Intelligent nanotherapeutic strategies for the delivery of CRISPR system

Abstract

CRISPR, as an emerging gene editing technology, has been widely used in multiple fields due to its convenient operation, less cost, high efficiency and precision. This robust and effective device has revolutionized the development of biomedical research at an unexpected speed in recent years. The development of intelligent and precise CRISPR delivery strategies in a controllable and safe manner is the prerequisite for translational clinical medicine in gene therapy field. In this review, the therapeutic application of CRISPR delivery and the translational potential of gene editing was firstly discussed. Critical obstacles for the delivery of CRISPR system in vivo and shortcomings of CRISPR system itself were also analyzed. Given that intelligent nanoparticles have demonstrated great potential on the delivery of CRISPR system, here we mainly focused on stimuli-responsive nanocarriers. We also summarized various strategies for CIRSPR-Cas9 system delivered by intelligent nanocarriers which would respond to different endogenous and exogenous signal stimulus. Moreover, new genome editors mediated by nanotherapeutic vectors for gene therapy were also discussed. Finally, we discussed future prospects of genome editing for existing nanocarriers in clinical settings.

Graphical abstract

Various strategies of CIRSPR-Cas9 system delivery via intelligent nanocarriers are summarized which respond to different endogenous and exogenous signal stimulus.

1. Introduction

As one of the most promising gene therapy weapons, clustered regularly interspaced short palindromic repeats (CRISPR) system in collaboration with Cas (CRISPR-associated) proteins have already become the revolutionized technology in various fields, including biology and chemistry¹. The 2020 Nobel Prize in Chemistry laureated the pioneering work in gene editing achieved by Emmanuelle Charpentier and Jennifer Doudna, which provided great inspire for researcher working on CRISPR genome editing filed. CRISPR is firstly found as a kind of immune defense mechanism against the invasion of genetic elements in bacteria or archaea^2,3. The CRISPR system is generally composed of crRNA/single guide RNA (sgRNA) and Cas endonuclease. As for CRISPR technologies, Cas9 is the most widely studied Cas protein with extensive applications. Recently, various delivery forms of Cas9 and novel feasible CRISPR systems, including CRISPR-Cas12a, CRISPR-Cas12b, CRISPR-Cas13a, CRISPR-dCas9, CRISPR-Casx and CRISPR-base editors, have been developed and optimized with the aim to improve the efficiency of gene editing and delivery process4, 5, 6, with the action mechanisms of various CRISPR system shown in Fig. 1. The current data have been achieved both in preclinical and clinical trials that used CRISPR-Cas9 for genome editing7, 8, 9. Nevertheless, there are some limitations for the application of CRISPR system in clinic, i.e., challenges for improving the efficient and safe delivery of CRISPR elements in vivo.

Figure 1.

Various well-known CRISPR system delivery patterns and action mechanisms. (A) Delivery types of CRISPR-Cas9 elements, i.e., DNA plasmid encoding both sgRNA and Cas9, the mixture composed of sgRNA and Cas9 mRNA, and the Cas9/sgRNA complex ribonucleoprotein. The working mechanisms of (B) CRISPR-Cas12a and Cas13a, (C) CRISPR-dCas9-based CRISPRa (activation) and CRISPRi (inhibition), and (D) CRISPR-ABE (adenine base editor) and CRISPR-CBE (cytosine base editor).

At present, the delivery of CRISPR system is mainly divided into two categories: viral vectors and non-viral vectors. Viral vectors, including lentivirus, adenovirus and adeno-associated virus (AAVs), have widely utilized to deliver CRISPR elements due to its high efficiency, but their undesired genome integration, immunogenetic responses and limited cargo loading hinder the further clinical applications^10,11. Compared with the viral vectors, the non-viral vectors (such as, biological membrane nanomaterials/liposomes and polymer- or metal-based nanocarriers) possess the advantages of lower immunogenicity and huge cargo size^12,13. The optimization of CRISPR delivery in vivo still face many challenges, including encapsulation of huge size of CRISPR system, targeting delivery, and enhanced endocytosis14, 15, 16. Taking CRISPR-Cas9 system as an example, three forms of CRISPR-Cas9 have been developed, i.e., the plasmid DNA encoding Cas9 and sgRNA, the mixture of Cas9 mRNA and sgRNA, and the mixture of Cas9 protein and sgRNA (Cas9 ribonucleoprotein, Cas9 RNP) (Fig. 1A)17, 18, 19. Compared with the long-time activated CRISPR forms (plasmid DNA and mRNA), CRISPR RNP shows excellent potential of reducing immunogenicity and off-target effects. Recently, in order to improve the precision of CRISPR system, researchers have developed CRISPR-mediated base editing (BE) systems for achieving repair of a single base in some genetic diseases (Fig. 1D)²⁰. In addition to gene editing, some CRISPR systems, such as CRISPR-dCas9 (Fig. 1C), have been developed for precise delivery of drugs, like doxorubicin (DOX)²¹. Therefore, based on the powerful functions of CRISPR system for disease correction, it is urgently needed to improve the in vivo delivery. Recently, intelligent delivery strategies, such as spatiotemporal delivery, have attracted increasing attentions and evidence has revealed the great superior performances for in vitro and in vivo delivery of CRISPR systems^4,22, but systematic summary of the intelligent delivery of CRISPR system is scarce. Comprehensive understanding of the manufacturing process of CRISPR system for intelligent delivery is meaningful for precise genome editing.

In this review, we systematically summarize the intelligent delivery approaches for CRISPR systems, and focus on the responsive strategies. Furthermore, we seek to review the recent progress of novel CRISPR systems (including CRISPR-Cas12a, dCas9, and CRISPR-base editors) delivery strategies, which are different from but complementary to the several recently published reviews that summarized related data on CRISPR-Cas9 spatiotemporal delivery^4,14,16,22. Additionally, the obstacles for the in vivo delivery of CRISPR elements and optimization strategies for improving delivery efficiency are discussed. For CRISPR-Cas9 intelligent delivery, we summarize various intelligent delivery methods for disease treatment, involving in active-targeting, micro or cytoplasmic environment responsive, and external stimuli-triggered strategies, and the latest research data are also analyzed, including works recently published by our groups^17,23. At the same time, we also pay attention to the novel CRISPR protein, which will be conducive to the research of CRISPR intelligent delivery system in the future.

2. CRISPR delivery and its therapeutic applications

CRISPR system, as one of the most advanced gene editing tools, is widely used in the treatment of various diseases, including immune-related diseases^16,24. In general, it is safer to knock out genes from diseased tissues (such as tumor) than to knock out genes from normal cells. The side effects of gene editing on germ cell have been considered as an important ethical issue in human gene editing therapy. Compared with traditional drugs and treatments, gene editing therapy in vivo will provide permanent gene-level changes, and its changes at the reproductive level will affect the genes of offspring. However, a recent study reported the CRISPR-Cas9 approach used for NTLA-2001 in the treatment of transthyretin amyloidosis. NLTA-2001, composed of liposome encapsulated CRISPR-Cas9, is used to target low-density lipoprotein (LDL) expressed on the surface of hepatocytes and knock out transthyretin gene which can significantly reduce the transthyretin level in serum (about 96%)²⁵. The development direction of in vivo knockout should be better targeting to reduce the side effect on cells rather than gene editing, and should target diseases that seriously threaten human life to reduce the ethical burden of gene editing technology in the future. At present, there have been a large number of researches on CRISPR for the treatment of diseases in vivo. Intelligent nano delivery system can edit genes more effectively and accurately, and introduce other functional roles into diseased cells. Hence, the researches to improve gene editing efficacy and nano delivery system are more in line with future development. With the progress of technology, the structure and function of nanoparticles (NPs) for clinical CRISPR system delivery will be more complex. Herein, we summarized the intelligent non-viral nano vectors used for CRISPR-Cas9 delivery in various disease therapies.

Exploration of different genes knocked-out by CRISPR-Cas9 technologies is beneficial to the development of related disease therapies and corresponding intelligent delivery strategies. Among those analyzed studies, intelligent CRISPR delivery system is widely used in the treatment of tumors, such as melanoma or cervical cancer. CRISPR-Cas9 system is also a mainstream technology employing for nanocarriers delivery cargo as the gene-editing therapeutic tools. The mechanism of CRISPR for disease treatment is to knock out or repair genes highly related to the disease, or combined with gene editing to improve the efficacy of synergistic treatment (Fig. 2A)²⁶. For example, beta-secretase 1 (Bace1) is a key gene in Alzheimer's disease (AD). Two research groups^27,28 used nanocarriers for CRISPR-Cas9 delivery to knock out Bace1 gene respectively, and the results showed good curative effects in vitro and in vivo, with the cognitive ability of model mice significantly improved. Some important pathways in tumors, such as β-catenin, EGFR, VEGFR, PLK1, KARS, P53, and cell cycle related genes (like, Cdk11 and Cdk5), have also been discussed29, 30, 31, 32, 33, 34, 35, 36. In addition, cancer stem cell related genes, such as NANOG, were used as the targeting gene for CRISPR system efficiently delivered with intelligent nanocarriers to the nucleus, which significantly weakened the cell viability of HeLa cells³⁷. Moreover, the intelligent CRISPR delivery system can be used as one of the therapeutic strategies for synergistic therapy involving in drugs in combination with genome editing (Fig. 2B)²⁹. For instance, the drug resistance can be reversed or the therapeutic effects of drugs can be enhanced by knocking out chemo-resistance related genes using CRISPR technology. Li et al.³⁸ used folic acid and chitosan as the nanocarrier to co-encapsulate DOX and Cas9 plasmid targeting SURVIVIN. The co-delivery system showed highly efficiency of gene editing and knockout of SURVIVIN also obviously improved the sensitivity to DOX so as to achieve a superior antitumor efficacy. Except for gene editing, the CRISPR system has also been used for precise nuclear delivery of drugs. Ma et al.²¹ employed the telomerase dCas9 system to achieve specific nuclear delivery of DOX by targeting telomeres, which significantly increased the concentrations of DOX in tumor nuclei (Fig. 2G).

Figure 2.

Joint therapeutic strategies involving CRISPR system. (A) Cationic polymer (HPAE-EB) delivered Cas9 plasmid for enhancing treatment effect. Reprinted with the permission from Ref. 26. Copyright © 2021 The Author(s). (B) Polyamidoamine-aptamer-modified hollow mesoporous silica nanoparticles (NPs) for co-delivery of sorafenib and Cas9 plasmid to achieve the combination of drug and gene therapy. Reprinted with the permission from Ref. 29. Copyright © 2020 American Chemical Society. (C) Cationic lipid-assisted poly(ethylene glycol)-b-poly(lactide-co-glycolide) (PEG-PLGA) NPs simultaneously co-delivered autoimmune diabetes-relevant peptide, Cas9 plasmid, and sgRNAs targeting CD80, CD86, and CD40 to carry out immunotherapy of immune cells. Reprinted with the permission from Ref. 39. Copyright © 2020 American Chemical Society. (D) Near-infrared (NIR)-triggered copper sulphide (CuS) NPs delivered Cas9 ribonucleoprotein (RNP) for gene editing of Ptpn2 to realize the combination of photothermal therapy (PTT) and immunotherapy. Reprinted with the permission from Ref. 42. Copyright © 2021 Elsevier Ltd. (E) Photothermal-controlled CuS NPs for the delivery of Cas9 RNP targeting heart shock protein Hsp90α improved mild-PTT. Reprinted with the permission from Ref. 17. Copyright © 2021 Wiley-VCH GmbH. (F) NIR-sensitive NPs delivered photosensitizer chlorin e6 and Cas9 RNP targeting Nrf2 for enhancing photodynamic therapy. Reprinted with the permission from Ref. 51. Copyright © 2020 The Author(s), some rights reserved. (G) Telomerase dCas9 nano system delivered doxorubicin to achieve precise nuclear delivery of doxorubicin for improving chemotherapeutic efficacy. Reprinted with the permission from Ref. 21. Copyright © 2021 American Chemical Society.

Delivery of CRISPR system using intelligent nanocarriers is a common strategy for ameliorating immune-related diseases. Diseases such as autoimmune type 1 diabetes (T1D) can also be treated by knocking out the relative genes of immune cells. Simultaneous knocking out of CD80, CD86 and CD40 genes of dendritic cells (DCs) using a cationic lipid-assisted PEG-PLGA NPs encapsulated Cas9 system triggered the generation and proliferation of autoantigen specific Tregs and relieved autoimmunity to islet components, thus inhibiting the development of T1D (Fig. 2C)³⁹. It has also been reported that direct knockout of CD40 can reduce immune rejection after organ transplantation⁴⁰. For tumors with immune heterogeneity, increasing immune sensitivity by CRISPR delivery platform can also be considered. Protein tyrosine phosphatase non-receptor type2 (PTPN2, also known as TC-PTP) is an emerging immune-targeting gene for tumor immunotherapy, which can be triggered by interferon-γ (IFN-γ) mediated antigen presentation and tumor growth inhibition to improve the effect of immunotherapy (Fig. 2D)^41,42. Programmed cell death ligand-1 (Pdl1) regulates the immune system and promotes self-tolerance by down-regulating the response of immune system to human cells and inhibiting the inflammatory activity of T cells⁴³. Accumulating studies have shown that nano delivery systems are used to knock out Pdl1 and/or Ptpn2, with high efficacy in inhibiting tumor growth of tumor xenograft models44, 45, 46, 47, 48, 49. Additionally, the combination of chemotherapeutic agents and knockout of immune-related genes by CRISPR technology can be realized using intelligent nano-strategies. Chemotherapeutic agents, such as DOX, paclitaxel (PTX), and hydroxycamptothecin, are the most commonly used small molecule drugs that can lead to immunogenic cell death (ICD), which elicits a strong anti-tumor immune response. Previous studies have demonstrated that genome editing of cyclin-dependent kinase 5 (Cdk5) using Cas9 plasmid in combination with PTX significantly inhibited the expression of Pdl1, which effectively converted “cold” into “hot” tumor and thus effectively inhibited tumor growth³⁵. Moreover, the intelligent nanocarriers induced its own therapeutic effects, mainly reflected in photothermal therapy (PTT), photodynamic therapy (PDT) and sonodynamic therapy (SDT), which can achieve synergistic treatment by knocking out genes sensitive to therapy. For example, knocking out the heat shock protein gene HSP90α can make the tumor more intolerant to high-temperature, which results in tumor thermal ablation (Fig. 2E)¹⁷. Similarly, the combination of knockout of MTH1 gene and SDT-triggered reactive oxygen species (ROS) can increase the sensitivity of SDT for enhanced tumor therapy⁵⁰. The synergistic effects of chlorin e6 (Ce6)-mediated PDT and gene-editing targeting NRF2 gene have been confirmed by previous evidence (Fig. 2F)⁵¹. In conclusion, the selection of CRISPR knockout targeting gene and the combination strategies are the committed steps for its therapeutic effects.

