Smart Strategies for Precise Delivery of CRISPR/Cas9 in Genome Editing.

The emergence of CRISPR/Cas technology has enabled scientists to precisely edit genomic DNA sequences. This approach can be used to modulate gene expression for the treatment of genetic disorders and incurable diseases such as cancer. This potent genome-editing tool is based on a single guide RNA (sgRNA) strand that recognizes the targeted DNA, plus a Cas nuclease protein for binding and processing the target. CRISPR/Cas has great potential for editing many genes in different types of cells and organisms both in vitro and in vivo. Despite these remarkable advances, the risk of off-target effects has hindered the translation of CRISPR/Cas technology into clinical applications. To overcome this hurdle, researchers have devised gene regulatory systems that can be controlled in a spatiotemporal manner, by designing special sgRNA, Cas, and CRISPR/Cas delivery vehicles that are responsive to different stimuli, such as temperature, light, magnetic fields, ultrasound (US), pH, redox, and enzymatic activity. These systems can even respond to dual or multiple stimuli simultaneously, thereby providing superior spatial and temporal control over CRISPR/Cas gene editing. Herein, we summarize the latest advances on smart sgRNA, Cas, and CRISPR/Cas nanocarriers, categorized according to their stimulus type (physical, chemical, or biological).

Smart arginine-equipped polycationic nanoparticles for p/CRISPR delivery into cells.

An efficient and safe delivery system for the transfection of CRISPR plasmid (p/CRISPR) into target cells can open new avenues for the treatment of various diseases. Herein, we design a novel nonvehicle by integrating an arginine-disulfide linker with low-molecular-weight PEI (PEI1.8k) for the delivery of p/CRISPR. These PEI1.8k-Arg nanoparticles facilitate the plasmid release and improve both membrane permeability and nuclear localization, thereby exhibiting higher transfection efficiency compared to native PEI1.8kin the delivery of nanocomplexes composed of PEI1.8k-Arg and p/CRISPR into conventional cells (HEK 293T). This nanovehicle is also able to transfect p/CRISPR in a wide variety of cells, including hard-to-transfect primary cells (HUVECs), cancer cells (HeLa), and neuronal cells (PC-12) with nearly 5-10 times higher efficiency compared to the polymeric gold standard transfection agent. Furthermore, the PEI1.8k-Arg nanoparticles can edit the GFP gene in the HEK 293T-GFP reporter cell line by delivering all possible forms of CRISPR/Cas9 system (e.g. plasmid encoding Cas9 and sgRNA targeting GFP, and Cas9/sgRNA ribonucleoproteins (RNPs) as well as Cas9 expression plasmid andin vitro-prepared sgRNA) into HEK 293T-GFP cells. The successful delivery of p/CRISPR into local brain tissue is also another remarkable capability of these nanoparticles. In view of all the exceptional benefits of this safe nanocarrier, it is expected to break new ground in the field of gene editing, particularly for therapeutic purposes.

Highly Photoluminescent Nitrogen- and Zinc-Doped Carbon Dots for Efficient Delivery of CRISPR/Cas9 and mRNA.

Safe and efficient delivery of CRISPR/Cas9 systems is still a challenge. Here we report the development of fluorescent nitrogen- and zinc-doped carbon dots (N-Zn-doped CDs) using one-step microwave-aided pyrolysis based on citric acid, branched PEI25k, and different zinc salts. These versatile nanovectors with a quantum yield of around 60% could not only transfect large CRISPR plasmids (∼9 kb) with higher efficiency (80%) compared to PEI25k and lipofectamine 2000 (Lipo 2K), but they also delivered mRNA into HEK 293T cells with the efficiency 20 times greater than and equal to that of PEI25k and Lipo 2K, respectively. Unlike PEI25k, N-Zn-doped CDs exhibited good transfection efficiency even at low plasmid doses and in the presence of 10% fetal bovine serum (FBS). Moreover, these nanovectors demonstrated excellent efficiency in GFP gene disruption by transferring plasmid encoding Cas9 and sgRNA targeting GFP as well as Cas9/sgRNA ribonucleoproteins into HEK 293T-GFP cells. Hence, N-Zn-doped CDs with remarkable photoluminescence properties and high transfection efficiency in the delivery of both CRISPR complexes and mRNA provide a promising platform for developing safe, efficient, and traceable delivery systems for biological research.

Synthesis and characterization of vitamin D3-functionalized carbon dots for CRISPR/Cas9 delivery.

Aim: To develop a novel nanovector for the delivery of genetic fragments and CRISPR/Cas9 systems in particular. Materials & methods: Vitamin D3-functionalized carbon dots (D/CDs) fabricated using one-step microwave-aided methods were characterized by different microscopic and spectroscopic techniques. The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide assay and flow cytometry were employed to determine the cell viability and transfection efficiency. Results: D/CDs transfected CRISPR plasmid in various cell lines with high efficiency while maintaining their remarkable efficacy at high serum concentration and low plasmid doses. They also showed great potential for the green fluorescent protein disruption by delivering two different types of CRISPR/Cas9 systems. Conclusion: Given their high efficiency and safety, D/CDs provide a versatile gene-delivery vector for clinical applications.

Nanotechnology against COVID-19: Immunization, diagnostic and therapeutic studies.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in early 2020 soon led to the global pandemic of Coronavirus Disease 2019 (COVID-19). Since then, the clinical and scientific communities have been closely collaborating to develop effective strategies for controlling the ongoing pandemic. The game-changing fields of recent years, nanotechnology and nanomedicine have the potential to not only design new approaches, but also to improve existing methods for the fight against COVID-19. Nanomaterials can be used in the development of highly efficient, reusable personal protective equipment, and antiviral nano-coatings in public settings could prevent the spread of SARS-CoV-2. Smart nanocarriers have accelerated the design of several therapeutic, prophylactic, or immune-mediated approaches against COVID-19. Some nanovaccines have even entered Phase IΙ/IIΙ clinical trials. Several rapid and cost-effective COVID-19 diagnostic techniques have also been devised based on nanobiosensors, lab-on-a-chip systems, or nanopore technology. Here, we provide an overview of the emerging role of nanotechnology in the prevention, diagnosis, and treatment of COVID-19.

Polyethylenimine-Functionalized Carbon Dots for Delivery of CRISPR/Cas9 Complexes.

Carbon dots (CDs) have become the focus of many studies due to their outstanding optical properties and good biocompatibility. We investigated their potential application to produce a smart and highly efficient yet nontoxic nanovector for gene delivery. This was achieved by conjugating PEI1.8k-functionalized CDs (synthesized by one-step microwave-assisted pyrolysis) with arginine-disulfide linkers to produce CD-PEI1.8k-Arg nanoparticles. This nanovector could deliver p-CRISPR (9.3 kb) into different types of cell lines with higher efficiency compared to native PEI1.8k or PEI25k. CD-PEI1.8k-Arg also maintained its outstanding transfection efficiency at a high serum concentration and low p-CRISPR dose, compared to PEI25k, which was ineffective under those conditions. Additionally, CD-PEI1.8k-Arg could knock out the GFP gene with great efficiency by delivering the required components of CRISPR/Cas9, including a plasmid encoding Cas9, sgRNA targeting GFP, and Cas9/sgRNA ribonucleoproteins (RNPs) into the HEK 293T-GFP cells. Moreover, the nanoparticles showed potential for the local delivery of p-CRISPR into brain tissue. The remarkable properties of CD-PEI1.8k-Arg could enable the development of a safe, highly efficient gene-delivery nanovector for the treatment of various diseases in the near future.