Advancing CRISPR/Cas9 Delivery: Supramolecular Nanoparticles and Nanosubstrate-Mediated Strategies
The clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9) system has revolutionized gene editing, transitioning from its natural function as an RNA-guided genetic adaptive immune system in prokaryotes to a robust site-specific gene editing method. This technology holds immense potential for treating various genetic diseases. However, effective translation into patient care faces several challenges, including minimizing off-target editing effects, ensuring proper delivery of gene-editing reagents into targeted cells, and optimizing editing efficiency. Current CRISPR/Cas9 delivery methods rely heavily on viral vectors, which present limitations in packaging capacity and potential safety concerns.
To overcome these limitations, researchers are exploring non-viral delivery methods. This article delves into a novel approach utilizing supramolecular nanoparticles (SMNPs) and a supramolecular nanosubstrate-mediated delivery (SNMD) strategy to enhance the efficiency of Cas9 ribonucleoprotein (RNP) delivery into target cells.
Components of the CRISPR/Cas9 System
The CRISPR/Cas9 system comprises two essential components: the Cas9 endonuclease and a short, single-guide RNA (sgRNA). These components form a Cas9•sgRNA ribonucleoprotein (RNP) complex. The RNP complex identifies and cleaves targeted DNA in the genome through a simple base-pairing mechanism, resulting in a double-strand break (DSB) at the specified location.
Following the creation of a DSB, endogenous DNA repair mechanisms, such as the non-homologous end joining (NHEJ) pathway, are activated. NHEJ often leads to insertions or deletions (indels), which can disrupt gene function. CRISPR/Cas9-mediated gene disruption is frequently used to knock down genes in cell lines and animal models. Alternatively, complete gene knockout can be achieved through CRISPR/Cas9-mediated gene deletion. This involves using a pair of sgRNAs targeting two ends of a specific gene in the presence of Cas9 protein to induce two DSBs at the targeted sites. Subsequent NHEJ-based DNA repair enables precise removal of the gene.
Overcoming Delivery Challenges with Non-Viral Vectors
Developing highly efficient intracellular delivery methods remains a major obstacle to the widespread application of CRISPR/Cas9 genome editing strategies both in vitro and in vivo. While physical strategies like microinjection and electroporation can deliver CRISPR/Cas9 reagents intracellularly by disrupting cellular membranes, they can also decrease cell viability and potentially cause premature cell differentiation, posing challenges for clinical applications. Viral-based approaches utilizing lentivirus (LV), adenovirus (AV), and adeno-associated virus (AAV)-derived vectors are commonly used for delivering gene-editing machinery because of their ease of construction, good production titer, and high transgene expression. However, AAVs have limited packaging capacity, and viral delivery raises concerns about insertional mutagenesis and immunogenicity.
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Significant effort has been devoted to exploring the design and application of non-viral vectors, including lipids, polymers, and nanoparticles, for delivering CRISPR/Cas9 reagents. The CRISPR/Cas9 system can be introduced as Cas9 DNA plasmid, Cas9 mRNA, or RNP. Direct delivery of RNP is the most straightforward strategy compared to non-viral methods of delivering Cas9 DNA plasmid and Cas9 mRNA. Non-viral delivery of RNP offers the advantage of rapid gene editing, bypassing the gene transcription and/or translation process. The transient gene editing also reduces off-target activity and avoids the risk of integrating CRISPR genes into the host genome.
Supramolecular Nanoparticles (SMNPs) for Efficient RNP Delivery
This research focuses on utilizing supramolecular nanoparticles (SMNPs) for non-viral delivery of CRISPR/Cas9 components. SMNPs are created via mixing three common molecular building blocks: adamantane-grafted polyamidoamine dendrimer (Ad-PAMAM), adamantane-grafted poly(ethylene glycol) (Ad-PEG), and β-cyclodextrin-grafted polyethyleneimine (CD-PEI). By leveraging multivalent molecular interactions between β-cyclodextrin (CD) and adamantane (Ad) motifs, modular control over the surface chemistry, sizes, and payloads of the nanoparticles can be easily achieved, offering a versatile platform for various diagnostic imaging and therapeutic applications.