3. Critical obstacles for gene-editing in vivo

As a precise gene-editing technology, the CRISPR has been underwent revolutionary development with vigorous vitality in various fields. In the past decade, a series of methods have been developed to deliver CRISPR system intracellularly with high efficiencies, but the development of safe and efficient nanocarriers for in vivo genome editing remains challenging in clinical practice. Herein, we summarize the major obstacles to CRISPR system delivery and the shortcomings that need to be optimized for delivery of CRISPR system itself (Fig. 3).

Figure 3.

Critical obstacles and optimization for CRISPR system delivery. Four key steps for realizing effective genome editing in vivo: efficient CRISPR system loading (huge-size CRISPR elements), accumulation of tumor regions (non-specific clearance of blood circulation), delivery to targeted cells (reduce non-target cell aggregation), and enhanced endocytosis and nuclear internalization (crucial process for genome editing of CRISPR system).

3.1. Cascading obstacles in delivery process

3.1.1. Cargo loading

Unlike classical biomolecular reagents (siRNA, therapeutic mRNA or protein), the CRISPR system (CRISPR plasmid, CRISPR mRNA/crRNA, or CRISPR RNP) has a large genetic size, which is difficult for nanocarriers to effectively envelope⁵². Take the CRISPR-Cas9 system as an example, it has been reported that the genetic size of the Cas9 protein is about 160 kDa, Cas9 mRNA is about 4500 nt, and the plasmid encoding Cas9 is 7–9 kb^10,14. Therefore, the large genetic size of Cas9 plasmid, mRNA, and protein makes it challenging to effectively package multiple elements into a single nanocarrier. For these challenges, various strategies have designed by researchers, such as, i. separated delivery systems conjugate with cell-penetrating peptide (CPP)⁵³; ii. using cationic nanomaterials (including cationic lipids, polymers, polypeptides, and gold nanocluster) for electrostatic adsorption of CRISPR complexes with sufficient negative charge⁵⁴; iii. single strand DNA-RNA base pairing used for condensing or controlling the release of CRISPR RNP components^17,55. Overall, the methods mentioned above can solve the loading problem of CRISPR system to some extent.

3.1.2. Blood circulation

The nanocarrier is disturbed by the complex environment of blood before arriving to the tumor regions. Additional modifications are needed to protect the nanocarriers packaging CRISPR system from degradation and enhance the blood circulation time. The core–shell multilayer nanostructure can ensure the stability of CRISPR elements by modifying the outer layer to avoid degradation, increase the circulation time and utilize the inner layer to condense the large cargo, which makes it a promising platform for effective systemic delivery in vivo¹². Multilayer shell construction has been designed to greatly decrease the non-specific uptake, undesired clearance and degradation of CRISPR components^46,56,57. The anionic outer layer is employed to enhance the circulation stability in blood and the degradation of CRISPR components in tumor region so as to form a cationic shell to increase the uptake of tumor cells.

3.1.3. Targeted delivery

Successful genome editing requires efficient targeted delivery of the CRISPR-Cas9 system into the desired organs, tissues and cells. Importantly, the physicochemical properties, such as size and surface charge of nanocarriers, greatly influence their biodistribution and metabolism. As for size, the nanocarriers with a diameter of 10–200 nm can be substantially accumulated in the tumor, inflammatory tissue or hepatic sinusoid due to their high vascular permeability58, 59, 60, 61, 62. Particles larger than 2 μm are usually captured by the pulmonary capillaries, and particles smaller than 5 nm are easily removed by the kidney. In the systemic administration of nanocarriers, a large proportion could be captured by the reticuloendothelial systems of the liver and spleen^59,63. For surface charge, the particles with positive charge are more easily absorbed by the liver and internalized by cells as compared with negatively charged or neutral nanocarriers⁶⁴. Notably, the formation of protein corona leads to a shift of the surface charge, thus making the surface charge of nanocarrier not always constant⁶⁵. To date, three categories of organ-targeted nanocarriers delivering CRISPR system have been summarized, including i. cationic lipid NPs⁶⁶; ii. specific ligand-conjugated nanovehicles to enhance receptor-mediated cell internalization^30,67,68; iii. biomimetic cell membrane coating nanocarriers, such as engineered cell membrane⁶⁹, and extracellular vesicles^70,71. Nevertheless, Chen et al.⁷² reported that the endogenous exosomes could export both sgRNA and Cas9 protein from CRISPR-Cas9 expressing cells, and unfortunately deliver the gene-editing activity to the adjacent and distant cells or tissues, thus making the off-target and safety concerns about CRISPR-Cas9 system complicate.

3.1.4. Cellular internalization

Endocytosis is the crucial pathway that CRISPR elements load nanocarrier entering into cells^72,73. Endosome trapping is the primary obstacle for successful delivery of CRISPR components, as the nanocarriers are highly likely to be entrapped in endosomes, which contain an acidic environment (around pH5) and multiple digestive enzymes, thus endocytosis may occur. The potential strategy is to directly enter into the cytoplasm, i.e., bypassing endosome after cell internalization, such as pore formation⁷⁴ and cytomembrane fusion⁷⁵, and disrupting the endosomal membrane to escape from the endosome, for instance, the proton sponge effect (also termed the pH-buffering effect)75, 76, 77, 78, 79 and hexagonal H_II conformation induced by cationic lipids⁶⁶. Moreover, the prerequisite for successful gene editing is that CRISPR-associated elements enter into the nucleus. Up to now, there are three major delivery modes for CRISPR system as we have mentioned: i. plasmid DNA encoding Cas and sgRNA; ii. the mixture of Cas mRNA and sgRNA; iii. the mixture of Cas protein and sgRNA (Cas RNP)^17,18. Except for the mentioned nuclear targeting AS1411 ligands mediated CRISPR-Cas9 plasmid gene editing⁷⁶, the efficiency of gene editing in the nucleus is generally related to the delivery forms of the CRISPR elements, as described below.

3.2. Defects of CRISPR system

Direct delivery of CRISPR RNP is the most straightforward but difficult delivery mode, as it is challenging to deliver a large size and negatively charged protein across the cytomembrane. For the presence of sgRNA in the RNP, the delivery also requires to protect the sgRNA from any possible degradation pathways. Additionally, it is more difficult to obtain the pure protein than plasmid or mRNA and the Cas protein extracted from the bacterial needs to remove the endotoxin before use so as to avoid endotoxin contamination. Addition of a non-native Cas protein from bacterial origin into mammal without dosage carefully monitored may trigger immunological response or cytotoxicity⁷⁶.

Another alternative form is mRNA (messenger RNA) encoding Cas protein, which was expressed by ribosomes in cytoplasm. This delivery mode may reduce the off-target effect to some extent and avoid the difficulty of crossing the nuclear membrane. As with the RNP delivery, mRNA is susceptible to be degraded by RNase both in vitro and in vivo. Chemical modification of mRNA has been developed to modulate their stability and expression in cells. Moreover, the presence of sgRNA and Cas protein at the same time is a prerequisite for effective genome editing. It has been demonstrated that delivery delay of the sgRNA up to 6 h after the mRNA may enhance the gene editing efficiency⁸⁰. Chemical modification of sgRNA may enhance its stability after delivery, which is also suitable for RNP delivery⁸¹. mRNA encoding CRISPR Cas protein co-delivered with viral genome constitutively expressing sgRNA can completely avoid the mentioned timing issue¹¹. For plasmid DNA encoding Cas protein or sgRNA, the expression time is increased as compared with the RNP and mRNA delivery modes, thus leading to enhanced off-target effects and triggering the immunogenic response⁸². Similarly, to achieve effective delivery, the plasmid DNA must penetrate both the cellular and nuclear membranes.

4. Intelligent delivery of CRISPR-Cas9 system for gene editing

The CRISPR-Cas9 technology is a promising method of gene therapy and has become a powerful genome editing tool for the treatment of multiple diseases¹⁷. As the wildly used vectors for cargo loading, nanocarriers have great potential in gene-therapy and it will be of great significance to improve its delivery efficiency for specific diseases treatment. Similar to nanomaterials for general cargo, nanocarriers packaging CRISPR-Cas9 system also need optimizations so as to improve the delivery efficiency. The optimization of nanocarriers is generally summarized as the following aspects, and we focus on describing the improvements for intelligent delivery of CRISPR-Cas9 elements. Three different methods of intelligent delivery of CRISPR-Cas9 for the treatment of multiple diseases are discussed here: active-targeting nanocarriers, micro- or cytoplasmic-environment responsive nanocomposites, and external stimuli-triggered nanoplatforms (Fig. 4).

Figure 4.

Schematic illustration of various strategies for intelligent nanocarriers as CIRSPR-Cas9 system delivery vectors.

4.1. Active-targeting nanocarriers

It is well known that the cellular engulfment of NPs is generally achieved by interaction with components of the extracellular matrix or plasma membrane. The process that NPs enrich in target tissues and cells can be divided into two major categories, i.e., passive targeting and active targeting⁸³. Passive targeting can be realized through controlling a specific size or shape of nanomaterials; and active targeting usually depends on the surface functionalization and surface charge to enhance the cellular uptake of NPs. Active delivery of nanocarriers packaging CRISPR-Cas9 also mainly relies on the modifications of materials with transmembrane delivery functions, such as substances binding to receptors or with high affinity to cytomembrane (details see Table 1^26,28, 29, 30, 31^{,33,36,37,39,44,45,51,74,76,}84, 85, 86, 87, 88, 89, 90, 91).

Table 1.

Summary of active-targeting CRISPR-Cas9 delivery nanocarriers for the treatment of various diseases.

