Non‐Viral CRISPR/Cas Gene Editing In Vitro and In Vivo Enabled by Synthetic Nanoparticle Co‐Delivery of Cas9 mRNA and sgRNA

CRISPR/Cas is a revolutionary gene editing technology with wide‐ranging utility. ¹ The safe, non‐viral delivery of CRISPR/Cas components would greatly improve future therapeutic utility. ^1e We report the synthesis and development of zwitterionic amino lipids (ZALs) that are uniquely able to (co)deliver long RNAs including Cas9 mRNA and sgRNAs. ZAL nanoparticle (ZNP) delivery of low sgRNA doses (15 nm ) reduces protein expression by >90 % in cells. In contrast to transient therapies (such as RNAi), we show that ZNP delivery of sgRNA enables permanent DNA editing with an indefinitely sustained 95 % decrease in protein expression. ZNP delivery of mRNA results in high protein expression at low doses in vitro (<600 pM) and in vivo (1 mg kg⁻¹). Intravenous co‐delivery of Cas9 mRNA and sgLoxP induced expression of floxed tdTomato in the liver, kidneys, and lungs of engineered mice. ZNPs provide a chemical guide for rational design of long RNA carriers, and represent a promising step towards improving the safety and utility of gene editing.     

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.     

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.     

Spatiotemporal Control of CRISPR/Cas9 Function in Cells and Zebrafish using Light‐Activated Guide RNA

We developed a new method for the conditional regulation of CRISPR/Cas9 activity in mammalian cells and zebrafish embryos using photochemically activated, caged guide RNAs (gRNAs). Caged gRNAs are generated by substituting four nucleobases evenly distributed throughout the 5′‐protospacer region with caged nucleobases during synthesis. Caging confers complete suppression of gRNA:dsDNA‐target hybridization and rapid restoration of CRISPR/Cas9 function upon optical activation. This tool offers simplicity and complete programmability in design, high spatiotemporal specificity in cells and zebrafish embryos, excellent off‐to‐on switching, and stability by preserving the ability to form Cas9:gRNA ribonucleoprotein complexes. Caged gRNAs are novel tools for the conditional control of gene editing, thereby enabling the investigation of spatiotemporally complex physiological events by obtaining a better understanding of dynamic gene regulation.     

Bioorthogonal Reaction-Mediated Tumor-Selective Delivery of CRISPR/Cas9 System for Dual-Targeted Cancer Immunotherapy

CRISPR system-assisted immunotherapy is an attractive option in cancer therapy. However, its efficacy is still less than expected due to the limitations in delivering the CRISPR system to target cancer cells. Here, we report a new CRISPR/Cas9 tumor-targeting delivery strategy based on bioorthogonal reactions for dual-targeted cancer immunotherapy. First, selective in vivo metabolic labeling of cancer and activation of the cGAS-STING pathway was achieved simultaneously through tumor microenvironment (TME)-biodegradable hollow manganese dioxide (H-MnO₂) nano-platform. Subsequently, CRISPR/Cas9 system-loaded liposome was accumulated within the modified tumor tissue through in vivo click chemistry, resulting in the loss of protein tyrosine phosphatase N2 (PTPN2) and further sensitizing tumors to immunotherapy. Overall, our strategy provides a modular platform for precise gene editing in vivo and exhibits potent antitumor response by boosting innate and adaptive antitumor immunity.     

A Conformational Restriction Strategy for the Control of CRISPR/Cas Gene Editing with Photoactivatable Guide RNAs**

The CRISPR/Cas system is one of the most powerful tools for gene editing. However, approaches for precise control of genome editing and regulatory events are still desirable. Here, we report the spatiotemporal and efficient control of CRISPR/Cas9- and Cas12a-mediated editing with conformationally restricted guide RNAs (gRNAs). This approach relied on only two or three pre-installed photo-labile substituents followed by an intramolecular cyclization, representing a robust synthetic method in comparison to the heavily modified linear gRNAs that often require extensive screening and time-consuming optimization. This tactic could direct the precise cleavage of the genes encoding green fluorescent protein (GFP) and the vascular endothelial growth factor A (VEGFA) protein within a predefined cutting region without notable editing leakage in live cells. We also achieved light-mediated myostatin (MSTN) gene editing in embryos, wherein a new bow-knot-type gRNA was constructed with excellent OFF/ON switch efficiency. Overall, our work provides a significant new strategy in CRISPR/Cas editing with modified circular gRNAs to precisely manipulate where and when genes are edited.     

