Enhanced Cytosolic Delivery and Release of CRISPR/Cas9 by Black Phosphorus Nanosheets for Genome Editing

A biodegradable two‐dimensional (2D) delivery platform based on loading black phosphorus nanosheets (BPs) with Cas9 ribonucleoprotein engineered with three nuclear localization signals (NLSs) at C terminus (Cas9N3) is successfully established. The Cas9N3‐BPs enter cells effectively via membrane penetration and endocytosis pathways, followed by a BPs biodegradation‐associated endosomal escape and cytosolic releases of the loaded Cas9N3 complexes. The Cas9N3‐BPs thus provide efficient genome editing and gene silencing in vitro and in vivo at a relatively low dose as compared with other nanoparticle‐based delivery platforms. This biodegradable 2D delivery platform offers a versatile cytosolic delivery approach for CRISPR/Cas9 ribonucleoprotein and other bioactive macromolecules for biomedical applications.     

CRISPR‐Cas: From the Bacterial Adaptive Immune System to a Versatile Tool for Genome Engineering

The field of biology has been revolutionized by the recent advancement of an adaptive bacterial immune system as a universal genome engineering tool. Bacteria and archaea use repetitive genomic elements termed clustered regularly interspaced short palindromic repeats (CRISPR) in combination with an RNA‐guided nuclease (CRISPR‐associated nuclease: Cas) to target and destroy invading DNA. By choosing the appropriate sequence of the guide RNA, this two‐component system can be used to efficiently modify, target, and edit genomic loci of interest in plants, insects, fungi, mammalian cells, and whole organisms. This has opened up new frontiers in genome engineering, including the potential to treat or cure human genetic disorders. Now the potential risks as well as the ethical, social, and legal implications of this powerful new technique move into the limelight.     

Thermo‐triggered Release of CRISPR‐Cas9 System by Lipid‐Encapsulated Gold Nanoparticles for Tumor Therapy

CRISPR/Cas9 system is a powerful toolbox for gene editing. However, the low delivery efficiency is still a big hurdle impeding its applications. Herein, we report a strategy to deliver Cas9‐sgPlk‐1 plasmids (CP) by a multifunctional vehicle for tumor therapy. We condensed CPs on TAT peptide‐modified Au nanoparticles (AuNPs/CP, ACP) via electrostatic interactions, and coated lipids (DOTAP, DOPE, cholesterol, PEG2000‐DSPE) on the ACP to form lipid‐encapsulated, AuNPs‐condensed CP (LACP). LACP can enter tumor cells and release CP into the cytosol by laser‐triggered thermo‐effects of the AuNPs; the CP can enter nuclei by TAT guidance, enabling effective knock‐outs of target gene (Plk‐1) of tumor (melanoma) and inhibition of the tumor both in vitro and in vivo. This AuNPs‐condensed, lipid‐encapsulated, and laser‐controlled delivery system provides a versatile method for high efficiency CRISPR/Cas9 delivery and targeted gene editing for treatment of a wide spectrum of diseases.     

CRISPR/Cas9-Mediated Genome Editing in Comfrey (Symphytum officinale) Hairy Roots Results in the Complete Eradication of Pyrrolizidine Alkaloids

Comfrey (Symphytum officinale) is a medicinal plant with anti-inflammatory, analgesic, and proliferative properties. However, its pharmaceutical application is hampered by the co-occurrence of toxic pyrrolizidine alkaloids (PAs) in its tissues. Using a CRISPR/Cas9-based approach, we introduced detrimental mutations into the hss gene encoding homospermidine synthase (HSS), the first pathway-specific enzyme of PA biosynthesis. The resulting hairy root (HR) lines were analyzed for the type of gene-editing effect that they exhibited and for their homospermidine and PA content. Inactivation of only one of the two hss alleles resulted in HRs with significantly reduced levels of homospermidine and PAs, whereas no alkaloids were detectable in HRs with two inactivated hss alleles. PAs were detectable once again after the HSS-deficient HRs were fed homospermidine confirming that the inability of these roots to produce PAs was only attributable to the inactivated HSS and not to any unidentified off-target effect of the CRISPR/Cas9 approach. Further analyses showed that PA-free HRs possessed, at least in traces, detectable amounts of homospermidine, and that the PA patterns of manipulated HRs were different from those of control lines. These observations are discussed with regard to the potential use of such a CRISPR/Cas9-mediated approach for the economical exploitation of in vitro systems in a medicinal plant and for further studies of PA biosynthesis in non-model plants.     