Classification	Studies description	Disease	Type	Gene	Ref.
Receptor-mediated targeted delivery	Rabies virus glycoprotein peptide was linked with polylysine and dopamine complex through PEG to prepare the CF-TBIO NPs for the delivery of Cas9 plasmid targeting Bace1 gene, which significantly improved the pathological damage and behavioral abilities of mice and provided accurate imaging signals.	Alzheimer's disease (bEnd.3 and Neuro 2a cells)	Plasmid	Bace1	28
	Polyamidoamine (PAMAM)-Apt coated hollow mesoporous silica nanoparticle for delivery of both Cas9 plasmid and sorafenib efficiently knocked out EGFR, thus resulting in tumor inhibition.	Hepatocellular carcinoma (HepG2 and Huh7 cells)	Plasmid	EGFR	29
	PEG-PEI cholesterol lipopolymer modified with OS cell-specific aptamer (LC09) for delivering Cas9 plasmid targeting VEGFA gene significantly inhibited orthotopic OS malignancy and tumor metastasis, angiogenesis and bone injury.	OS (K7M2 and Saos2 cells)	Plasmid	VEGFA	30
	Linker-Apt and nuclear targeting peptide (TAT)-modified gold nanorods were used to achieve effective nuclear targeting delivery of Cas9 RNP targeting PLK1 gene, which significantly inhibited tumor cell proliferation.	Breast cancer (MCF-7 cells)	RNP	PLK1	31
	PBA-modified cationic liposomes delivered Cas9 mRNA targeting HPV18E6 gene by interacting with sialic acid receptor, which effectively prohibited HeLa cell growth.	Cervical cancer (Hela cells)	mRNA	HPV18E6	33
	AS1411 aptamer modified carboxymethyl chitosan nanoparticles were linked with endosomolytic peptide KALA for the delivery of CRISPR-Cas9 plasmid targeting CDK11 gene, which significantly inhibited tumor development and metastasis.	Breast cancer (MCF-7 cells)	Plasmid	CDK11	36
	By using Dopamine as the skeleton carrier modifying with HA as cell targeting ligand and dexamethasone as nuclear localization signal, PDA/DEX-PEI@HA nanoparticles facilitated the precise CRISPR/Cas9 targeting delivery and the NANOG protein of HeLa cells were successfully knocked out, which significantly reduced the proliferation and migration of tumor cells.	Cervical cancer (Hela cells)	Plasmid	NANOG	37
	TPP-COOH targeting mitochondria bound to positively charged PEI and adsorbed HA on outermost layer to deliver Cas9 plasmid targeting Ptpn2, and then Ce6 triggered PDT was used to achieve ICD combined immunotherapy for cancer treatment.	Melanoma (B16F10 cells)	Plasmid	Ptpn2	44
	AS1411 aptamer and TAT-NLS peptide conjugated with HA composed into nanoparticle for CRISPR-Cas9 plasmid delivery, which could efficiently knock out β-catenin, thus leading to tumor growth inhibition and immune activation caused by down-regulation of PD-L1.	Lung cancer (H1299 cells)	Plasmid	β-catenin	45
	iRGD-labeled nanocomposites were used to enhance Cas9 RNP delivery by interacting with Integrin αv, which achieved highly gene-editing of Nrf2 and enhanced synergistic PDT.	Nasopharyngeal carcinoma (CNE-2 cells)	RNP	Nrf2	51
	Carboxymethyl chitosan containing biotin and AS1411 ligand were employed to deliver Cas9 plasmid to knock out CDK11 gene in tumor, which resulted in down-regulation of various genes highly involved in tumor development.	Breast cancer (MCF-7 cells)	Plasmid	CDK11	76
	Three peptides were utilized to modify liposomes, including pH-sensitive H-peptide, EGFR-targeting P-peptide and nucleus-directing R-peptide, to co-deliver epirubicin and CRISPR-Cas9 system targeting HUR gene, which reduced drug resistance and tumor metastasis.	Head and neck cancer (SAS cells)	Plasmid	HUR	84
	TAT peptide modified Au nanoparticles coated with lipids to form a cationic nanoparticle for delivering Cas9 plasmid so as to realize controlled PLK1 gene knockout and melanoma inhibition.	Melanoma (A375 cells)	Plasmid	PLK1	85
	TAT peptide-labeled reductase sensitive liposomes were used to directionally knock out PLK1 gene by delivering Cas9 RNP, which slowed down the progression of melanoma.	Melanoma (A375 cells)	RNP	PLK1	86
Bio-derived membranes	Tumor derived exosomes were employed to increase the cellular uptake of CRISPR system for knockout of PARP-1 gene in melanoma and enhanced apoptosis and the sensitivity of cisplatin.	Ovarian cancer (SKOV3 cells)	Plasmid	PARP-1	87
	Chimeric-antigen receptor (CAR) modified exosomes were unitized to deliver CRISPR-Cas9 system for knocking out MYC gene in tumors so as to achieve effective therapeutic effect.	Burkitt lymphoma (Raji cells)	Plasmid	MYC	88
Positively charged molecule	Highly branched poly (β-amino ester) polymer encapsulated Cas9 RNP targeting COL7A1 was employed to treat human recessive dystrophic epidermolysis bullosa (RDEB), and the results showed that the genomic deletion of exon 80 was increased to over 40% in RDEB keratinocytes.	RDEB (HEK293 and keratinocytes)	RNP	COL7A1	26
	Cationic lipid-assisted poly PEG-PLGA NPs were used to encapsulate autoimmune diabetes associated peptide, Cas9 plasmid and three gRNAs targeting costimulatory molecules (CD80, CD86 and CD40), which effectively entered into DCs and thus triggered the generation and expansion of autoantigen-specific Tregs. Moreover, this NP prevented autoimmunity in islet components and inhibited the development of autoimmune type 1 diabetes.	Autoimmune type 1 diabetes (BMDCs)	Plasmid &sgRNAs	CD80&CD86&CD40	39
	Cationic α-helical polypeptide based PEGylated nanoparticles were utilized to deliver Cas9 plasmid targeting PLK1 so as to achieve highly genome-editing and suppress tumor growth.	Cervical cancer (Hela cells)	Plasmid	PLK1	74
	Cationic lipid-assisted poly PEG-PLGA nanoparticles were unitized to achieve targeted delivery of Cas9 plasmid for knocking out BCR-ABL gene, thus improving the survival.	Chronic myeloid leukemia (K562 cells)	Plasmid	BCR-ABL	89
	Optimized cationic lipid nanoparticles were used to encapsulate Cas9 mRNA and sgRNA targeting Antithrombin, which recovered bleeding-associated phenotypes in both hemophilia A and hemophilia B.	Hemophilia A and B (in mouse)	mRNA &sgRNA	Antithrombin	90
	Cationic liposome hydrogel nanoparticles were used to deliver Cas9 RNP targeting PLK1 gene, which achieved a higher gene-editing efficiency than lipo2000 and increased the survival of tumor bearing mice.	Brain cancer (U87 cells)	RNP	PLK1	91

EGFR, epithelial growth factor receptor; HA, hyaluronic acid; ICD, immunogenic cell death; OS, osteosarcoma; PBA, phenylboronic acid; PDT, photodynamic therapy; RDEB, recessive dystrophic epidermolysis bullosa; RNP, ribonucleoprotein; ROS, reactive oxygen species; TNBC, triple-negative breast cancer; TPP-COOH, 3-(carboxypropyl) triphenylphosphonium bromide; VEGFA, vascular endothelial growth factor A.

4.1.1. Receptor-mediated targeted delivery

As one of the most common signal-transduction processes both in vitro and in vivo, “receptor–ligand interaction” has become an effective means to enhance cellular uptake^92,93. Due to the high affinity between receptor and ligand, the ligand-modifying NPs can be easily adsorbed on the surface of target cells. After binding to the receptor of the target cells, the NPs are subsequently endocytosed under the control of Clathrin, a process known as cellular endocytosis. The NPs can be encapsulated by vesicles and enter a series of more acidic organelles, such as late endosomes and lysosomes, thereby leading to the structural changes of NPs^94,95. Hence, the selection of materials targeting specific receptors as nanocarriers that load CRISPR-Cas9 components remains an important potential research direction. In this part, we will introduce the CRISPR-Cas9 delivery nano system for enhancing genome editing through “receptor–ligand interaction”, as well as potential strategies for combination of modified nanomaterials. Meanwhile, the potential problems in practical applications of receptor-mediated targeting delivery will also be discussed.

4.1.1.1. Hyaluronic acid

Multifunctional bio-derived compounds have emerged as potential research objects of CRISPR-Cas9 delivery system, with characteristics of easy availability, excellent bio-safety and biocompatibility, and are suitable as biological targets of nanocarriers. Hyaluronic acid (HA), a kind of endogenous macromolecular substance widely used in medical beauty industry, is a negatively charged and nosulfated glycosaminoglycan composed of repeated disaccharide units of d-glucuronic acid and N-acetyl-d-glucosamine, with outstanding biocompatibility, biodegradability, hydrophilicity and nonimmunogenicity^96,97. It has reported that HA is a well-known ligand for cellular surface receptors, such as CD44, CD168 (also termed receptor for hyaluronan-mediated motility, RHAMM), and lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1)⁹⁸. Among them, the interaction of CD44 (high-expression in most tumors, especially in tumor stem cells)⁹⁹ and HA is widely employed in CRISPR-Cas9 delivery¹⁰⁰.

HA has been widely used in traditional polymer NPs and can be used to encapsulate CRISPR-Cas9 for gene editing. At present, the immune checkpoint blockade is a promising strategy for tumor immunotherapy¹⁰¹. As previously mentioned, PTPN2 is a recently reported tyrosine phosphatase involved in regulation of multiple signaling process triggered by growth factor or cytokine receptors^102,103. Yang et al.⁴⁴ used negatively charged HA as the backbone to combine the plasmid system expressing CRISPR-Cas9 system that targets Ptpn2 gene with a modified inorganic mitochondria-targeting Ce6, terming HPR@CCP NPs (Fig. 5A). The HPR@CCP showed a high genome disrupting efficiently and significant PDT efficacy, and the disruption of Ptpn2 gene could enhance the efficacy of immunotherapy through regulating the IFN-γ and TNF-α signaling, thereby increasing the proliferation of tumor killer CD8⁺ T cells.

Figure 5.

Receptor-mediated intelligent delivery of CRISPR-Cas9 system. (A) Hyaluronic acid modified HPR@CCP nanoparticles (NPs) interacted with CD44 receptor on cytomembrane for effective delivery of Cas9 plasmid targeting ptpn2. Reprinted with the permission from Ref. 44. Copyright © 2020 Elsevier Ltd. (B) Biotin moiety-based nano vector improved Cas9 plasmid endocytosis through interaction with biotin receptor. Reprinted with the permission from Ref. 76. Copyright © 2018 American Chemical Society. (C) Phenylboronic acid-conjugated PBA-BADP NPs enhanced the cell uptake of Cas9 mRNA by interacting with sialic acid receptors. Reprinted with the permission from Ref. 33. Copyright © 2019 American Chemical Society. (D) Intercellular adhesion molecule-1 (CD54) antibody-modified nanolipogel system was used for the delivery Cas9 plasmid to achieve highly genome editing efficiency. Reprinted with the permission from Ref. 130. Copyright © 2019 National Academy of Sciences. (E) mHph3-and iRGD peptides-conjugated liposomes delivered Cas9 protein for facilitating penetration of the blood–brain barrier (BBB). Reprinted with the permission from Ref. 91. Copyright © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. (F) Angiopep-2 peptide-coated nanocarriers was used to delivery Cas9 RNP and realize BBB penetration by interaction with LRP-1 of BBB epithelial cells and gliomas. Reprinted with the permission from Ref. 144. Copyright © 2022 The Authors, some rights reserved. (G) PAMAM-aptamer-conjugated hollow mesoporous silica nanoparticle was used for the delivery of Cas9 plasmid through interaction of EpCAM on cytomembrane. Reprinted with the permission from Ref. 29. Copyright © 2020 American Chemical Society.

HA is also reported as a nanocarrier modification for double-gene knockout by CRISPR-Cas9 system. Previous study¹⁰⁴ has shown that Pdl1 plays a crucial role in immune evasion and ultimately results in poor prognosis of tumor. The researchers⁴⁶ used HA to assemble a programmed tumor microenvironment (TME) unlocking nano-matryoshka NPs, which packages a huge CRISPR-Cas9 plasmid simultaneously encoding Pdl1 and Ptpn2 gene. The nanocomposite can realize nano-matryoshka-like release of Cas9 plasmid in the TME with abundant enzyme and preternatural intracellular oxidative stress. The highly efficient gene editing of both Pdl1 and Ptpn2 attenuated the immunosurveillance evasion and further enhanced the adaptive immunity, which disrupted Pd1/Pdl1 interaction and promoted tumor susceptibility to CD8⁺ T cells through deleting Ptpn2. Although HA can enhance cellular uptake of tumor cells with low cytotoxicity, it fails to assist in endosome escape of NPs⁹⁶. Therefore, the combination strategies need to be developed for promoting endosome escape of HA-modified NPs. In addition, as CD44 is highly expressed in liver, the HA-modified nanocarriers should be further designed to reduce liver-targeting.

4.1.1.2. Biotin

With the rapid growth of tumors, there is a higher demand for the corresponding substances supporting tumor proliferation, which results in the increasing of receptors on the surface of cytomembrane for transporting these substances^105,106. In addition, these substances have been studied as molecular targets to enhance cellular uptake in the field of nanomaterials. Biotin, also called Vitamin H, is a type of endogenous substance essential for the rapid growth of tumors¹⁰⁷. Liu et al.⁷⁶ combined biotinylated carboxymethyl chitosan with biotin ligands and aptamer-incorporated carboxymethyl chitosan with AS1411 ligands to construct a calcium-based dual-targeting nano vector for Cas9 plasmid nuclear targeting. The cytomembrane targeting capacity of biotin and AS1411 as well as the nuclear targeting of AS1411 made it possible for highly efficiency of Cas9 plasmid into the nucleus. Efficient Cdk11 gene knockout (>90%) and significantly down-regulated expression of other proteins related to tumorigenesis had achieved, including MMP-9 (∼90%), VEGF (>40%), and SURVIVIN (∼70%) (Fig. 5B). Similar to biotin, folic acid and glucose analogs have been widely applied in nano delivery systems in the previous studies^108,109. For CRISPR delivery system, folic acid was used to graft acrylate-methyl modified chitosan for a pH-sensitive nanocarrier, which coated Cas9 plasmid, DOX and thus possessed comparable anti-tumor efficiency³⁸. When using such ligands as active-targeting elements, attention should be paid to their off-target effects to the cells with high expression of receptors other than tumors, such as immune cells.

4.1.1.3. Phenylboronic acid

A certain of bio-derived products with natural excellent affinity properties cannot be developed for nanocarriers surface modifications due to its complex structures or lacking of chemical synthesis sites. On this condition, the artificially synthesized chemical small molecule is a preferred option. Phenylboronic acid (PBA) is a sialic acid analog which can specifically bind to sialic acid receptors, and sialic acid usually presents in cytomembranes and is positively correlated with tumor growth110, 111, 112. Tang et al.³³ designed a PBA conjugated cationic lipid, called PBA-BADP, which was self-assembled into nanocomplexes by electrostatic interaction with Cas9 mRNA, thereby resulting in enhanced cellular uptake. The overexpressed HPV18E6 and GFP genes in cervical cancer were selected as the targeted gene of Cas9 mRNA, and the results indicated that PBA-BADP NPs could effectively knock out HPV18E6 gene (with 18.7% indel) and GFP gene (with 14% indel) in HeLa cells, respectively (Fig. 5C). Additionally, PBA can conjugate polyethyleneimine (PEI) and 2,3-dimethylmaleic anhydride (DMMA)-modified poly (ethylene glycol)-b-polylysine (mPEG113-b-PLys100/DMMA) for the delivery of CRISPR-dCas9 system, which will be described in details in Section 5.3⁵⁶. Consideration of CRISPR-Cas9 nuclear delivery is critical for efficient genome editing, HA, biotin, or PBA modified nanocarriers can enhance the endocytosis, but lack the mechanism of endosome escape. Hence, the multifunctional nanocarriers heightening cellular uptake, endosome escape, and nuclear entry are required for effective CRISPR delivery, and multiple targeted modifications need to be developed. What's more, the wide expression of sialic acid receptors on cell membranes makes it difficult to achieve efficient targeting using PBA only.

