CRISPR/Cas9 and Agrobacterium tumefaciens virulence proteins synergistically increase efficiency of precise genome editing via homology directed repair in plants.

CRISPR/Cas9 genome editing and Agrobacterium tumefaciens-mediated genetic transformation are widely-used plant biotechnology tools derived from bacterial immunity-related systems, each involving DNA modification. The Cas9 endonuclease introduces DNA double-strand breaks (DSBs), and the A. tumefaciens T-DNA is released by the VirD2 endonuclease assisted by VirDl and attached by VirE2, transferred to the plant nucleus and integrated into the genome. Here, we explored the potential for synergy between the two systems and found that Cas9 and three virulence (Vir) proteins achieve precise genome editing via the homology directed repair (HDR) pathway in tobacco and rice plants. Compared with Cas9T (Cas9, VirD1, VirE2) and CvD (Cas9-VirD2) systems, the HDR frequencies of a foreign GFPm gene in the CvDT system (Cas9-VirD2, VirD1, VirE2) increased 52-fold and 22-fold, respectively. Further optimization of the CvDT process with a donor linker (CvDTL) achieved a remarkable increase in the efficiency of HDR-mediated genome editing. Additionally, the HDR efficiency of the three rice endogenous genes ACETOLACTATE SYNTHASE (ALS), PHYTOENE DESATURASE (PDS), and NITROGEN TRANSPORTER 1.1 B (NRT1.1B) increased 24-, 32- and 16-fold, respectively, in the CvDTL system, compared with corresponding Cas9TL (Cas9T process with a donor linker). Our results suggest that collaboration between CRISPR/Cas9 and Agrobacterium-mediated genetic transformation can make great progress towards highly efficient and precise genome editing via the HDR pathway.

Gold Nanorod Assemblies: The Roles of Hot-Spot Positioning and Anisotropy in Plasmon Coupling and SERS.

Plasmon-coupled colloidal nanoassemblies with carefully sculpted "hot-spots" and intense surface-enhanced Raman scattering (SERS) are in high demand as photostable and sensitive plasmonic nano-, bio-, and chemosensors. When maximizing SERS signals, it is particularly challenging to control the hot-spot density, precisely position the hot-spots to intensify the plasmon coupling, and introduce the SERS molecule in those intense hot-spots. Here, we investigated the importance of these factors in nanoassemblies made of a gold nanorod (AuNR) core and spherical nanoparticle (AuNP) satellites with ssDNA oligomer linkers. Hot-spot positioning at the NR tips was made possible by selectively burying the ssDNA in the lateral facets via controlled Ag overgrowth while retaining their hybridization and assembly potential at the tips. This strategy, with slight alterations, allowed us to form nanoassemblies that only contained satellites at the NR tips, i.e., directional anisotropic nanoassemblies; or satellites randomly positioned around the NR, i.e., nondirectional nanoassemblies. Directional nanoassemblies featured strong plasmon coupling as compared to nondirectional ones, as a result of strategically placing the hot-spots at the most intense electric field position of the AuNR, i.e., retaining the inherent plasmon anisotropy. Furthermore, as the dsDNA was located in these anisotropic hot-spots, this allowed for the tag-free detection down to 10 dsDNA and a dramatic SERS enhancement of 1.6 × 108 for the SERS tag SYBR gold, which specifically intercalates into the dsDNA. This dramatic SERS performance was made possible by manipulating the anisotropy of the nanoassemblies, which allowed us to emphasize the critical role of hot-spot positioning and SERS molecule positioning in nanoassemblies.

Engineering an orchestrated release avalanche from hydrogels using DNA-nanotechnology.

Most medical therapies require repeated, sequential administration of therapeutic agents in well-defined intervals and over extended time windows. Typically, the patient is in charge of applying the individual drug doses, and insufficient patient compliance reduces the efficiency of the treatment. Therefore, the development of a smart delivery mechanism releasing therapeutic agents in a pre-defined, time-controlled fashion would be beneficial for many medical treatments. Here, we present a DNA-mediated release cascade which allows for precisely controlling the sequential delivery of several different nanoparticles. By using complementary DNA sequences, nanoparticle aggregates are created, embedded into distinct layers of a hydrogel and released by triggering aggregate dispersal. This mechanism is compatible with physiological conditions as the release cascade is initiated by exposing the nanoparticle-loaded gel to physiological salt concentrations. Moreover, we show that the reservoir hydrogel can be enriched with biopolymers to receive charge-selective release properties towards small molecules - without interfering with the DNA-based release cascade. Owing to the excellent reproducibility, precision and effectiveness of the presented mechanism, a similar DNA-mediated release avalanche may lead to the development of autonomous and robust delivery systems, which minimize the possibility of pharmaceutical therapy failure due to patient non-compliance.

Controlled nanoparticle release from a hydrogel by DNA-mediated particle disaggregation.

For many pharmaceutical applications, it is important that different drugs are present in the human body at distinct time points. Typically, this is achieved by a sequential administration of different therapeutic agents. A much easier alternative would be to develop a drug delivery system containing a whole set of medically active compounds which are liberated in an orchestrated and controlled manner. Yet, such a controlled, sequential release of drugs from a carrier system that can be used in a physiological situation is difficult to achieve. Here, we combine two molecular mechanisms, i.e. a build-up of osmotic pressure by the depletion of a control molecule and triggered disaggregation of nanoparticle clusters by synthetic DNA sequences. With this approach, we gain spatio-temporal control over the release of molecules and nanoparticles from a gel environment. The strategy presented here has strong implications for developing complex drug delivery systems for wound healing applications or for the sustained release of pharmaceuticals from a drug-loaded gel and will lower the need for multiple drug administrations.