Active Intracellular Delivery of a Cas9/sgRNA Complex Using Ultrasound‐Propelled Nanomotors

Direct and rapid intracellular delivery of a functional Cas9/sgRNA complex using ultrasound‐powered nanomotors is reported. The Cas9/sgRNA complex is loaded onto the nanomotor surface through a reversible disulfide linkage. A 5 min ultrasound treatment enables the Cas9/sgRNA‐loaded nanomotors to directly penetrate through the plasma membrane of GFP‐expressing B16F10 cells. The Cas9/sgRNA is released inside the cells to achieve highly effective GFP gene knockout. The acoustic Cas9/sgRNA‐loaded nanomotors display more than 80 % GFP knockout within 2 h of cell incubation compared to 30 % knockout using static nanowires. More impressively, the nanomotors enable highly efficient knockout with just 0.6 nm of the Cas9/sgRNA complex. This nanomotor‐based intracellular delivery method thus offers an attractive route to overcome physiological barriers for intracellular delivery of functional proteins and RNAs, thus indicating considerable promise for highly efficient therapeutic applications.     

Dual‐Pore Mesoporous Carbon@Silica Composite Core–Shell Nanospheres for Multidrug Delivery

Monodispersed mesoporous phenolic polymer nanospheres with uniform diameters were prepared and used as the core for the further growth of core–shell mesoporous nanorattles. The hierarchical mesoporous nanospheres have a uniform diameter of 200 nm and dual‐ordered mesopores of 3.1 and 5.8 nm. The hierarchical mesostructure and amphiphilicity of the hydrophobic carbon cores and hydrophilic silica shells lead to distinct benefits in multidrug combination therapy with cisplatin and paclitaxel for the treatment of human ovarian cancer, even drug‐resistant strains.     

Tumour‐Targeted Drug Delivery with Mannose‐Functionalized Nanoparticles Self‐Assembled from Amphiphilic β‐Cyclodextrins

Multivalent mannose‐functionalized nanoparticles self‐assembled from amphiphilic β‐cyclodextrins (β‐CDs) facilitate the targeted delivery of anticancer drugs to specific cancer cells. Doxorubicin (DOX)‐loaded nanoparticles equipped with multivalent mannose target units were efficiently taken up via receptor‐mediated endocytosis by MDA‐MB‐231 breast cancer cells that overexpress the mannose receptor. Upon entering the cell, the intracellular pH causes the release of DOX, which triggers apoptosis. Targeting by multivalent mannose significantly improved the capability of DOX‐loaded nanoparticles to inhibit the growth of MDA‐MB‐231 cancer cells with minimal side effects in vivo. This targeted and controlled drug delivery system holds promise as a nanotherapeutic for cancer treatment.     

Self‐Assembled Micelles Based on PEG‐Polypeptide Hybrid Copolymers for Drug Delivery

A series of amphiphilic poly(L ‐leucine)‐block‐poly(ethylene glycol)‐block‐poly(L ‐leucine) (PLL‐PEG‐PLL) hybrid triblock copolymers have been synthesized. All the blocks in this system have good biocompatibility and low toxicity. The PLL‐PEG‐PLL copolymers could self‐assemble into micelles with PLL blocks as the hydrophobic core and PEG blocks as the hydrophilic shell, which were characterized by FT‐IR, ¹H NMR, and transmission electron microscopy analysis. The critical micellar concentration of the copolymer was 95.0 mg · L⁻¹. The circular dichroism spectrum shows that the PLL segments adopt a unique α‐helical conformation, which is found to play an important role in controlling the drug release rate. The drug release could be effectively sustained by encapsulation in the micelles. The copolymers may have potential applications in drug delivery.

     

Optical Control of a CRISPR/Cas9 System for Gene Editing by Using Photolabile crRNA

Currently CRISPR/Cas9 is a widely used efficient tool for gene editing. Precise control over the CRISPR/Cas9 system with high temporal and spatial resolution is essential for studying gene regulation and editing. Here, we synthesized a novel light-controlled crRNA by coupling vitamin E and a photolabile linker at the 5′ terminus to inactivate the CRISPR/Cas9 system. The vitamin E modification did not affect ribonucleoprotein (RNP) formation of Cas9/crRNA/tracrRNA complexes but did inhibit the association of RNP with the target DNA. Upon light irradiation, vitamin E-caged crRNA was successfully activated to achieve light-induced genome editing of vascular endothelial cell-growth factor A (VEGFA) in human cells through a T7E1 assay and Sanger sequencing as well as gene knockdown of EGFP expression in EGFP stably expressing cells. This new caging strategy for crRNA could provide new methods for spatiotemporal photoregulation of CRISPR/Cas9-mediated gene editing.     