4.1.1.4. Protein

Proteins derived from endogenous organisms to act as biological targets for nanocarriers possess the biggest advantage, i.e., its biocompatibility¹¹³. These macromolecular proteins can target the receptors on cytomembrane surface to promote endocytosis¹¹⁴. At present, the most classic protein anticancer drug on the market is PTX albumin. Human serum albumin (HSA) NPs show eminent characteristics, including biodegradability, non-immunogenicity and high stability. It has reported that HSA-modified NPs can target cancer cells through gp60 (a 60 kDa glycoprotein) and SPARC binding affinity¹¹⁵. For CRISPR-Cas9 delivery, HSA was reported to be employed as the surface modification of nanocarriers, and Cas9 plasmid targeting Pdl1 was mixed with stearyl PEI by a double emulsion method. In vitro results showed that a significant inhibition on PDL1 (21.95%) expression was observed in case of codelivery of Cas9 plasmid and siRNA in CT26 cells⁴⁷. Bovine serum albumin (BSA) can be also used to increase the cellular endocytosis efficiency of Cas9 plasmid DNA. The BSA-coating nanodiamond-based Cas9 DNA delivery system could effectively induce RS1 c.625C > T mutation, thus establishing the disease model of X-linked retinoschisis¹¹⁶. As a ligand of transferrin receptors 1 (TfR1, overexpressed on the surface of numerous tumor cells), H-chain apoferritin has also been applied to increase the endocytosis of CRISPR-Cas9 system¹¹⁷. The tetralysine-modified H-chain apoferritin as the nanocarrier packaging Cas9 RNP elements showed a satisfied genome editing efficiency (28.96%). In addition, Cas9 protein is also used to increase cell uptake. Wu et al.⁴⁸ used Cas9 protein to make a protein crown nanocarriers and the results showed that the protein crown of Cas9 directly increased the uptake of nanocarriers and realized endosome escape. Recently, Zhang et al.¹¹⁸ used human retrotransposon PEG10 to load Cas9 system. PEG10 was participated in RNA transport between cells in vivo, which modified the system with endogenous protein syncytin so as to increase cell membrane fusion and realize effective delivery of Cas9 in vitro. Compared with traditional virus-like particles (VLPs), this system shows no immunogenicity and has great application value.

Gelatin, as a natural protein derived from collagen hydrolysis, has widely used for the modification materials of nanocarriers, with high biocompatibility and biodegradability¹¹⁹. As we all known, gelatin is obtained by partial hydrolysis of collagen in skin, white connective tissue and bone of animals, and its protein components include various amino acids linked together by amide bonds, which mainly consists of glycine, proline, hydroxyproline and glutamate¹²⁰. The functional groups of amino acids and the N-terminal and C-terminal groups bring the amphoteric properties of gelatin. In acidic solutions, the polymer is positively charged, while in alkaline solutions, the polymer is negatively charged, which makes it ideal as a delivery vehicle for biomolecules¹²¹. In addition, it has been reported that gelatin-based biodegradable scaffolds can be used to prolong the delivery of adenoviruses and prodrugs in the TME, thus slowing the inactivation process in vivo and enhancing the therapeutic effect¹²². Previous study has confirmed that murine bone marrow DCs have a significant uptake effect on gelatin-modified NPs, so they are suitable for targeting antigens to DCs and ideal immune adjuvants¹²³. It has been reported that the first nanoparticles recommended as carriers for pharmaceutical applications were consisted of gelatin and albumin¹²⁴. Given that gelatin can bind to naked DNA or DNA-liposomes to increase gene expression, the cationic gelatin hydrogels containing plasmid DNA is fabricated to enhance gene expression of implanted DNA in muscle^125,126. Gelatin has been extensively reported used as the delivery tools for gene therapy, such as siRNA, mainly because this natural polymer derived from collagen contains integrin-binding cell adhesion peptide and metalloproteinase-sensitive peptide sequences, which allows the cell to trigger degradation and facilitating biological mechanisms, such as lysosomal escape^127,128. For the delivery of CRISPR-Cas9 system, gelatin has potential for achieving good circulation of nanocarriers in vivo due to its good biocompatibility. Since the preparation conditions of gelatin-based nanocarriers require high temperature and thermal gelation of gelatin usually leads to its brittleness, most of them are used as drugs, DNA carriers or microspheres. Therefore, it is urgent to design a scheme to mitigate the brittleness of gelatin so as to broaden its applications.

In addition, monoclonal antibodies of protein have also been used as the modification materials for nano system delivering CRISPR-Cas9 components. Monoclonal antibodies can be matched to become the target of biomarkers expressed on the surface of specific cells, such as tumor cell, macrophage, etc. Intercellular adhesion molecule-1 (ICAM-1), also called CD54, has demonstrated as the molecular target and biomarker for triple-negative breast cancer (TNBC)¹²⁹. ICAM-1 antibody was used for the delivery of Cas9 plasmid targeting Lipocalin 2 (LCN2), a well-known oncogene of breast cancer, by covalently conjugating a tumor-targeted nanolipogel system (tNLG). The results showed that the engineered tNLG could actively target TNBC and significantly inhibited tumor growth up to 77% with high genome editing efficiency of LCN2 (>81%) in an orthotopic TNBC model (Fig. 5D)¹³⁰. Rosenblum et al.¹³¹ engineered a lipid nanocarrier with epithelial growth factor receptor (EGFR) targeted the delivery of Cas9 mRNA and sgRNAs against PLK1, a tumor survival associated gene. Intraperitoneal injection of this antibody-targeted deliver nanocarriers demonstrated a captivating effect with ∼80% gene editing in vivo and inhibited ovarian tumor growth by up to 80%. Up to now, most of protein elements have utilized to increase the targeting of liposomes. Screening proteins with high specific expression in diseases as intelligent elements for CRISPR delivery will achieve high efficacy and low toxicity in the treatment of diseases. However, the protein coating increases the original size of the nanostructure, which makes it difficult to be further modified with harder endocytosis. At the same time, the organic solvents used in the synthesis process of nanocarriers damage the proteins' structure, which also limits the application of macromolecular proteins in CRISPR intelligent delivery.

4.1.1.5. Peptide

Compared with proteins/antibodies, short peptides show excellent properties of lower molecular weight, more simple structure, and easier chemical grafting, which makes it possible as the surface modification of nanocarriers to enhance the gene editing capacity. mHph3 peptide is consisted of mHph1 CPP fused with a CM18 fragment and internalizing RGD (iRGD), which is a modified form of Arg-Gly-Asp peptide and has high affinity to αvβ3/αvβ5 integrins and neuropeptide-1. Herein, these two different peptides were conjugated with the liposome-templated hydrogel NPs for the delivery of Cas9 protein targeting PLK1. It was demonstrated that the protein expression of PLK1 was significant inhibited by 60.4% and the survival of tumor-bearing mouse was obviously improved (Fig. 5E)⁹¹. Deng et al.⁵¹ employed iRGD as the modification materials of nanocarriers to enhance the cellular uptake by interaction with Integrin αv, thereby improving the CRISPR-Cas9-based genome editing. Additionally, CPP is composed of 10–30 amino acid residues and can interact with the cell membrane in multiple ways¹³², with the potential to delivery CRISPR-Cas9 components. For instance, Pepfect14 CPP was used to deliver Cas9 RNP and showed a high gene editing rate (∼80%) in HEK-293T cells¹³³. Moreover, three CPPs, including pH-sensitive H-peptide, EGFR-targeting P-peptide, and nucleus directing R-peptide were applied to modify the solid lipid nanocarrier and liposome for the delivery of Cas9 plasmid. The CPP modified nanocarriers could successfully knock out HUR oncogene and inhibit cell proliferation, metastasis, and resistance in oral squamous cell carcinoma cells⁸⁴. Cationic peptides were also used directly to couple with Cas9 protein for intelligent delivery of CRISPR-Cas9 system¹³⁴. Non-cationic peptides have the advantages of less off-target effects when used as nanocarriers. Hence, short peptides with high specificity can be screened through peptide libraries, which makes it possible to target cell populations with different gene expression.

4.1.1.6. Viral mimics

Recently, peptide- and small-protein-based structural virus mimics have been attracted increasing attention in terms of nanomaterial for the delivery of proteins and nucleic acid¹³⁵. It has been reported that the classical filamentous tobacco mosaic virus (TMV) mimics are developed by de novo synthetic methods¹³⁶. Human immunodeficiency virus (HIV) has a well-known cell-penetrating ability and simian virus 40 (SV40) shows a strongly capacity to bind nucleoporins of cell^137,138. Kong et al.¹³⁹ designed a peptidyl-VLPs (pVLPs) by mimicking the HIV and SV40 viruses, and encapsulated the Cas9 plasmid DNA to form biodegradable pVLPs with excellent cell/nucleus-targeting capacity and biocompatibility. This pVLPs could target the chemokine receptor CCR5 on cytomembrane by the V3 peptide of HIV virus (304RKSIHIGPGRAFYTTG321) and provided excellent nuclear localization for T antigen through the nuclear localization peptide (NLS, PKKKRKV) of SV40, and finally indicating a good delivery efficiency of Cas9 plasmid in CCR5-positive HeLa tumor cells (also known as Magi cells). Moreover, crossing the blood–brain barrier (BBB) is a crucial process for effective treatment of neurodegenerative diseases140, 141, 142, 143. Shen et al.²⁸ designed a traceable nano-biohybrid complexes through connecting the rabies virus glycoproteins (RVG) peptides with the polylysine and dopamine complex by PEG, which could permeate BBB by specific binding to the RVG receptors to efficiency delivery the Cas9 plasmid targeting Bace1 gene (the primary and rate-limiting enzyme for amyloid-β (Aβ) regeneration). The results indicated that this nanocomplex could knock out Bace1 gene and reduce amyloid-β, thereby improving the cognitive capacities of 2xTg-AD mice. Additionally, the low-density lipoprotein receptors are also used for BBB delivery of Cas9 system as a classical nanocarrier. For the overexpression of LRP-1 in BBB epithelial cells and gliomas, the nanocarrier targeting peptide angiopep-2 was prepared, which significantly increased the knockout efficiency of PLK1 gene in glioma cells of orthotopic GBM mouse models (Fig. 5F)¹⁴⁴. Similar to viral vectors, viral mimics-mediated nanocarriers should also be concerned about their immunogenicity.

4.1.1.7. Aptamers

As a class of emerging artificial ligands, aptamers, also known as “chemical antibodies”, has distinctive advantages, including small size, low cost, customized modification, uniform synthesis, and template nature, as compared with antibodies¹⁴⁵. Aptamers (with small molecular weight of 5–15 kDa) are composed of short chain nucleic acid sequences, and have the possibility of secondary structure formation. Previous researches have used aptamers-modified nanocarriers to delivery CRISPR-Cas9 system. For instance, MUC1-specific aptamers combined with AS1411 aptamers were employed for nuclear delivery of Cas9 plasmid to knock out focal adhesion kinase in MUC1 overexpressed tumor cells, thereby resulting in inhibition on tumor metastasis and cancer stemness and reverse of immune suppression¹⁴⁶. Previous evidence has reported that downregulation of β-catenin inhibits Pdl1 expression in tumor cells¹⁴⁷. Thus, AS1414 aptamers conjugated with TAT-NLS peptide and HA to form a polymer was used to deliver the Cas9 plasmid targeting β-catenin⁴⁵. The results of this study showed that the polymer significantly inhibited β-catenin with gene-editing efficiency of 40.2% and successfully reversed tumor-immunoescape and immunosuppression by improving the tumor cell killing capacity of CD8⁺ T cells⁴⁵. Similar to scFv, the selection of new specific-target aptamers is of great significance for the nano-targeting delivery system of CRISPR-Cas9 elements. Zhang et al.²⁹ utilized an anti-EpCAM (epithelial cell adhesion molecules) aptamers and polyamidoamine noncovalent conjugated onto the surface of the hollow mesoporous silica nanoparticle (HMSN) for the delivery of Cas9 plasmid to knock out EGFR gene. The engineering HMSN knock out gene specificity and effectively (with >60% EGFR-editing efficiency) without off-target effects and inhibited tumor growth up to 85% in hepatocellular carcinoma (Fig. 5G). Similar to other libraries, aptamers are also biological targets with strong affinity to specific components and weak affinity to other components, so that more meaningful specific aptamers are developed, especially for the delivery of CRISPR-Cas9 system.