Supramolecular Nanosubstrate-Mediated Delivery (SNMD) Strategy
Inspired by existing substrate-mediated delivery approaches, a supramolecular nanosubstrate-mediated delivery (SNMD) strategy was developed to improve the delivery efficiency of SMNP vectors. This strategy utilizes Ad-grafted silicon nanowire substrates (Ad-SiNWS) for dynamic assembly and local enrichment of SMNPs. As cells settle onto the substrates, their membranes form intimate contacts with the nanowires, creating transient defects that facilitate intracellular uptake of SMNPs. Furthermore, multiple rounds of delivery can be performed on the same batch of cells by sequentially adding SMNPs without regenerating or reloading the substrates after each use.
The study demonstrates that SNMD facilitates the delivery of Cas9•sgRNA RNPs into cells attached to Ad-SiNWS, enabling both CRISPR/Cas9-mediated gene disruption and deletion. The SMNPs were prepared via a self-assembly approach.
Optimizing SMNP Formulation for Enhanced Cellular Uptake
To obtain an optimized formulation of RNP⊂SMNPs, an enhanced green fluorescent protein (EGFP)-labeled Cas9 protein and sgRNA complex (EGFP-labeled RNP) was employed as a cargo in a quantitative fluorescent imaging study. Through small-scale combinatorial screening, an optimal formulation of EGFP-labeled RNP⊂SMNPs that yielded the highest cellular uptake was identified and subjected to time-dependent imaging studies using the U87 cell line as a model system. The optimal formulation identified was subsequently used to deliver a Cas9 protein and green fluorescent protein (GFP) gene-targeting sgRNA complex (Cas9•sgRNA-GFP, i.e., RNP-GFP) into a GFP-expressing U87 cell line (GFP-U87). The RNP-GFP is designed to disrupt GFP gene expression via the introduction of a frameshift mutation resulting from the formation of a CRISPR/Cas9-mediated DSB followed by DNA repair via NHEJ.
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Application: Deletion of Exons in the Dystrophin Gene
Finally, the researchers demonstrated the utility of SNMD for the CRISPR/Cas9-mediated large deletion of exons 45-55 (708 kb) of the dystrophin gene in human cells, including cardiomyocytes (AC16), induced pluripotent stem cells (iPSCs), and mesenchymal stem cells (MSCs). Approximately 60% of mutations causing Duchenne muscular dystrophy (DMD), a severe inherited genetic muscle wasting disease, occur within exons 45-55 of the dystrophin gene. Effective gene editing at these locations represents an attractive target for gene therapies. In addition, given that approximately 90% of DMD patients suffer from cardiomyopathies, the capability to correct cardiomyocytes directly or stem cell-derived products may offer a solution to a primary cause of morbidity and mortality in this population. It has been demonstrated that CRISPR/Cas9-mediated in-frame deletion of exons 45-55 could produce an internally deleted dystrophin protein, resulting in rescue from the disease phenotype.
In this study, a pair of RNP complexes, i.e., Cas9 protein and intron 44-targeting sgRNA complex (RNP-44) and Cas9 protein and intron 55-targeting sgRNA complex (RNP-55), were encapsulated in separate SMNP vectors to form RNP-44⊂SMNPs and RNP-55⊂SMNPs, respectively. After deletion of exons 45-55 of the dystrophin gene, the 3′ end of intron 44 and the 5′ end of intron 55 join together to create a new chimeric intron via NHEJ.
The SNMD strategy was employed for CRISPR/Cas9-mediated GFP gene disruption by introducing a Cas9 protein and a GFP-targeting sgRNA (RNP-GFP) into a GFP-U87 cell line. The RNP-GFP cargo is designed to disrupt GFP gene expression by establishing a frameshift mutation via the formation of double-strand breaks (DSBs) and subsequent DNA repair via the non-homologous end joining (NHEJ) pathway. The SNMD strategy was also used for CRISPR/Cas9-mediated deletion of exons 45-55 of the dystrophin gene by delivering RNP-44⊂SMNPs and RNP-55⊂SMNPs into human cells, including cardiomyocytes (AC16), induced pluripotent stem cells (iPSCs), and mesenchymal stem cells (MSCs).
