DNA Nanomaterials for Delivery of Clustered Regularly Interspaced Short Palindromic Repeats/Cas Systems

The clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein (CRISPR/Cas) system has been exploited as an efficient gene editing tool with precise site specificity and high efficiency; the delivery of the CRISPR/Cas system is critical for the efficacy of therapeutics and remains challenging. DNA nanomaterials are widely utilized as promising delivery carriers for gene agents because of their excellent molecular recognition capabilities, sequence programmability, and biocompatibility. Herein, recent advances in DNA nanomaterials for the delivery of CRISPR/Cas systems are summarized. Based on their construction strategy, DNA nanomaterial‐based carriers are categorized as branched DNA‐based nanostructures or rolling circle amplification (RCA)‐based DNA nanostructures. Representative studies on the design of DNA nanomaterials as CRISPR/Cas system delivery carriers are highlighted. The current challenges and opportunities for the development of DNA nanomaterials for the delivery of CRISPR/Cas systems are also discussed. It is envisioned that with the development of DNA nanotechnology, DNA nanomaterials will open up new possibilities for CRISPR/Cas‐based therapeutics.

molecular recognition. [14]For instance, Li et al. constructed a DNA-polymer nanomaterial via a cascade hybridization chain reaction, which enabled the precise and efficient delivery of siRNA. [15]In particular, their sequence programmability endows DNA nanomaterials with dynamic assembly behaviors, thus facilitating the controlled release of gene agents under the stimuli of a specific endogenous environment. [16]14a] Moreover, functional units can be programmed into DNA nanomaterials to realize desired biological functions, such as targeting units to enhance cellular internalization and reduce toxicity. [17]Yao et al. designed a tumor-targeting DNA nanocomplex containing polyvalent DNAzymes via rolling circle amplification (RCA) to realize a combined gene/chemodynamic therapy. [18]n this review, the recent progress in DNA nanomaterials for the delivery of CRISPR/Cas systems is summarized and discussed.According to the construction strategy, DNA nanomaterial-based carriers are categorized as branched DNA-based nanostructures and RCA-based DNA nanostructures (Figure 1).Representative studies on the design and construction of DNA nanomaterials for delivery of the CRISPR/Cas system are highlighted.Finally, the challenges and prospects of using DNA nanomaterials for the delivery of CRISPR/Cas system are discussed in detail.

Branched DNA-Based Nanostructures
As a typical molecular structure of DNA, branched DNA can be extended as a building block in the desired manner or linked to functional sequences via base pairing to construct branched DNA-based nanostructures. [19]19b,20] Zhu et al. constructed a Y-DNA-based nanostructure for delivery of the CRISPR/Cas9 system to achieve efficient gene editing. [21]NAzyme and sgwere sequentially connected via base pairing at the end of Y-DNA.Subsequently, Y-DNA was assembled on streptavidin, and the Cas9 protein was loaded through the hybridization of sgRNA and Cas9, thus creating a selfassembling CRISPR/Cas9 delivery nanostructure.
Unlike linear DNA, extended branched DNA can be rationally designed to endow branched DNA-based nanostructures with multiple loading sites for gene agents. [22]Ding et al. developed a noncationic DNA-crosslinked nanogel to deliver Cas9/sgRNA ribonucleoprotein (RNP), thereby realizing effective target gene editing. [23]DNA-grafted polycaprolactone (DNA-g-PCL) was prepared via a copper-free click reaction to provide crosslinking sites for nanogel formation.The grafted DNA was designed to be complementary to the sgRNA, allowing Cas9/sgRNA RNP to be successfully loaded onto DNA-g-PCL at a molar ratio of 1:30.Then, the remaining grafted DNA that was not occupied by Cas9/sgRNA RNP was crosslinked by a DNA linker to form the nanogel (Nanogel-Cas9/sgRNA) (Figure 2A).After cellular internalization and lysosome escape, the Cas9/sgRNA RNP was released in response to endogenous RNase H for subsequent gene editing.Confocal laser scanning microscopy (CLSM) images demonstrated the colocalization of Cas9/sgRNA RNP and nuclei, indicating the successful entry of Cas9/sgRNA RNP into the nuclei (Figure 2B).Western blotting (WB) analysis showed that Nanogel-Cas9/sgRNA effectively downregulated the expression of enhanced green fluorescent protein (EGFP) (Figure 2C).Notably, the T7E1 assay showed that Nanogel-Cas9/sgRNA had a genome editing efficiency of 18.7%, which verified the effectiveness of nanogel-based gene editing in vitro (Figure 2D).
Because of the multibranched structure of branched DNA, diverse DNA materials can be precisely fabricated, which can improve the stability of gene agents. [24]Liu et al. reported a branched DNA-based nanostructure for the delivery of sgRNA/Cas9/Antisense. [25] β-cyclodextrin was designed to covalently couple nucleic acid sequences to fabricate a branched DNA structure.The branched DNA structure with seven DNA arms was introduced as an adamantane-conjugated nucleic acid aptamer for targeting and an adamantine-modified peptide for endosomal escape via supramolecular host-guest recognition.The 3' end of sgRNA was extended to hybridize with antisense nucleic acids that were modified by disulfide bonds (referred to as Linkers).Through the coassembly of branched DNA, Cas9/ sgRNA RNP, and linkers, a branched DNA-based nanostructure was prepared (the molar ratio of branched DNA and linkers was 1:7) (Figure 3A).Serum stability analysis suggested that more than 40% of the assembled structures treated with 10% FBS for 48 h remained, verifying that the assembled structures significantly prevented nuclease attack compared with sgRNA and linkers (Figure 3B).The genomic modification efficiency was explored through T7E1 experiments, and the results showed that the nanostructure elicited 42% genetic cleavage, indicating the effective gene editing efficiency of the nanostructure (Figure 3C).The in vivo biodistribution assay demonstrated that the nanostructure specifically targeted and accumulated at tumor sites with the assistance of a DNA aptamer (Figure 3D).Through the combined effects of gene editing and gene silencing, the nanostructure demonstrated a remarkable synergistic therapeutic effect in vivo (Figure 3E).