An RNA‐Guided Cas9 Nickase‐Based Method for Universal Isothermal DNA Amplification

We have developed an ingenious method, termed Cas9 nickase‐based amplification reaction (Cas9nAR), to amplify a target fragment from genomic DNA at a constant temperature of 37 °C. Cas9nAR employs a sgRNA:Cas9n complex with a single‐strand nicking property, a strand‐displacing DNA polymerase, and two primers bearing the cleavage sequence of Cas9n, to promote cycles of DNA replication through priming, extension, nicking, and displacement reaction steps. Cas9nAR exhibits a zeptomolar limit of detection (2 copies in 20 μL of reaction system) within 60 min and a single‐base discrimination capability. More importantly, the underlying principle of Cas9nAR offers simplicity in primer design and universality in application. Considering the superior sensitivity and specificity, as well as the simple‐to‐implement, rapid, and isothermal features, Cas9nAR holds great potential to become a routine assay for the quantitative detection of nucleic acids in basic and applied studies.     

Microfluidic Cell Deformability Assay for Rapid and Efficient Kinase Screening with the CRISPR‐Cas9 System

Herein we report a CRISPR‐Cas9‐mediated loss‐of‐function kinase screen for cancer cell deformability and invasive potential in a high‐throughput microfluidic chip. In this microfluidic cell separation platform, flexible cells with high deformability and metastatic propensity flowed out, while stiff cells remained trapped. Through deep sequencing, we found that loss of certain kinases resulted in cells becoming more deformable and invasive. High‐ranking candidates identified included well‐reported tumor suppressor kinases, such as chk2, IKK‐α, p38 MAPKs, and DAPK2. A high‐ranking candidate STK4 was chosen for functional validation and identified to play an important role in the regulation of cell deformability and tumor suppression. Collectively, we have demonstrated that CRISPR‐based on‐chip mechanical screening is a potentially powerful strategy to facilitate systematic genetic analyses.     

Metal–Organic‐Framework‐Templated Polyelectrolyte Nanocapsules for the Encapsulation and Delivery of Small‐Molecule–Polymer Conjugates

Herein, we report a strategy for exploiting nanoscale metal–organic frameworks (nano‐MOFs) as templates for the layer‐by‐layer (LbL) assembly of polyelectrolytes. Because small‐molecule drugs or imaging agents cannot be efficiently encapsulated by polyelectrolyte nanocapsules, we investigated two promising and biocompatible polymers (comb‐shaped polyethylene glycol (PEG) and hyperbranched polyglycerol‐based PEG) for the conjugation of model drugs and imaging agents, which were then encapsulated inside the nano‐MOF‐templated nanocapsules. Furthermore, we also systemically explored the release kinetics of the encapsulated conjugates, and examined how the encapsulation and/or release processes could be controlled by varying the composition and architecture of the polymers. We envision that our nano‐MOFs‐templated nanocapsules, through combining with small‐molecule–polymer conjugates, will represent a new type of delivery system that could open up new opportunities for biomedical applications.     

Dual Stimuli‐Responsive Self‐Assembled Supramolecular Nanoparticles

Supramolecular nanoparticles (SNPs) encompass multiple copies of different building blocks brought together by specific noncovalent interactions. The inherently multivalent nature of these systems allows control of their size as well as their assembly and disassembly, thus promising potential as biomedical delivery vehicles. Here, dual responsive SNPs have been based on the ternary host–guest complexation between cucurbit[8]uril (CB[8]), a methyl viologen (MV) polymer, and mono‐ and multivalent azobenzene (Azo) functionalized molecules. UV switching of the Azo groups led to fast disruption of the ternary complexes, but to a relatively slow disintegration of the SNPs. Alternating UV and Vis photoisomerization of the Azo groups led to fully reversible SNP disassembly and reassembly. SNPs were only formed with the Azo moieties in the trans and the MV units in the oxidized states, respectively, thus constituting a supramolecular AND logic gate.     