Optimizing Cellular Uptake of EGFP-labeled RNP⊂SMNPs
To identify RNP⊂SMNP formulations that achieve optimal cell-uptake performance, SNMD was applied to conduct a three-step optimization process. An EGFP-labeled Cas9 protein (EGFP-Cas9, GenScript, New Jersey) and sgRNA complex (EGFP-labeled RNP) was used to monitor the cellular uptake in U87 cells using fluorescence microscopy-based quantitative analysis. Three batches of EGFP-labeled RNP⊂SMNPs were formulated by systemically modulating i) the weight ratios (wt%) of SMNP to EGFP-labeled RNP (from 100:1 to 100:8), ii) SMNP size (from110 to 200 nm), and iii) the surface coverage of a membrane penetration peptide, TAT (from 2% to 10%).
For cellular-uptake studies, each batch of EGFP-labeled RNP⊂SMNPs containing 1.0 μg of EGFP-Cas9 was added to a well (in a 24-well plate), in which an Ad-SiNWS (1×1 cm2) was immersed with 1.0 mL of Dulbecco’s modified Eagle’s medium (DMEM). Due to the supramolecular assembly process, EGFP-labeled RNP⊂SMNPs were quickly enriched and grafted onto the Ad-SiMWS from the medium. Prior to settling the cells onto the Ad-SiMWS, U87 cells were synchronized in serum-free DMEM overnight (10 h) to the G0/G1 phases of the cell cycle. Thereafter, approximately 1×105 U87 cells were introduced into each well. The delivery efficiency of EGFP-labeled RNP into U87 cells was quantified by fluorescence microscopy 24 h after treatment.
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The researchers first evaluated how the weight ratios of SMNPs to EGFP-labeled RNP affect the intracellular delivery performance. The highest percentage (47%) of EGFP-positive U87 cells was obtained at a ratio of 100:4. Based on this ratio, they then studied the influence of the sizes of RNP⊂SMNPs. By adjusting the mixing ratio of Ad-PAMAM and CD-PEI, they were able to obtain EGFP-labeled RNP⊂SMNPs with varying sizes from 110 to 200 nm as measured by dynamic light scattering (DLS). They found that 120 nm EGFP-labeled RNP⊂SMNPs showed the best cellular uptake, achieving ~60% EGFP-positive cells. TAT-grafted EGFP-labeled RNP⊂SMNPs were prepared with TAT coverage ranging between 2% to 10%. EGFP-labeled RNP⊂SMNPs with 8% TAT exhibited the highest cell-uptake performance up to 75%. Thus, the formulation that yielded the optimal SMNP configuration of 120 nm EGFP-labeled RNP⊂SMNPs with 8% TAT coverage was identified.
Time-Dependent Intracellular Trafficking of EGFP-labeled Cas9
After cellular uptake and dynamic disassembly of EGFP-labeled RNP⊂SMNPs, the EGFP-labeled Cas9 protein is expected to traffic into the targeted nucleus to facilitate gene editing. To characterize the transport of EGFP-labeled RNP into cell nuclei using the SNMD strategy, a time-dependent quantitative imaging study was conducted on individual U87 cells under the optimal delivery conditions. Control conditions, i.e., U87 cells treated with EGFP-labeled RNP⊂SMNPs (without Ad-SiNWS), U87 cells treated with EGFP-labeled RNP (without SMNP vectors), and U87 cells treated with EGFP-labeled RNP encapsulated using the commercial Lipofectamine CRISPRMAX system were tested in parallel to SNMD treated cells. The results highlight the critical role of both functional components of the SNMD strategy (i.e., the SMNP vectors and the Ad-SiNWS). Multiple cellular uptake studies were performed in parallel and terminated at 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0, 6.0, 24, and 48 h after treatment. Single-cell image analyses to quantify EGFP-labeled Cas9 signals in the micrographs, histograms of single-cell EGFP-Cas9 uptake were obtained for the respective times, indicating that the highest cell uptake (92% of EGFP-positive U87 cells) occurred at 3.0 h.
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