RCA-Based DNA Nanostructures
RCA is an isothermal enzyme amplification platform that generates ultralong ssDNA catalyzed by DNA polymerase. [26]An ultralong RCA chain with myriad duplicates can be programmed to load a specific sgRNA.Through the rational design of the RCA template, functional sequences such as DNA aptamer, [27] ASOs, and DNAzyme [18] can be integrated on RCA products, enabling RCA-based DNA nanostructures for the delivery of the CRISPR/ Cas system and other threptic agents.Reproduced with permission. [23]Copyright 2019, Royal Society of Chemistry.Reproduced with permission. [25]Copyright 2019, American Chemical Society.
Sun et al. reported a biocompatible and programmable RCAbased DNA nanoclew for delivery of the CRISPR/Cas9 system. [28]Through RCA, the nanoclew formed tightly wound DNA with sequences complementary to the sgRNA, allowing the loading of Cas9/sgRNA RNP (Figure 4A).To achieve endosomal escape, a layer of cationic polymer polyethyleneimine (PEI) was wrapped around the nanoclew to make it positively charged.Upon uptake by cells, Cas9/sgRNA RNP was released from the endosome to the cytoplasm due to the "proton sponge effect."With the assistance of nuclear localization signal peptides, Cas9/sgRNA RNP entered the nucleus for gene editing.EGFP tumor-bearing mice were used as the model.EGFP xenograft tumor section images showed that %25% of the cells demonstrated no EGFP expression (Figure 4B), verifying the promising application of the RCA-based DNA nanostructure for delivery of the CRISPR/Cas9 system.
In addition to the extensively investigated CRISPR/Cas9 system, the CRISPR/Cas12a system has since been developed for gene editing.Cas12a, also known as cpf1, is a single crRNAguided enzyme containing a RuvC domain that recognizes the PAM sequence of TTN. [29]CRISPR/Cas12a targets dsDNA adjacent to PAM sequences, generating a DNA end with 5 0 -overhang using a single RuvC nuclease. [30]Sun et al. utilized this RCAbased DNA nanoclew to deliver the CRISPR/Cas12a system. [31]e CRISPR/Cas12a system included the Cas12a protein and shorter crRNA. [32]Based on the above studies, a Cas12a/ crRNA RNP (targeting the Pcsk9 gene) was loaded onto the nanoclew to regulate cholesterol levels.After being coated with PEI, the nanoclew was further modified with galactose and 2,3dimethylmaleic anhydride to obtain a hepatocyte-targeted charge reversal layer (Figure 4C).With the recognition between galactose and the ASGP receptor, the nanoclew was selectively internalized into hepatocytes.Subsequently, the nanoclew successfully escaped from the endosome owing to a charge switch from negative to positive in the acidic endosomal environment of the lysosomes, thus releasing Cas12a/crRNA RNP for gene editing.Base deletion around the target site caused by Cas12a/crRNA-exon3 was measured using deep sequencing (Figure 4D).The size of the missing nucleotides varied greatly (Figure 4E), with the 19 bp deletion occurring most frequently; representative deletions are shown in Figure 4F.These results verified that the delivered RNP had high specificity.
The combination of gene editing and gene silencing is expected to improve therapeutic efficacy.DNAzyme is a ssDNA sequence with catalytic activity that is a promising nucleic acid drug. [33]Li et al. developed a proton-activatable DNA nanosystem to codeliver Cas9/sgRNA RNP and DNAzyme for combined gene therapy. [34]An ultralong ssDNA with a repeated  A-B) Reproduced with permission. [28]Copyright 2015, Wiley-VCH.C-F) Reproduced with permission. [31]Copyright 2020, The American Association for the Advancement of Science.
DNAzyme sequence, sgRNA recognition sequence, and HhaI cleavage sites was generated via RCA, which was the scaffold of the nanosystem.After the Cas9/sgRNA RNP was loaded via base pairing, the RCA chain was further compressed into nanoparticles (DNC) in the presence of Mn 2þ .Mn 2þ served as a cofactor of DNAzyme to enhance its activity.The acid-responsive degraded polymer-coated HhaI was then assembled on the surface of the DNC to form a proton-activatable DNA nanosystem (H-DNC).After accumulation in the tumor tissue, the polymer coating on the surface of HhaI was degraded in the acidic lysosomal environment to release HhaI (Figure 5A), thereby cleaving specific sites in the DNC to release Cas9/sgRNA RNP, DNAzyme, and Mn 2þ .The combination of gene editing and gene silencing could result in efficient and synergistic cancer therapy.CLSM images showed that compared to DH-DNCtreated cells (the thermally desaturated HhaI used in H-DNC), there was more colocalization of Cas9 and nuclei in H-DNCtreated cells, demonstrating that the proton-activable HhaI was conducive for the release of Cas9/sgRNA RNP (Figure 5B).H-D E C E with EGR-1 silencing DNAzyme and PLK1 gene recognition sgRNA was designed and prepared.WB analysis showed that both EGR-1 and PLK1 expression were significantly decreased, suggesting the excellent efficiency of gene silencing and gene editing (Figure 5C).The gene editing efficiency of the delivered Cas9/sgRNA RNP was further evaluated using the T7EI assay, which showed %40% genetic cleavage in H-D E C E -treated cells (Figure 5D).The cellular apoptosis results showed that the apoptosis rate reached 75.26% in H-D E C Etreated cells, confirming that the combination of Cas9/sgRNA RNP and DNAzyme had a synergistic effect on cell apoptosis (Figure 5E).Tumor growth curves showed that H-D E C E -treated mice exhibited the most effective tumor suppression, indicating a synergistic therapeutic effect in vivo (Figure 5F).