A Photolabile Semiconducting Polymer Nanotransducer for Near‐Infrared Regulation of CRISPR/Cas9 Gene Editing

Noninvasive regulation of CRISPR/Cas9 gene editing is conducive to understanding of gene function and development of gene therapy; however, it remains challenging. Herein, a photolabile semiconducting polymer nanotransducer (pSPN) is synthesized to act as the gene vector to deliver CRISPR/Cas9 plasmids into cells and also as the photoregulator to remotely activate gene editing. pSPN comprises a ¹O₂‐generating backbone grafted with polyethylenimine brushes through ¹O₂‐cleavable linkers. NIR photoirradiation spontaneously triggers the cleavage of gene vectors from pSPN, resulting in the release of CRISPR/Cas9 plasmids and subsequently initiating gene editing. This system affords 15‐ and 1.8‐fold enhancement in repaired gene expression relative to the nonirradiated controls in living cells and mice, respectively. As this approach does not require any specific modifications on biomolecular components, pSPN represents the first generic nanotransducer for in vivo regulation of CRISPR/Cas9 gene editing.     

Micellar Nanocarriers Assembled from Doxorubicin‐Conjugated Amphiphilic Macromolecules (DOX–AM)

Amphiphilic macromolecules (AMs) have unique branched hydrophobic domains attached to linear PEG chains. AMs self‐assemble in aqueous solution to form micelles that are hydrolytically stable in physiological conditions (37 °C, pH 7.4) over 4 weeks. Evidence of AM biodegradability was demonstrated by complete AM degradation after 6 d in the presence of lipase. Doxorubicin (DOX) was chemically conjugated to AMs via a hydrazone linker to form DOX–AM conjugates that self‐assembled into micelles in aqueous solution. The conjugates were compared with DOX‐loaded AM micelles (i.e., physically loaded DOX) on DOX content, micellar sizes and in vitro cytotoxicity. Physically encapsulated DOX loading was higher (12 wt.‐%) than chemically bound DOX (6 wt.‐%), and micellar sizes of DOX‐loaded AMs (≈16 nm) were smaller than DOX–AMs (≈30 nm). In vitro DOX release from DOX–AM conjugates was faster at pH 5.0 (100%) compared to pH 7.4 (78%) after 48 h, 37 °C. Compared to free DOX and physically encapsulated DOX, chemically bound DOX had significantly higher cytotoxicity at 10^?7 M DOX dose against human hepatocellular carcinoma cells after 72 h. Overall, DOX–AM micelles showed promising characteristics as stable, biodegradable DOX nanocarriers.

     

Self‐Assembled PAA‐Based Nanoparticles as Potential Gene and Protein Delivery Systems

A series of nanoparticles is prepared via layer‐by‐layer assembly of oppositely charged, synthetic biocompatible polyamidoamine polymers as potential carriers. Particle size, surface charge and internal chain mobility are quantified as a function of the polymer type and number of layers. The effect of addition of surfactant is examined to simulate the effects of nanoparticle dissolution. The cyctotoxicity of these particles (in epithelia and murine cell lines) are orders of magnitude lower than polyethyleneimine controls. Stable nanoparticles may be prepared from mixtures of strongly, oppositely charged polymers, but less successfully from weakly charged polymers, and, given their acceptable toxicity characteristics, such modularly designed constructs show promise for drug and gene delivery.

     

Highly Efficient Chirality Transfer from Diamines Encapsulated within a Self‐Assembled Calixarene–Salen Host

A calix[4]arene host equipped with two bis‐[Zn(salphen)] complexes self‐assembles into a capsular complex in the presence of a chiral diamine guest with an unexpected 2:1 ratio between the host and the guest. Effective chirality transfer from the diamine to the calix–salen hybrid host is observed by circular dichroism (CD) spectroscopy, and a high stability constant K_2,1 of 1.59×10¹¹ M ^?2 for the assembled host–guest ensemble has been determined with a substantial cooperativity factor α of 6.4. Density functional calculations are used to investigate the origin of the stability of the host–guest system and the experimental CD spectrum compared with those calculated for both possible diastereoisomers showing that the M,M isomer is the one that is preferentially formed. The current system holds promise for the chirality determination of diamines, as evidenced by the investigated substrate scope and the linear relationship between the ee of the diamine and the amplitude of the observed Cotton effects.