4.1.2. Bio-derived membranes

Many natural biological cell membranes with low immunogenicity and good targeting can also be used for the delivery of CRISPR system148, 149, 150, 151, 152. Extracellular vesicles (EVs) are membrane-enclosed nanoscale particles released by many cell types and have been widely studied as delivery platforms for delivering various drugs, protein, and nucleic acid153, 154, 155, 156, 157. Among EVs, exosomes are the most commonly developed and widely used in delivery systems due to their appropriate size (40–160 nm)¹⁵⁸. Compared with liposome, metal, and polymer NPs, exosomes have superior properties of natural chamber for cargo loading, cell active-targeting, and natural intercellular information transmission159, 160, 161. Furthermore, exosomes can be modified by nanotechnology to make a delivery system with excellent tissue distribution. The most common method to create effective exosome is the addition of artificial liposomes. For example, Lin et al.¹⁶² fused exosome with liposomes of Lipofectamine 2000, a common commercially transfection reagent, to form a hybrid exosome, and the results demonstrated a high delivery efficiency of Cas9 plasmid in mesenchymal stem cells and chondrocytes (Fig. 6A)¹⁶³. Induction of receptive cells to produce engineered exosomes-packaged Cas9 RNP by chemical or biological approaches is also a promising strategy for effective gene editing. For instance, Yao et al.⁷¹ utilized the specific interaction between RNA aptamers and aptamers-binding proteins to enrich CRISPR RNP into exosomes (Fig. 6B). Cas9 protein was also recruited via chemically induced dimerization, and sgRNA could tethered by a viral RNA packaging signal and two self-cleaving riboswitches, and subsequently packaged into exosomes¹⁶⁴. The CAR-T technology has been used to modify chimeric antigen receptors on exosomes to achieve higher cellular uptake⁸⁸. Generally, exogenous RNP is not easy to be encapsulated into exosomes. The new proteins designed by protein heterodimerization techniques made part of their regions bind to Cas9 protein and exosomes proteins, respectively, thus increasing the loading efficiency of RNP¹⁶⁵. Effective encapsulation of human-derived exosomes can reduce the immunogenicity of the delivery system, and have the potential to develop an excellent delivery platform for clinical reagents, including CRISPR-Cas9 components. Whereas, such exosomes are difficult to be obtained and few methods have been reported to encapsulate therapeutic agents into exosomes, including CRISPR elements. The large-scale preparation of agents-loading exosomes is difficult, even with the commonly used electro-transfection technology, which leads to a high cost in industrial production.

Figure 6.

Bio-derived membranes modified intelligent nanocarriers for the delivery of CRISPR-Cas9 system. (A) Chondrocyte-targeting exosomes (CAP-Exo) fused with liposomes to form hybrid CAP-Exo for the effective delivery of Cas9 plasmid. Reprinted with the permission from Ref. 163. Copyright ©2022 Ivyspring International Publisher. (B) The specific interaction between RNA aptamers and aptamers-binding protein was utilized to enrich CRISPR RNP into exosomes. Reprinted with the permission from Ref. 71. Copyright © 2021 The Authors. (C) The tumor cytomembrane coated with zeolitic imidazolate frameworks was employed to deliver Cas9 RNP to meet the needs of cell-specific targeting. Reprinted with the permission from Ref. 69. Copyright © 2020 American Chemical Society.

Mammalian cytomembrane nano delivery system has been widely studied, especially for cancer cell membrane. The biomimetic nanocarriers based on tumor cell membrane coating technology have been applied in cancer therapy and vaccination166, 167, 168, 169. Due to the retained cytomembrane, these nanocarriers inherit the antigenic profile of the source cancer cells, and are suitable for the development of personalized medicines with low intrinsic immunogenicity, homotypic targeting, and complex antigenic profile^170,171. The cytomembrane of human breast cancer cell MCF-7 was used as the shell to coat the zeolitic imidazolate frameworks for the delivery of CRISPR-Cas9. When enhanced green fluorescent protein (EGFP) gene was used as the target of gene editing, such biomimetic nanocarriers could reduce the expression of EGFP by three folds (Fig. 6C)⁶⁹. Additionally, the biofilm-, biofilm modification- or biomimetic biofilm-delivery of CRISPR have certain application prospects. Nevertheless, its development is limited by the preparation process, which is concluded as following: i, it is difficult to extract the cell membrane through ultracentrifugation due to low yield; ii, the stability of the prepared nanocarriers is poor and it is difficult to prepare the nanostructures with regular shape.

4.1.3. Positively charged molecules

4.1.3.1. Cationic peptide

In addition to increase cellular uptake by binding to receptors, some molecules also have the ability to enhance non-specific uptake, such as R7L10 peptide. The R7L10 short peptide is composed of 7-arginines and 10-leucines, and the polyarginine short peptide is considered to be a classic CPP¹⁷². It is generally believed that the penetration effect of oligoarginine is triggered by its strong positive charge and easy combination with negatively charged cytomembranes. Strong positive charge also leads to the escape of NPs in endosomes. After being linked with the highly hydrophobic leucine short peptide, it becomes amphiphilic polymer, which can be prepared into NPs in aqueous phase. Park et al.²⁷ used R7L10 peptide to encapsulate the CRISPR-Cas9 system targeting Bace1, and the results revealed that it effectively inhibited Aβ-associated pathologies and ameliorated cognitive deficits in Alzheimer's mouse model. Many cationic viral peptides with multiple arginine also applied to enhance the cellular uptake for the delivery of CRISPR system, such as the transactivating translator (TAT) peptide of HIV-1 virus. For example, TAT-modified gold nanoclusters could cause gene editing of PCSK9 (Proprotein Convertase Subtilisin/Kexin Type 9), a key regulator of low-density lipoprotein metabolism, in vitro by ∼ 60%⁶⁸. Another TAT-modified gold nanoclusters for the delivery of sgRNA plasmid/Cas9 protein complex could significantly reduce PLK1 protein expression (>70%) in A375 cells (Fig. 7A)⁸⁶. Furthermore, peptide dendrimer Z22 and Endo-Porter composed of many polyarginines have been used for the delivery of CRISPR-Cas9 in previous studies^173,174. It has also been reported that single arginine was used to increase the cellular uptake of CRISPR-Cas9 components^175,176.

Figure 7.

Positively charged intelligent nanocarriers for the delivery of CRISPR-Cas9 elements. (A) Cationic transactivating translator (TAT) peptide-modified gold nanoclusters were used for the delivery of Cas9 plasmid. Reprinted with the permission from Ref. 86. Copyright ©2017 The Authors. (B) Cationic lipid-assisted nanoparticle (CLAN) efficiently delivered Cas9 mRNA and sgRNA both in vitro and in vivo. Reprinted with the permission from Ref. 40. Copyright © 2019 Elsevier Ltd. (C) DOTAP, a permanent cationic supplemental component, was added into traditional lipid nanoparticle for the delivery of Cas9 RNP. Reprinted with the permission from Ref. 188. Copyright © 2020 The Author(s). (D) Yarn-like DNA nanoclews coated with polyethyleneimine were used for the delivery of Cas9 RNP. Reprinted with the permission from Ref. 55. Copyright © 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. (E) Cationic carboxylated branched poly(β-amino ester)s with pH sensitivity was employed to encapsulate Cas9 RNP so as to achieve excellent cellular uptake, endosomal escape, and functional intracellular release of Cas9 RNP. Reprinted with the permission from Ref. 192. Copyright © 2019 The Authors, some rights reserved.

Except for arginine-rich short peptides, lysine-rich short peptides are also used to enhance the cellular uptake. The researchers used dNP2 peptide derived from transmembrane protein of BBB to deliver the CRISPR-Cas9 system¹⁷⁷. The peptide consists of multiple (KIKKVKKKGRK) repeats and has high targeting to cell membrane in vivo and in vitro, even for brain. KALA, as a common cationic membrane penetrating peptide, is usually utilized for gene editing delivery³⁶. Another nuclear localization sequence (PKKKRKV) of simian SV40 virus is also widely used¹⁷⁸. Except for its cationic properties, it can also strongly bind to nucleoporin. Therefore, it is used to efficiently deliver Cas9 protein into nucleus. Cationic peptide is a cell membrane penetrating peptide with relatively low toxicity and accessibility, but it has weak specificity for failing to target a specific membrane marker. Cationic peptide combined with nanotechnology can improve its tissue specificity. Synthetic peptide analogs also have good application prospects. Wang et al.⁷⁴ synthesized a helical polypeptide that used multiple cationic groups (pyridine and aminomethyl groups) to modify glutamate. Its special structure could adsorb plasmid DNA and siRNA, and resist enzymatic hydrolysis due to its special modification. Due to its positive electricity, it could escape from the endosome and achieve a high knockout rate. Nanocarriers with strong positive charge can usually induce endosome proton sponge effect and realize endosome escape. Hence, compared with non-cationic membrane penetrating peptides, cationic peptides can absorb protons and escape from endosomes. However, cationic peptides still have a non-negligible disadvantage, i.e., low specificity, which can be improved by matching with other intelligent elements to achieve effective and accurate delivery of CRISPR system.

4.1.3.2. Cationic liposome

Liposomes are the analog of cytomembranes, whose hydrophilic ends are inserted into water, and the hydrophobic ends form a bilayer through hydrophobic force¹⁷⁹. Liposomes have been used for gene transfection, among which cationic liposomes, such as, cationic oligopeptides, can increase the cellular uptake through an electrostatic effect^180,181. The difference between cationic lipid-assisted nanoparticle (CLAN) and other nanocarriers is that the methods of liposomes entering into cells may prefer membrane fusion rather than endocytosis^182,183. The targeting of CLAN is also related to its surface charge. Zhang et al.⁴⁰ used a poly (ethylene glycol)-block-poly(lactide-co-glycolide)-based CLAN to delivery Cas9 mRNA and sgRNA targeting the costimulatory molecule CD40, and found that CLAN successfully delivered CRISPR complex into DCs with effective genome editing of CD40 (Fig. 7B). Moreover, Cheng et al.¹⁸⁴ obtained multiple permanently cationic, anionic, zwitterionic, and ionizable sort lipids by changing the ratio of different liposome materials, called Selective ORgan Targeting (SORT). It was found that nanocarriers with different feeding ratios had different surface charges and organ targeting. Through specific liposome formulation, SORT liposomes targeting liver could completely knock out PCSK9 in serum and protein by delivering Cas9 complex targeting PCSK9 gene. Hence, optimization of CLAN allows targeted gene editing in different organs, such as, surface charge and surface modification. For example, optimized CLAN was used to target the delivery of CRISPR-Cas9 to macrophages¹⁸⁵, B cells, neutrophils, and monocytes¹⁸⁶.

CLAN can provide alterable cargo packaging capacity and also realize the co-delivery of RNA. Miller et al.¹⁸⁷ employed zwitterionic amino lipids as the non-viral system for the first successful co-delivery of Cas9 mRNA and sgRNA, thus reducing protein expression by >90% in cells. Furthermore, the researchers used CLAN to simultaneously load autoimmune diabetes-related peptide, Cas9 plasmid, and three kinds of sgRNAs targeting CD80, CD86, and CD40, which enhanced the cellular uptake of DCs and led to the generation and expansion of autoantigen-specific Tregs³⁹. Similar to other types of cationic nanocarriers, liposomes usually use electrostatic force to wrap CRISPR-Cas9 system, which may lead to unstable binding process and difficulty in controllable size, uniformity, and stability of nanostructures. Therefore, Wei et al.¹⁸⁸ developed a generalizable engineering approach to add permanently cationic component, such as DOTAP, into traditional lipid nanoparticle, which could solve the above problems (Fig. 7C). At present, most of liposomes studied are synthetic compounds, so they may have some potential toxicity as compared with the biomimetic nanocarriers.

4.1.3.3. Other positively charged materials

Other positively charged substances are also used for the delivery of CRISPR-Cas9, such as polyamide and polyurethane. As a classical polyamide, polyethyleneimine (PEI) is a cationic polymer that has been widely employed for CRISPR-Cas9 delivery to enhance cellular uptake and promote proton-sponge effect on endosome escape by using pH-responsive high-density ionizable amines¹⁸⁹. Sun et al.⁵⁵ reported a biologically inspired yarn-like DNA nanoclews (DNA NCs), which synthesized by rolling circle amplification (RCA), coated with PEI for the delivery of Cas9 RNP, to investigate the potential of gene editing efficiency and found that PEI-coated DNA NCs could enhance cellular internalization and endosome escape of Cas9 RNP, thus significantly inhibiting GFP expression up to 36% in vitro and ∼25% in vivo (Fig. 7D). PEI-β-cyclodextrin (PC) cationic polymer was used for Cas9 plasmid delivery and showed efficient genome editing at two genome loci, namely, hemoglobin subunit beta (19.1%) and rhomboid 5 homolog 1 (RHBDF1) (7.0%) in HeLa cells¹⁹⁰. Our recent studies also employed PEI to form a shell encapsulating metallic gold nanorods (AuNRs) or semiconductor copper sulfide (CuS) to realize the effective delivery of Cas9 RNP nuclear^17,28. Moreover, lipid-containing oligoaminoamides (lipo-OAAs) was screened from a compound library of sequence-defined oligo (ethylenamino) amides containing structural motifs for Cas9 RNP delivery, which had stable nanostructure formation, cellular uptake, and endosomal release¹⁹¹. Similar to the short peptide library, it takes amino polyethylene as the skeleton and can screen these substances with potential research prospects.

Poly(β-amino ester)s (PBAEs) are a kind of cationic polyurethane with low cytotoxicity and pH sensitivity, so that they have potential value for the delivery of negatively charged nucleic acids. Rui and Green¹⁹² developed a carboxylated branched PBAEs which could self-assemble into NPs for encapsulating Cas9 RNP. This hyperbranched PBAEs showed excellent properties, including enhanced cellular uptake, efficient endosomal escape and functional intracellular release of Cas9 RNP, which led to robust genome-editing efficiency with gene knock out of 75% and knock in of 4% in several cell types (Fig. 7E). PBAEs was also employed to deliver CRISPR/short hairpin RNA (shRNA) targeting HPV16 E7 gene, which showed an obvious downregulation of HPV16 E7 expression and inhibited the growth of cervical cancer cells and xenograft tumors in nude mice¹⁹³. In addition, arginine mimetic materials can also deliver CRISPR-Cas9 system with high efficiency and low toxicity. Researchers used aminoguanidine modified polyethylene glycol (PEG) as a hydrophilic shell for encapsulating periodic mesoporous organosilicas and Cas9 RNP, and the study results demonstrated decreased GFP expression (about 40%) in GFP-positive HT1080 cells¹⁹⁴.