Conclusion
The CRISPR/Cas system has revolutionized gene editing and has shown great potential for precision medicine applications.Compared with other cancer therapy strategies, such as chemodynamic therapy (CDT) and photodynamic therapy (PDT), which use the reactive oxygen species (ROS)-related mechanism for cancer therapy, [35] the CRISPR/Cas system knocks out specific disease-causing genes, which makes it more precise, effective, and flexible.However, several potential limitations, such as off-target effects, immunogenicity, and the possibility of host genome integration, need to be addressed for clinical translation of the CRISPR/Cas system.The development of biocompatible and efficient carriers for the delivery of CRISPR/Cas systems to avoid potential risks is essential for safe clinical use.
DNA nanomaterials are highly designable because of the unique sequence programmability of DNA, and thus these Reproduced with permission. [34]Copyright 2022, Wiley-VCH.materials have great promise for the delivery of gene agents.This review focuses on recent progress in DNA nanomaterials including branched DNA-based nanostructures and RCA-based DNA nanostructures for the delivery of the CRISPR/Cas system (Table 1). 1) Branched DNA-based nanostructures with a welldefined topology of branched arms can be accurately designed to load a CRISPR/Cas system at a desired ratio.2) RCA-based DNA nanostructures can be rationally designed using functional units to load the CRISPR/Cas system and other gene agents for combined therapy.3) Both branched DNA-based nanostructures and RCA-based DNA nanostructures possess size adjustability, which can effectively deliver the CRISPR/Cas system into cells.
Several aspects must be considered to further promote the application of DNA nanomaterials in the delivery of CRISPR/ Cas systems. 1) DNA has excellent biocompatibility and degradability, and DNA-based nanomaterials have demonstrated good biosafety.However, the long-term biosafety of DNA nanomaterials as CRISPR/Cas carriers should be considered.Particularly, the presence of immunostimulatory sequences in DNA nanomaterials may elicit immune responses.The rational design of DNA sequences is critical for ensuring the biological security of DNA nanomaterials.2) Owing to the large steric hindrance and low atomic utilization of DNA molecules, the standardized manufacturing of DNA-based CRISPR/Cas delivery nanomaterials remains challenging, which is important for further clinical applications.Optimization of the synthesis and assembly conditions for DNA nanomaterials may improve atom utilization.
3) The CRISPR/Cas system is easily degraded by nucleases and proteases, which affects the efficiency of gene editing.The stability of the nanocarriers must be considered to enhance their resistance to nucleases and proteases.Chemical modification of DNA nanomaterials is a possible strategy for improving their stability under physiological conditions.4) The delivery of the CRISPR/Cas system into target cells/tissues plays an important role in clinical applications.DNA nanomaterials can be programmed with cell-targeting aptamers or modified with cell-targeting peptides for targeted delivery of the CRISPR/Cas system, thereby reducing the toxicity to non-targeted cells/ tissues.5) Effective and controlled CRISPR/Cas system release from nanocarriers into the cytoplasm in a spatiotemporal manner has great potential for decreasing the off-target effects of gene editing.Therefore, DNA nanomaterials should be rationally designed with endosome/lysosome escape units to ensure subsequent gene editing in the nuclei.Moreover, DNA nanomaterials can be designed as stimuli-responsive nanocarriers in response to endogenous stimuli (e.g., pH, ATP H 2 O 2 , enzymes) or exogenous stimuli (e.g., light, temperature, magnetic fields) for spatiotemporal delivery of the CRISPR/Cas system.
The CRISPR/Cas system currently has a wide range of promising applications in diagnosis [36] and bioimaging [37] in addition to gene therapy.We believe that with the development of DNA nanotechnology, DNA nanomaterials will play an important role in the delivery of CRISPR/Cas systems, broadening their potential in the biomedical field.CRISPR/Cas9 DNAzyme MCF-7 cells Yes [34]