Spatiotemporal Control of CRISPR/Cas9 Function in Cells and Zebrafish using Light-Activated Guide RNA

We developed a new method for the conditional regulation of CRISPR/Cas9 activity in mammalian cells and zebrafish embryos using photochemically activated, caged guide RNAs (gRNAs). Caged gRNAs are generated by substituting four nucleobases evenly distributed throughout the 5′-protospacer region with caged nucleobases during synthesis. Caging confers complete suppression of gRNA:dsDNA-target hybridization and rapid restoration of CRISPR/Cas9 function upon optical activation. This tool offers simplicity and complete programmability in design, high spatiotemporal specificity in cells and zebrafish embryos, excellent off-to-on switching, and stability by preserving the ability to form Cas9:gRNA ribonucleoprotein complexes. Caged gRNAs are novel tools for the conditional control of gene editing, thereby enabling the investigation of spatiotemporally complex physiological events by obtaining a better understanding of dynamic gene regulation.     

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.     

Engineering CRISPR/Cas-based nanosystems for therapeutics,diagnosis and bioimaging

CRISPR/Cas system has been utilized to rationally manipulate intracellular genes, and it has been engineered as versatile and efficient gene editing tools with precise site-specificity and excellent targeting ability for therapeutics, diagnostics, and bioimaging. Here, the evolution and application of CRISPR/Cas systems were sketched chronologically. Landmark works were exemplified to illustrate the design principles of CRISPR/Cas systems. Furthermore, the delivery vectors of CRISPR/Cas system especially DNA nanomaterials-based vectors were categorized and illuminated. DNA nanomaterials are suitable for CRISPR/Cas system delivery via base pairing due to its sequence programmability and biocompatibility. Then the applications of CRISPR/Cas in diagnosis and genomic imaging were highlighted. At the end of the review, the challenges and opportunities of CRISPR/Cas systems were deeply discussed. We envision that the grant advances on CRISPR/Cas systems will promote the development of interdisciplinary fields in chemistry, biology and medicine.     

Self‐Assembled DNA Nanoclews for the Efficient Delivery of CRISPR–Cas9 for Genome Editing

CRISPR–Cas9 represents a promising platform for genome editing, yet means for its safe and efficient delivery remain to be fully realized. A novel vehicle that simultaneously delivers the Cas9 protein and single guide RNA (sgRNA) is based on DNA nanoclews, yarn‐like DNA nanoparticles that are synthesized by rolling circle amplification. The biologically inspired vehicles were efficiently loaded with Cas9/sgRNA complexes and delivered the complexes to the nuclei of human cells, thus enabling targeted gene disruption while maintaining cell viability. Editing was most efficient when the DNA nanoclew sequence and the sgRNA guide sequence were partially complementary, offering a design rule for enhancing delivery. Overall, this strategy provides a versatile method that could be adapted for delivering other DNA‐binding proteins or functional nucleic acids.     

A DNA Origami-Based Gene Editing System for Efficient Gene Therapy in Vivo

DNA nanostructures have played an important role in the development of novel drug delivery systems. Herein, we report a DNA origami-based CRISPR/Cas9 gene editing system for efficient gene therapy in vivo. In our design, a PAM-rich region precisely organized on the surface of DNA origami can easily recruit and load sgRNA/Cas9 complex by PAM-guided assembly and pre-designed DNA/RNA hybridization. After loading the sgRNA/Cas9 complex, the DNA origami can be further rolled up by the locking strands with a disulfide bond. With the incorporation of DNA aptamer and influenza hemagglutinin (HA) peptide, the cargo-loaded DNA origami can realize the targeted delivery and effective endosomal escape. After reduction by GSH, the opened DNA origami can release the sgRNA/Cas9 complex by RNase H cleavage to achieve a pronounced gene editing of a tumor-associated gene for gene therapy in vivo. This rationally developed DNA origami-based gene editing system presents a new avenue for the development of gene therapy.     

