-gold, where head-to-head dimerized S protein was estimated for d ≤ 80 nm and a parallel in opposite directions formation for d = 100 nm.

Examination of Adsorption Orientation of Amyloidogenic Peptides Over Nano-Gold Colloidal Particle Surfaces.

The adsorption of amyloidogenic peptides, amyloid beta 1-40 (Aß1-40), alpha-synuclein (α-syn), and beta 2 microglobulin (ß2m), was attempted over the surface of nano-gold colloidal particles, ranging from d = 10 to 100 nm in diameter (d). The spectroscopic inspection between pH 2 and pH 12 successfully extracted the critical pH point (pHo) at which the color change of the amyloidogenic peptide-coated nano-gold colloids occurred due to aggregation of the nano-gold colloids. The change in surface property caused by the degree of peptide coverage was hypothesized to reflect the ΔpHo, which is the difference in pHo between bare gold colloids and peptide coated gold colloids. The coverage ratio (Θ) for all amyloidogenic peptides over gold colloid of different sizes was extracted by assuming Θ = 0 at ΔpHo = 0. Remarkably, Θ was found to have a nano-gold colloidal size dependence, however, this nano-size dependence was not simply correlated with d. The geometric analysis and simulation of reproducing Θ was conducted by assuming a prolate shape of all amyloidogenic peptides. The simulation concluded that a spiking-out orientation of a prolate was required in order to reproduce the extracted Θ. The involvement of a secondary layer was suggested; this secondary layer was considered to be due to the networking of the peptides. An extracted average distance of networking between adjacent gold colloids supports the binding of peptides as if they are "entangled" and enclosed in an interfacial distance that was found to be approximately 2 nm. The complex nano-size dependence of Θ was explained by available spacing between adjacent prolates. When the secondary layer was formed, Aß1-40 and α-syn possessed a higher affinity to a partially negative nano-gold colloidal surface. However, ß2m peptides tend to interact with each other. This difference was explained by the difference in partial charge distribution over a monomer. Both Aß1-40 and α-syn are considered to have a partial charge (especially δ+) distribution centering around the prolate axis. The ß2m, however, possesses a distorted charge distribution. For a lower Θ (i.e., Θ <0.5), a prolate was assumed to conduct a gyration motion, maintaining the spiking-out orientation to fill in the unoccupied space with a tilting angle ranging between 5° and 58° depending on the nano-scale and peptide coated to the gold colloid.