Dendrimer polyamide macromolecules are also widely studied as cationic polymer materials for CRISPR delivery. Liu et al.⁵⁴ conjugated electron-deficient PBA ligand onto a cationic polymer with high grafting ratio to form a cationic PBA-rich dendrimer for the delivery of Cas9 RNP. The cationic dendrimer showed a high binding affinity with different types of proteins based on the combination of nitrogen-boronate complexation and cation–π and ionic interactions. Further studies about genome editing found that the PBA-rich dendrimer could enhance intracellular delivery of Cas9 RNP into various cell lines and showed high efficiency in CRISPR-Cas9 gene editing. Chitosan (CS), an inexpensive and ideal cationic biopolymer, has widely applied to the nanodelivery of drug with characteristics of biocompatibility, biodegradability and non-cytotoxicity¹⁹⁵. The positively charged RFP@CS NPs were applied to achieve genome editing by directly encapsulating Cas9 RNPs for gene knock out and donor DNA for gene knock in, respectively. It was found that RFP@CS could efficiently realize genome knock out in HEK293T, RAW264.7, HeLa, U2OS, and A549 cells, and showed an impressive efficiency of simultaneously delivery Cas9 RNPs and DNA donors into cells for HDR editing¹⁹⁶. Although synthetic polymer cationic materials increase the endocytosis of targeting cells, the relative cytotoxicity should not be ignored. At present, polymer cationic materials or modifications from biological sources are more in line with the requirements of intelligent delivery of CRISPR with high biocompatibility.

4.2. Micro- or cytoplasmic-environment responsive nanocomposites

It is well-known that bare, small-size, or positively charged nanocarriers are usually easily removed when used to deliver substance in vivo. A certain of materials, such as PEG, have been used to modify nanocarriers in order to reduce the cargo clearance during delivery with decreased cellular uptake and endosomal escape efficacy of NPs^52,197,198. Importantly, effective genome editing needs CRISPR-Cas9 system enter into the nucleus with pore size only 10–40 nm¹⁹⁹. The defects of CRISPR technology, such as off-target effects and immunogenicity, also puts forward higher requirements for its delivery nanomaterials. Therefore, intelligent nanocarriers for CRISPR system localization release have more potential application prospects.

Specific inherent characteristics of normal or pathological tissues environment can be utilized and developed into special functional delivery systems. Most of the pathological tissues have inflammatory environment, which increased the content of ROS. The increased ROS concentration in disease regions, such as tumors, results in upregulation of ROS decomposition-related enzymes. The increase of reductase in tumor cells will lead to the reduction of the whole tumor area together with hypoxia²⁰⁰. Accompanied by hypoxia and increased lactate, the pathological tissues show a lower pH condition. Most nanocarriers need to undergo a process of cellular internalization followed by endosomes escape, in which the pH value gradually decreases²⁰¹. Therefore, both high concentration of reductase and low pH are important elements for controlled delivery of CRISPR-Cas9 system (Table 2^{23,32,34,35,46,84,202}). For instance, the microenvironment-responsive nano delivery system has been developed widely for tumor therapy due to the significant particularity of TME. The design basis of TME-responsive nanocarriers is to target an increasing number of substances in the TME. Generally, different tumors have the common TME properties, such as unrestricted tumor growth induced by hypoxia and low pH203, 204, 205. Additionally, specifically expressed genes, such as CD68 in monocytes and macrophages¹⁸⁶ and miRNA in tumor cells²⁰⁶, can be designed as the intelligent elements for CRISPR delivery. In addition to respond to the pathological microenvironment, there are also reports of knocking down focal adhesion kinase to relieve stiff and fibrous stroma of tumor and increase the cellular uptake of Cas9 delivery system²⁰⁷. However, since it is not a standard model of micro- or cytoplasmic-environment responsive nanocarriers, we have no further discussion in this review.

Table 2.

Summary of micro/cytoplasmic environment or external stimuli-triggered CRISPR-Cas9 delivery nanocarriers for the treatment of various diseases.

Intelligent type	Classification	Studies description	Disease	Type	Gene	Ref.
Micro or cytoplasmic environment responsive nanocomposites	pH	The characteristics of nanocarriers that negatively charged in alkaline and positively charged in acid were utilized to self-assemble gold nanoclusters with Cas9 RNP, which realized E6 oncogene knockout at a low pH, thus inducing tumor cell apoptosis.	Cervical cancer (Hela cells)	RNP	E6	34
		A pH-sensitive poly (lactic co glycolic acid) (PEI-PLGA) was used to co-deliver CRISPR-Cas9 system targeting Cdk5 and paclitaxel, which achieved combined immunotherapy through the reduction of PDL1 and the ICD effect of paclitaxel.	Colorectal (CT26 cells) and melanoma (B16F10 cells)	Plasmid	Cdk5	35
		Three peptides modified liposomes, including pH-sensitive H-peptide, EGFR-targeting P-peptide and nucleus-directing R-peptide, were used to co-deliver epirubicin and Cas9 plasmid for gene-editing of HUR, thus reducing drug resistance and tumor metastasis.	Head and neck cancer (SAS cells)	Plasmid	HUR	84
		A pH-responsive 2,5-dihydro-2,5-dioxofuran-3-acetic acid bridged with polylysine and PEG were utilized to deliver CRISPR-Cas9 system dual-targeting STAT3 and RUNX1. The cationic complexes realized effectively genome-editing under weak acid conditions, and thereby significantly inhibited tumor growth.	Tumor heterogeneity (U87MG cells)	RNP	STAT3 &RUNX1	202
	Redox	Azobenzene-4,4′-dicarboxylic acid modified gold nanoparticles were used to achieve hypoxia-responsive Cas9 RNP release through azo bonds, and gene-editing of HSP90α enhanced the sensitivity of PTT.	Lung cancer (A549 cells)	RNP	HSP90α	23
		Hyaluronic acid (HA) decorated nanocomplexes composed of Ad-SS-GD and CP were used to realize controlled Cas9 RNP release in the reductive intracellular milieu, which showed high gene-editing activity on mutant KRAS gene and resulted in obvious tumor growth and metastasis inhibition.	Colorectal cancer (SW-480 cells)	RNP	Mutant KRAS	32
		ROS reductase, metalloproteinase and hyaluronidase-sensitive materials were used to make a multi sensitivity cascade response nanoparticle, which successfully knocked out the tumor Ptpn2 and Pdl1 genes to realize long-term immune memory.	Melanoma (B16F10 cells)	Plasmid	Pdl1 &Ptpn2	46
External stimuli-triggered nanoplatforms	Photo- or photothermal- controlled	DNA double strand was employed as a heat sensitive element to deliver Cas9 RNP targeting HSP90α gene, and thereby improved the efficacy of PTT triggered by copper sulphide nanoparticles.	Melanoma (A375 cells)	RNP	HSP90α	17
		4-(hydroxymethyl)-3-nitrobenzoic acid (ONA) photosensitive molecules were used to control the release of Cas9 RNP by upconversion nanoparticle, which could convert NIR light into ultraviolet, and thus achieved controlled genome-editing of PLK1 and inhibition of tumor growth.	Lung cancer (A549 cells)	RNP	PLK1	241
		The protector DNAs stably hybridizing with sgRNA were assembled on gold nanorod and thus realized controlled release of CRISPR system by NIR laser generated heat, which successfully knocked out PLK1 and induced apoptosis of tumor cells.	Lung cancer (A549 cells)	RNP	PLK1	242
	Microneedle-assisted	A dissolvable microneedle patch consisted of HA and collagen tripeptide was developed for the transdermal delivery of polymer-encapsulated Cas9 RNP targeting Nlrp3 and polymeric NPs containing dexamethasone, which alleviated skin inflammations and improved the efficacy of glucocorticoid therapy in mouse models of psoriasis and atopic dermatitis.	Inflammatory skin disorders (DC2.4 and 3T3 cells)	RNP	Nlrp3	243
	Ultrasound responsive	A sono-controllable and ROS-sensitive sonosensitizer-integrated metal–organic framework was utilized to deliver CRISPR-Cas9 system in a controlled manner. Ultrasound-triggered ROS interrupted the sulfide bond to realize controllable genome-editing of MTH1 gene, and resulted in tumor growth inhibition.	Lung cancer (A549 cells)	RNP	MTH1	50
		A microbubble-nanoliposomal particle was constructed to wrap Cas9 RNP targeting SRD5A2 gene, which increased the intake of hair follicle cells and achieved effective treatment of androgenic alopecia.	Androgenic alopecia (human DPC)	RNP	SRD5A2	244
		Liposome polymer hybrid nanoparticles were used to deliver the CRISPR-Cas9 system targeting O6-methylguanine-DNA methyltransferase (MGMT) gene. Based on the evidence that focused ultrasound-microbubbles increased the permeability of BBB, knockout of MGMT improved the drug sensitivity of temozolomide.	Glioblastoma (T98G cells)	Plasmid	MGMT	245

Ad-SS-GD, disulfide-bridged biguanidyl adamantine; BBB, blood–brain barrier; CP, β-cyclodextrin-conjugated low-molecular-weight polyethyleneimime; EGFR, epithelial growth factor receptor; HA, hyaluronic acid; ICD, immunogenic cell death; NIR, near-infrared; OS, osteosarcoma; RNP, ribonucleoprotein; ROS, reactive oxygen species.

4.2.1. pH

Unrestricted tumor growth results in widespread hypoxia in the tumor regions, so acidity is one of its remarkable characteristics. It is well-known that the pH of tumor regions can reach 6.0–6.5 and the pH of lysosomes is about 5.5²⁰¹. Therefore, some special functions can be realized in the tumor areas by using materials whose structure can be changed under weak acid conditions^208,209. Effective escape from low pH of endosomes is the prerequisite for functional Cas9 protein²¹⁰. Most of the cationic materials can actively deliver CRISPR-Cas9 and promote endosome escape through low pH-triggered proton-sponge effects. In general, cation, such as polyurethane-based nanocarriers, has considered to own certain pH sensitivity, and its pH sensitivity is caused by its absorption of protons, which is similar to the principle of endosome escape³⁸. Except for PBAEs of polymer cation that we have described, the multifunctional pH-sensitive amino lipids were also reported by Sun et al.²¹¹ for the delivery of Cas9 plasmid. The amino lipids showed pH-responsive hemolysis and efficient genome editing with downregulation of GFP up to 80% in NIH3T3-GFP cells.

Most of classic cationic materials have certain cytotoxicity and high plasma clearance, and some strategies have been developed to improve the biocompatibility and stability of cationic materials. PEG modification is the most commonly used method with a certain shielding side effect on charges and often needs to be used in conjunction with other acid-sensitive linkages. Liu et al.²⁰² synthesized a pH-sensitive 2,5-dihydro-2,5-dioxofuran-3-acetic acid (CA) to bridge the polylysine-g-poly (ethylene glycol) (PLys100-CAmPEG₇₇) and self-assemble nanostructure for the delivery of Cas9 RNP, called nanoRNP. This nanoRNP could effectively release cationic nanocores packaging Cas9 RNP in TME of low pH (∼6.5). Further results of genome editing efficiency showed that nanoRNP could effectively knock out EGFP with expression downregulation of 40%, signal transducer and activator of transcription 3 (STAT3) of 55%, and Runt-associated transcription factor 1 (RUNX1) of 59% in U87MG cells, respectively (Fig. 8A). Additionally, the PEG corona was covalently linked with the cationic copolymer poly(ethyleneimine)-poly (lactic-co-glycolic acid) (PEI-PLGA) by acid cleavable bond, for the co-delivery of Cas9 plasmid and chemotherapeutic agent, PTX³⁵. Cas9 plasmid specifically knocked out Cdk5 and PTX induced ICD effects, which could downregulate the expression of Pdl1, reduce the population of Tregs and enhance the polarization of M2 to M1 in the TME.

Figure 8.

Micro- or cytoplasmic-environment-responsive nanocomposites for the delivery of CRISPR-Cas9 system. (A) pH-responsive 2,5-dihydro-2,5-dioxofuran-3-acetic acid was used to synthesize the polylysine-g-poly (ethylene glycol) for the delivery of Cas9 RNP and effective release of Cas9 RNP in tumor microenvironment (TME) of low pH. Reprinted with the permission from Ref. 202. Copyright © 2019 American Chemical Society. (B) Acrylate or imidazole-based monomers were employed to introduce the glutathione-degradable crosslinker for the delivery of Cas9 RNP. Reprinted with the permission from Ref. 67. Copyright © 2019 The Author(s). (C) ATP-sensitivity nanoscale zeolitic imidazole frameworks (ZIF-90) delivered Cas9 protein with high efficiency. Reprinted with the permission from Ref. 225. Copyright © 2019 American Chemical Society. (D) DNA nanoflowers were developed to deliver the miR-21 for controlled release of Cas9 RNP. Reprinted with the permission from Ref. 206. Copyright © 2020 Elsevier Ltd. (E) The sgRNA sequences incorporated with a Dicer substrate sequence siRNA was polymerized by rolling circle transcription to form nanocarriers with Cas9 proteins, called poly-RNP, which achieved controlled release of Cas9 RNP by the endogenous double-strand RNA specific ribonuclease in cytoplasm. Reprinted with the permission from Ref. 235. Copyright © 2017 Elsevier B.V. (F) PUN@Cas-PT, a programmable unlocking nano-matryoshka system, was used for the accurate delivery of Cas9 plasmid through metalloproteases and hyaluronidase (HAase)-responsive, endogenous ROS-sensitive, and iRGD modification. Reprinted with the permission from Ref. 46. Copyright © 2021 The Authors.