Photocontrol of CRISPR/Cas9 function by site-specific chemical modification of guide RNA

The function of CRISPR/Cas9 can be conditionally controlled by the rational engineering of guide RNA (gRNA) to target the gene of choice for precise manipulation of the genome. Particularly, chemically modified gRNA that can be activated by using specific stimuli provides a unique tool to expand the versatility of conditional control. Herein, unlike previous engineering of gRNA that generally focused on the RNA part only but neglected RNA–protein interactions, we aimed at the interactive sites between 2′-OH of ribose in the seed region of gRNA and the Cas9 protein and identified that chemical modifications at specific sites could be utilized to regulate the Cas9 activity. By introducing a photolabile group at these specific sites, we achieved optical control of Cas9 activity without disrupting the Watson–Crick base pairing. We further examined our design through CRISPR-mediated gene activation and nuclease cleavage in living cells and successfully manipulated the gene expression by using light irradiation. Our site-specific modification strategy exhibited a highly efficient and dynamic optical response and presented a new perspective for manipulating gRNA based on the RNA–protein interaction rather than the structure of RNA itself. In addition, these specific sites could also be potentially utilized for modification of other stimuli-responsive groups, which would further enrich the toolbox for conditional control of CRISPR/Cas9 function.

The CRISPR/Cas9 function is optically controlled in living cells by the site-specifically caged guide RNA based on the RNA–protein interaction.     

Proteomic and Bioinformatic Investigation of Altered Pathways in Neuroglobin-Deficient Breast Cancer Cells

Neuroglobin (NGB) is a myoglobin-like monomeric globin that is involved in several processes, displaying a pivotal redox-dependent protective role in neuronal and extra-neuronal cells. NGB remarkably exerts its function upon upregulation by NGB inducers, such as 17β-estradiol (E2) and H₂O₂. However, the molecular bases of NGB’s functions remain undefined, mainly in non-neuronal cancer cells. Human MCF-7 breast cancer cells with a knocked-out (KO) NGB gene obtained using CRISPR/Cas9 technology were analyzed using shotgun label-free quantitative proteomics in comparison with control cells. The differential proteomics experiments were also performed after treatment with E2, H₂O₂, and E2 + H₂O₂. All the runs acquired using liquid chromatography–tandem mass spectrometry were elaborated within the same MaxQuant analysis, leading to the quantification of 1872 proteins in the global proteomic dataset. Then, a differentially regulated protein dataset was obtained for each specific treatment. After the proteomic study, multiple bioinformatics analyses were performed to highlight unbalanced pathways and processes. Here, we report the proteomic and bioinformatic investigations concerning the effects on cellular processes of NGB deficiency and cell treatments. Globally, the main processes that were affected were related to the response to stress, cytoskeleton dynamics, apoptosis, and mitochondria-driven pathways.     

Highly efficient and safe genome editing by CRISPR-Cas12a using CRISPR RNA with a ribosyl-2′-O-methylated uridinylate-rich 3′-overhang in mouse zygotes

The CRISPR-Cas12a system has been developed to harness highly specific genome editing in eukaryotic cells. Given the relatively small sizes of Cas12a genes, the system has been suggested to be most applicable to gene therapy using AAV vector delivery. Previously, we reported that a U-rich crRNA enabled highly efficient genome editing by the CRISPR-Cas12a system in eukaryotic cells. In this study, we introduced methoxyl modifications at C2 in riboses in the U-rich 3′-overhang of crRNA. When mixed with Cas12a effector proteins, the ribosyl-2′-O-methylated (2-OM) U-rich crRNA enabled improvement of dsDNA digestibility. Moreover, the chemically modified U-rich crRNA achieved very safe and highly specific genome editing in murine zygotes. The engineered CRISPR-Cas12a system is expected to facilitate the generation of various animal models. Moreover, the engineered crRNA was evaluated to further improve a CRISPR genome editing toolset.Subject terms: Gene targeting, Genetic engineering     

A Smart DNAzyme–MnO2 Nanosystem for Efficient Gene Silencing

DNAzymes hold promise for gene‐silencing therapy, but the lack of sufficient cofactors in the cell cytoplasm, poor membrane permeability, and poor biostability have limited the use of DNAzymes in therapeutics. We report a DNAzyme–MnO₂ nanosystem for gene‐silencing therapy. MnO₂ nanosheets adsorb chlorin e6‐labelled DNAzymes (Ce6), protect them from enzymatic digestion, and efficiently deliver them into cells. The nanosystem can also inhibit ¹O₂ generation by Ce6 in the circulatory system. In the presence of intracellular glutathione (GSH), MnO₂ is reduced to Mn²⁺ ions, which serve as cofactors of 10–23 DNAzyme for gene silencing. The release of Ce6 generates ¹O₂ for more efficient photodynamic therapy. The Mn²⁺ ions also enhance magnetic resonance contrast, providing GSH‐activated magnetic resonance imaging (MRI) of tumor cells. The integration of fluorescence recovery and MRI activation provides fluorescence/MRI bimodality for monitoring the delivery of DNAzymes.