Figure 1 .
Figure 1.Schematic illustration of DNA nanomaterials for delivery of the CRISPR/Cas system.DNA nanomaterial-based carriers are categorized as branched DNA-based nanostructures and RCA-based DNA nanostructures.

Figure 2 .
Figure 2. Branched DNA-based nanostructure for the delivery of Cas9/sgRNA RNP.A) Preparation of a DNA-crosslinked nanogel (nanogel-Cas9/sgRNA) for the delivery of Cas9/sgRNA RNP.B) Colocalization of Cas9 (Cy5, red) and DNA linkers (FAM, green) with different times during the intracellular process of nanogel-Cas9/sgRNA; scale bar: 20 μm.C) WB analysis of EGFP levels in HeLa-EGFP cells treated with various formulations.D) T7EI assay to indicate gene mutation of EGFP in HeLa-EGFP cells treated with various formulations.Reproduced with permission.[23]Copyright 2019, Royal Society of Chemistry.

Figure 3 .
Figure 3. Branched DNA-based nanostructure for the delivery of sgRNA/Cas9/Antisense. A) Molecular design and preparation of the nanostructure.B) Serum stability of the nanostructure for different durations and the corresponding statistical analysis by Image J; data are represented as mean AE s.d.(n = 3).C) T7EI assay to indicate indel mutations of PLK1.D) Dynamic biodistribution after treatments in 24 h.E) Tumor growth curves with various treatments; data are represented as mean AE s.d.(n = 3); *P < 0.05, ***P < 0.001.Reproduced with permission.[25]Copyright 2019, American Chemical Society.

Figure 4 .
Figure 4. RCA-based DNA nanostructures for delivery of the CRISPR/Cas system.A) Preparation of an RCA-based DNA nanoclew for genome editing in vivo.B) CLSM images of U2OS.EGFP cells in tumor sections; scale bar: 50 μm.C) Preparation of an RCA-based DNA nanoclew for the delivery of CRISPR/Cas12a.D-F) Deep sequencing for nucleotide deletion distribution around the target site (D), distribution of different sizes of deleted nucleotide fragments (E), and the most common sequences of deleted fragments (F).A-B) Reproduced with permission.[28]Copyright 2015, Wiley-VCH.C-F) Reproduced with permission.[31]Copyright 2020, The American Association for the Advancement of Science.

Table 1 .
Summary of DNA nanomaterials for delivery of the CRISPR/Cas system.