Other compounds capable of absorbing protons can also be used as acid-responsive elements. Nanoscale zeolitic imidazole frameworks (ZIFs) have been reported for intelligent deliver of CRISPR-Cas9 system due to proton-sponge effects of imidazole linker in endosomes²¹². ZIF-8 capsulating Cas9 RNP enhanced endosome escape and resulted in reduction of GFP expression up to 37% over 4 days. Ju et al.³⁴ modified gold nanoclusters (AuNCs) with carboxylic acid ions to form a negatively charged shell, which easily self-assembled and attached positively charged Streptococcus pyogenes Cas9 (SpCas9) endonuclease under physiological conditions. The assembly and disassembly process of SpCas9-AuNCs in low pH conditions enabled the delivery of SpCas9 into cytoplasm and nucleus. SpCas9-AuNCs was also employed to effectively knock out HPV18 E6 oncogene in cervical cancer cells with high biocompatibility. Ren et al.²¹³ used the methoxy-poly(ethylene glycol)-b-poly(2-(azepan-1-yl)ethyl methacrylate) (mPEG-PC7A) to assemble nanocarriers. The designed PC7A group accepted the proton and changed into a hydrophilic group in the endosome environment as it was a hydrophobic group at physiological pH. Additionally, some cationic liposomes can produce certain deformation after absorbing protons, and facilitate membrane hexagonal transformation, thereby resulting in endosome escape²¹⁴. Although relatively single the pH-responsive elements are, there are promising in nanocarriers design for tumor targeted delivery and endosomal escape. However, the variable TME makes it difficult for simple pH-responsive elements to achieve effective acid-sensitivity mediated nano-delivery. Since the pH range of tumor cells is similar to the endosomes of normal cells, this pH-responsive elements targeting endosomes is widely applicable with the potential to become the main development direction in the future.

4.2.2. Redox

As high concentrations of ROS in TME, the corresponding scavenger enzymes also increased, mainly including glutathione (GSH) reductase^215,216. Disulfide bond is the most classical reduction sensitive bond for GSH and has been used to deliver CRISPR-Cas9 elements. For instance, Rui et al.²¹⁷ synthesized a reducible branched ester-amine quadpolymers (rBEAQs) by using oligomeric branched cationic polymer with disulfide bond as the connecting arm. The hyperbranched rBEAQs packaged Cas9 plasmid/sgRNA by electrostatic adsorption and could trigger reduced cargo release in cytosolic environment, subsequently achieve 40% gene editing of GFP in HEK-293T cells. In addition, acrylate or imidazole-based monomers have been used to introduce the GSH-degradable crosslinker for the delivery of Cas9 RNP. Chen and coworkers⁶⁷ took Cas9 RNP as the core and modified it with a mixture of cationic and anionic acrylate monomers, an imidazole-containing monomer, GSH-degradable crosslinker, and acrylate mPEG and acrylate PEG conjugated ligands to form a nanocapsule (NC) (Fig. 8B). The results revealed that the GSH-responsive NC could effectively escape from endosome and showed a high efficiency of genome-editing up to 79.1 ± 0.6% without any apparent cytotoxicity in HEK cells. Recently, our research also modified CuS NPs with thiol-modified DNA to enhance Cas9 RNP release responded by GSH in tumor cytoplasm¹⁷.

In addition, some nitrogen-containing compounds also have reductase sensitivity. Our other studies have reported a hypoxia-responsive azobenzene linker, azobenzene-4, 4′-dicarboxylic acid (p-AZO) for the delivery of Cas9 RNP²³. The N N double bond of p-AZO could be reduced and yield aniline derivative in hypoxia-induced imbalance of cellular redox states of tumor regions^218,219. In our research²⁴, the thiol-modified PEG coating Au nanorods (AuNRs) as the core and crosslinked with Cas9 RNP with p-AZO, and finally wrapped with PEI. It was found that the Cas9 RNP could be effectively released in hypoxia TME and enhance 980 nm near-infrared (NIR)-triggered mild-PTT when delivering Cas9 RNP targeting HSP90α, a classical heat shock protein. 2-Nitro-1h-imidazole is also an effective reductase sensitive bond^220,221. After reduction, it causes bond fracture or change of material hydrophobicity, but it is not used in CRISPR delivery system at present. Compared with nitrogen-containing compounds, disulfide bonds often have lower cytotoxicity. Therefore, as a component of intelligent gene editing system, its safety needs to be further verified. In addition, such sensitive elements can only be used in diseases with elevated reductase, such as tumors or inflammation.

4.2.3. ATP

As the primary energy source for cellular activity, the ubiquitous biomolecular adenosine triphosphate (ATP) has attracted accumulating attentions due to the huge differences between intracellular concentrations (1–10 mmol/L) and extracellular environment (<5 μmol/L)^222,223. The integrity of nanoscale zeolitic imidazole frameworks (ZIF-90) have shown to be disrupted by intracellular ATP, with the mechanisms of the competitive coordination between ATP and Zn²⁺ inducing the collapse of ZIF-90 in cytoplasm²²⁴. Wang et al.²²⁵ designed a ZIF-90/protein nanocomplex constructed by imidazole-2-carboxaldehyde (2-ICA), Zn²⁺, and Cas9 nuclease (Fig. 8C). The ZIF-90/protein nanocomplex can be disintegrated and release Cas9 proteins in the presence of ATP, and effectively escape from endosome through the protonation of 2-ICA within the acidic endosome that drives a proton sponge effect. Further genome editing efficiency study showed that it knocked out the GFP expression up to 35% in HeLa-GFP cells. Nevertheless, ATP-sensitive elements appear to have a promising effect in controlling release of the CRISPR system, whereas the extracellular targeting modifications should be required to achieve accurate delivery, since the extracellular ATP is non-specific.

4.2.4. miRNA

As an important members of non-coding RNA, microRNAs (miRNAs) with the length between 21 and 24 nucleotides have been extensively studied in various physiological and pathological processes^158,226. Multiple miRNAs have been reported to be overexpressed in tumors and participated in the crucial process of tumor occurrence and progression^227,228. A miRNA-triggered CRISPR-Cas9 platform had been designed and the study results demonstrated that the CRISPR-Cas9 platform could be turned on by specific endogenous or exogenous miRNAs and applied to be miRNA sensors and cell-type-specific gene editing tools²²⁹. Based on the mentioned research, Shi et al.²⁰⁶ developed an DNA nanoflowers (DNF) containing MUC1 aptamer by rolling circle transcription (RCT) for the delivery of miR-21 (a well-known miRNA which was overexpressed in tumors and could promote tumor progression^230,231) responsive Cas9 RNP (Fig. 8D). In this study, the DNA nanomaterials were used as DNA aptamers for miRNA-responsive structure for Cas9 RNP release by toehold-mediated strand displacement²³². The results demonstrated that the DNF delivery platform could effectively release Cas9 RNP triggered by miR-21 in cytoplasm, and induced EGFP downregulation (∼21%) in HeLa-EGFP cells. Nevertheless, the huge size of DNF (around 500 nm) limited its applications, and thus pony-size nanocarriers for miRNA-responsive CRISPR-Cas9 platform delivery should be developed. Moreover, the development of miRNA-responsive element with high specificity also faces huge challenges in term of selecting the appropriate miRNA in the clinical applications.

4.2.5. Nuclease

As a promising RNA polymerization technique, RCT enables the synthesis of long RNA strands with repetitive sequences²³³. Compared with the monomeric RNAs, RCT possesses the potential of producing polymeric RNA so as to form much stronger nanocomplexes with cationic polymers²³⁴. Multiple responsive methods can be developed through designing the template sequence for RCT, such as miRNA-²⁰⁶ or ribonuclease-responsive²³⁴. For CRISPR-Cas9 delivery, the sgRNA sequences incorporated with a Dicer substrate sequence siRNA was polymerized by RCT and formed nanocarriers with Cas9 proteins, called poly-RNP (Fig. 8E)²³⁵. The polymeric RNP of poly-RNP NPs would be cleaved into monomeric RNP by the endogenous double-strand RNA specific ribonuclease in cytoplasm. Moreover, the ploy-RNP NPs enhanced the serum stability and genome editing due to the increased production of monomeric active RNPs, thus inducing GFP gene disruption of 58% in GFP-expressed HeLa cells.

DNAzyme (DNA-based catalysts), first reported in 1994, has great potential of RNA cleavage²³⁶. Up to now, multiple types of DNAzyme have been reported to be used for catalyzing RNA/DNA cleavage, ligation and other reactions²³⁷. RNA-cleaving DNAzymes triggered by metal ions have been extensively used as therapeutic agents for gene therapy^238,239. Based on the RNA cleaving properties of DNAzyme, Zhu et al.²⁴⁰ designed a DNAzyme-controlled genome editing system through conjugating Cas9 RNP with streptavidin-based DNA tetrad and DNAzyme. A Y-shaped DNA was linked to sgRNA via DNAzyme based on the principle of complementary base pairing, thus realizing the control of DNAzyme. The results showed that this nanosystem could effectively release Cas9 RNP through Mn²⁺ triggered DNAzyme cleavage and process an excellent genome editing efficiency in HeLa or MDA-MB-231 cells. Whereas, the cellular DNAzyme with high catalytic activity is dependent on the intracellular sufficient cofactors, such as Pb²⁺, Zn²⁺, and Mn²⁺. Therefore, inorganic metal nanocarriers in combination of DNAzyme system or DNAzyme-triggered CRISPR system are also a feasible strategy to achieve controllable delivery.

4.2.6. Hierarchical-responsive

At present, the delivery nanocarriers of CRISPR-Cas9 gradually become more intelligent and diversified. A variety of responsive elements can often be used simultaneously to obtain better delivery effects both in vitro and in vivo. Recently, a novel delivery system termed programmable unlocking nano-matryoshka system based on the characterizations of TME was developed by Yang et al.⁴⁶ for the delivery of Cas9 plasmid co-coding Pdl1 and Ptpn2 (Fig. 8F). This matryoshka-like PUN@Cas-PT NPs consisted of metalloproteases and hyaluronidase (HAase)-responsive corona, and endogenous ROS-sensitive core, and then prolonged blood circulation by surface PEGylation and enhanced nucleus localization through iRGD modification. The successful nucleus localization of Cas9 plasmid processed highly efficient genome editing of Pdl1 and Ptpn2, and thus triggered cascade amplified adaptive immune response. To date, increasing nano-delivery systems tend to achieve multi-level precise delivery, so that potential hierarchical-response should be developed for precise delivery of CRISPR systems due to its obvious off-target effects and immunogenicity. For multi-responsive nanocarriers, the cytotoxicity and side effects of intelligent elements should also be considered to meet the clinical requirements.

4.3. External stimuli-triggered nanoplatforms

Exogenous stimulus-responsive nanoplatforms can control the gene editing efficiency in a spatiotemporal manner. Compared to the active-targeting or micro-/cytoplasmic environment-responsive nanocarriers, this nanoplatform has great potential for precise delivery of CRISPR-Cas9. Herein, we summarize the recent progress of external stimulus nanocarriers for the delivery of gene editing, including light irradiation, ultrasound/magnetic and microneedle, as shown in Table 2^17,50,241, 242, 243, 244, 245.

4.3.1. Photo/thermal-controlled

Photo or photothermal-responsive gene editing delivery triggered by light irradiation provides a remote control of CRISPR-Cas9 system with high spatiotemporal resolution. Photo-responsive molecules have been used for precise and remote temporal and spatial control of gene editing, such as o-nitrobenzyl moieties. Pan et al.²⁴¹ designed a NIR upconversion-activated genome editing platform that constructed by photo cleavable 4-(hydroxymethyl)-3-nitrobenzoic acid covalently linked Cas9 RNP and upconversion nanoparticle (UCNP) (Fig. 9A). This platform realized precise release of Cas9 RNP in cytoplasm by using UCNP to convert NIR to high-energy UV, and surface coating PEI to improve endosome escape, and thus achieved gene editing of PLK1 gene up to 32.2% in A549 cells upon NIR irradiation. Photo-cleavable linker (PC-linker) has great potential for UV illumination-controlled intelligent delivery. The binding-mediated protein corona (BMPC) was developed for the delivery of Cas9 RNP and realized UV light-responsive release of Cas9 RNP by using PC-linker with two cleavage sites to connect a short thiol-labeled oligonucleotide (SH-DNA) modified gold nanoparticles (AuNPs) to sgRNA⁴⁸. This UV-sensitive BMPC platform solved the problems of traditional protein corona, i.e., irreversible binding246, 247, 248 and protein leakage^249,250, and demonstrated successful gene editing in HeLa and HEK-293T cells with high stability and low cytotoxicity. Alternatively, Pu et al.²⁵¹ constructed a photolabile semiconducting polymer nanotransducer (pSPN) for remote controlled genome editing using 680 nm NIR irradiation. This pSPN nano delivery achieved NIR regulation of Cas9 plasmid using a ¹O₂-generating backbone and PEI brushes grafted with ¹O²-cleavable linkers. For gene editing efficiency, it showed that this nanocarriers afforded 15- and 1.8-fold enhancement in knock-out efficiency relative to the nonirradiated controls in HeLa cells and mice, respectively. Nevertheless, nanocarriers triggered by light at a specific wavelength usually have a poor photo/thermal stability, which needs to be protected from the corresponding light when stored or used.

Figure 9.

Spatiotemporal delivery of CRISPR-Cas9 system by external stimuli. (A) Photo cleavable 4-(hydroxymethyl)-3-nitrobenzoic acid was covalently linked with Cas9 RNP and upconversion nanoparticle (UCNP) for precise release of Cas9 RNP in cytoplasm by using UCNP to convert NIR into high energy UV. Reprinted with the permission from Ref. 241. Copyright © 2019 The Authors, some rights reserved. (B) Cationic gold nanorod was used to delivery Cas9 plasmid fused with heat-inducible promotor, Hsp70, so as to achieve photothermal-controlled Cas9 plasmid activation. Reprinted with the permission from Ref. 260. Copyright © 2021 Wiley-VCH GmbH. (C) The T-CC-NPs realized controllable cytoplasmic release of Cas9 RNP by NIR-triggered Ce6-mediated photodynamic therapy. Reprinted with the permission from Ref. 51. Copyright © 2020 The Author(s), some rights reserved. (D) Au nanorods was employed to delivery controllable Cas9 RNP in a NIR laser-activated manner, in which protector DNA was stably hybridized with the target binding domain of sgRNA. Reprinted with the permission from Ref. 242. Copyright © 2020 American Chemical Society. (E) Microbubbles conjugating nanoliposome was used for intelligent delivery of Cas9 RNP through ultrasound-activated method. Reprinted with the permission from Ref. 244. Copyright © 2019 Elsevier Ltd. (F) Ultrasound-propelled gold nanowire was used for effective deliver of Cas9 RNP, which could be achieved by ultrasound irradiation mediated plasma-membrane penetration. Reprinted with the permission from Ref. 267. Copyright © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. (G) Magnetic nanoparticle was synthesized based on FeCl₃·6H₂O for controlled release of Cas9 plasmid in a magnetic strength-dependent manner. Reprinted with the permission from Ref. 273. Copyright © 2020 The Author(s). (H) Microneedle patches were used to assist supramolecular polymer containing Cas9 RNP for transdermal delivery. Reprinted with the permission from Ref. 243. Copyright © 2021 The Authors, some rights reserved.

Generally, photo or photothermal-responsive nanocarriers possess multiple functions for the treatment of diseases, involving in PTT or PDT that has been approved to be used in clinical practice252, 253, 254. Previous studies have indicated that PTT/PDT can induce ICD with the properties of non-invasiveness, spatial specificity and reversibility^255,256. It is well-known that during the progress of ICD, tumor-associated antigens released from the dying tumor cells promote DCs maturation, thereby inducting the activation of T cells to elicit antitumor immune responses^257,258. Tang et al.²⁵⁹ developed a Cas9 plasmid fused with heat-inducible promotor, Hsp70, for programmable gene-editing. Based on the above research, they recently designed a photothermal genome editing platform by using a cationic gold nanorod (AuNR) for the delivery of photothermal-controlled Cas9 plasmid targeting Pdl1 (Fig. 9B)²⁶⁰. This nanoplatform realized mild hyperthermia to induce both ICD and Pdl1 knock out by irradiation of the second near-infrared-window (NIR-II) light. Further gene editing efficiency showed 39.7% indel frequency in vitro and 19.3% in vivo. The Pdl1 and ICB combination strategies obviously inhibited primary and metastatic tumors and exhibited long-term immune memory effects against both rechallenged and recurrent tumors.

The ROS generated by light-triggered PDT can destroy the membrane of endosome in cytoplasm, which results in endosome escape of nanocarriers. Deng et al.⁵¹ used the nitrilotriacetic acid (NTA)-modified liposomes (NTA-SS-PEG-PCL) and a photosensitizer Ce6 self-assembly formed nanocarriers for encapsulating Cas9 RNP, and then the surface was coated with iRGD-modified cationic copolymer (iRGD-PEG-pAsp), called T-CC-NPs. The T-CC-NPs enhanced cellular internalization via integrin-iRGD binding and promoted endosome escape by Ce6 generating ROS upon NIR irradiation, and thus successfully released Cas9 RNP into cytoplasm in a NIR-responsive manner. The genome editing of NRF2 gene (critical gene in the cell antioxidant response) and Ce6-mediated PDT showed a synergistic effect in the treatment of cancers (Fig. 9C)⁵¹. Additionally, Verteporfin (VP), an FDA-approved ophthalmological drug, is used as a photosensitive molecule inserted into the lipid bilayer for the delivery of Cas9 RNP in a light-controlled manner²⁶¹. VP reacted with available oxygen molecules and generated singlet oxygen within a fraction of a second immediately under the light irradiation at 690 nm, thereby oxidizing unsaturated liposomes to release Cas9 RNP. For delivery efficiency, Cas9 RNP showed 77% genome editing in zebrafish²⁶².

As a natural genetic material, DNA serves as protector DNA for controlled delivery of drugs²⁶³ and gene editing^75,79. Peng et al.²⁴² utilized AuNRs as the carriers to construct a NIR laser activated genome-editing nanomachine by being decorated with dozens of protector DNAs that stably hybridized with the target binding domain of sgRNA (Fig. 9D). This NIR-activated nanomachine could achieve precise release of sgRNA ins the targeted cells and demonstrated highly efficient gene editing of EGFP and EMXI genes in A549 and HEK-293T cells, with ∼70% downregulation of PLK1 mRNA levels in A549 cells. Furthermore, our recent study¹⁷ designed a NIR-controlled synergistic therapeutic nanoplatform including a protector SH-DNA for binding sgRNA and CuS NPs, and finally modifying PEI for promoting endosome escape in living cells. For the excellent photothermal conversion performance of CuS NPs, this NIR light-controlled nanoplatform could precisely release Cas9 RNP in cytoplasm by photothermal-triggered chain unwinding of SH-DNA/sgRNA. Synergistic integration of NIR triggered mild-PTT and genome-editing of HSP90α could achieve enhanced inhibition of tumor growth.

4.3.2. Ultrasound/magnetic-responsive

Ultrasound (US)-activated microbubbles (MBs) strategy has been employed to increase the permeability of BBB and cytomembrane for the delivery of therapeutic agents, with the properties of non-invasion, reversibility, and site-specifity^264,265. Yang et al.²⁴⁵ utilized the MBs to be conjugated with lipid-polymer hybrid nanoparticles (LPHNs) and cRGD peptide to construct MBs-LPHNs_pCas9/MGMT-cRGD complexes for the delivery of Cas9 plasmids targeting O6-methylguanine-DNA methyltransferase (MGMT) gene, a crucial gene for Temozolomide (TMZ) resistance. This complex could effectively cross the BBB under US irradiation and enhanced the cellular uptake by targeting integrin αvβ3 receptors, thereby achieving excellent gene-editing and restoring the sensitivity of GBM cells to TMZ. Furthermore, MB was also used to enhance the delivery efficiency of Cas9 RNP by conjugating nanoliposome (NL) for the treatment of androgenic alopecia (Fig. 9E)²⁴⁴. This MB-based nanocarriers with topical administration induced increased delivery efficiency of Cas9 RNP to the dermal papilla cells (DPC) by MB cavitation produced sonoporation under high acoustical wave US frequency (1–5 MHz)²⁶⁶. It demonstrated high gene editing efficiency up to 71.6% when targeting steroid type II 5-alpha-reductase (SRD5A2), which was responsible for the pathogenesis of male pattern baldness, and showed ∼70% mRNA levels downregulation in vivo.

Metal-based nanomotors have great potential of developing spatiotemporally controlled genome-editing delivery, which converts external energy into mechanical motion. The US-propelled gold nanowire (AuNW) was reported as the active nanomotors for the delivery of Cas9 RNP (Fig. 9F)²⁶⁷. The AuNW-based nanomotors was self-assembled by conjugating cysteine residues within Cas9 RNP onto the surface of AuNW through a reversible disulfide linkage, which resulted in Cas9 RNP release in cytoplasm by intracellular GSH triggering thiol bridge degradation. The results indicated that this nanomotors could directly penetrate into plasma-membrane with 5 min under US irradiation and thus achieved high genome editing efficiency of 80% Gfp knockout in Gfp-expressed B16F10 cells. Moreover, US can also be used to control the intracellular release of CRISPR system. For example, the metal organic frameworks (MOFs) NPs that generate ROS by ultrasonic control can disrupt sulfide bonds after receiving acoustic energy so as to realize the controllable release of CRISPR elements⁵⁰.

Recently, magnetic nanoparticle (MNP) has been developed for spatial controlled delivery of therapeutic reagents, including gene therapy268, 269, 270, 271. For example, MNP was synthesized basing FeCl₃·6H₂O and surface functionalized with PEI for the delivery of Cas9 plasmid, and achieved controlled gene editing in HEK cells and rat primary cortical neurons in a time-dependent and magnetic strength-dependent manner²⁷². PEI-modified Fe₂O₃-based MNP was also used to deliver Cas9 plasmid and realized effective gene editing in HEK293 cell line (Fig. 9G)²⁷³. In addition, MNP also shows potential for the delivery of CRISRP elements when combined with viral vector. In spite of unique properties of transducing gene expression in mammalian cells without integrating into host genome, baculoviral vectors (BV) is ineffective in vivo due to the presence of serum components to inactivate BV transduction. Hence, Zhu et al.²⁷⁴ employed MNP to constructed a nanocomplex by using BV and realized magnetic field-driven BV transduction and controlled release of CRISPR-Cas9 system in various tissues. This MNP-BV nanoplatform with large DNA loading capacity could activate BV transduction in vivo in the presence of magnetic field, and thus had the potential to facilitate multiplexed and controlled gene editing in vivo.

Combination of magnetic and US responsive strategies are potential methods for achieving accurate tissue localization and enhanced cellular endocytosis. Dong et al.²⁷⁵ designed a Cas9 plasmid loadable magnetic/US sensitive nanodroplets, called PMUNDs. PMUNDs were successfully accumulated in tumor regions through leaky tumor vessels driven by magnetic field, which enhanced the efficiency of entering into cytoplasm by US caused sonoporation. Although magnetic or US responsive manners present excellent performance for the delivery of CRISPR system, its disadvantages of strong cytotoxicity and difficult metabolism cannot be neglected, especially for MNP, and thus potential magnetic and/or US responsive nanocarriers with high biocompatibility should be developed.

4.3.3. Microneedle-assisted

As a promising delivery tool, microneedle (MN) patches consist of miniaturized needles for perforating the stratum corneum and delivering therapeutic agents to the dermis in a minimally invasive manner276, 277, 278. MN system for transdermal drug delivery has been widely used for the detection and treatment of various diseases279, 280, 281, 282. Wan et al.³² previously designed a supramolecular polymer (SP), called CP/Ad-SS-GD, which consisted of β-cyclodextrin-conjugated PEI (CD-β-PEI, CP) and biguanidyl group and adamantine linked by disulfide bond (Ad-SS-GD) for the delivery of Cas9 RNP. Based on the previously mentioned evidence, they developed a dissolvable MN patch using HA and collagen tripeptide (CTP) as the matrix materials, which embedded with Cas9 RNP loaded SP and SP packaging dexamethasone (Dex) for the transdermal co-delivery of two nanoformulations (Fig. 9H)²⁴³. Upon insertion into the skin, the MN could be dissolved rapidly and resulted in two SP nanoformulations internalized by keratinocytes and surrounding immune cells, and thus achieved Dex and Cas9 RNP targeting Nlrp3 gene release in cytoplasm. With the capacity of inducing dilation of nuclear pores, Dex promoted nuclear entry of Cas9 RNP and disrupted Nlrp3 inflammasomes, which enhanced the sensitivity of glucocorticoid therapy in the inflammatory subcutaneous layers. This platform provides innovative insights into the rational design of transdermal delivery for CRISPR-Cas9 system.

5. Nanotherapeutic delivery of new CRISPR genome editors for gene therapy

Except for classical CRISPR-Cas9 gene editing technology, a serials of novel genome editors, including the CRISPR-associated protein Cas12a (previous known as Cpf1), CRISPR-Cas13a, CRISPR/nuclease-inactivated Cas9 (dCas9), and other CRISPR-base editors (Fig. 1B–D), have been developed for the treatment of multiple diseases as delivered by nanocarriers.

5.1. CRISPR-Cas12a (Cpf1)

As an attractive alternative to CRISPR-Cas9, the Cas12a (an endonuclease from the type V-A CRISPR system) is composed of single CRISPR RNA (crRNA) with T-rich protospacer adjacent motif (PAM) sequence to bind and cleave double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) targets, which has been applied in both in vivo genome editing and in vitro DNA assembly^6,283. The improved delivery methods for Cas9 RNP have been employed to enhance the delivery of Cas12a RNP. For example, a cationic PAsp (DET) coated Gold-based NPs (CRISPR-Gold) was designed to deliver Cas12a RNP, which caused rapid release of RNP by endogenous glutathione⁷⁹. The CRISPR-Gold vehicle, as a new brain-targeted therapeutic strategy, could be used for neurons, astrocytes and microglia genome editing. Thomas et al.²⁸⁴ employed an engineered CPP peptide 6His-CM18-PTD4 to deliver Cas12a RNP for gene editing of human stem cells, especially natural killer (NK) cells, and HeLa cancer cells. This 6His-CM18-PTD4 peptide showed robust gene editing by fused Cas12a RNP with the 6x histidine-rich domain, the endosomolytic peptide CM18, and the CPP of PTD4. Three kinds of engineered amphiphilic peptides, termed Shuttle10 (S10), Shuttle18 (S18), and Shuttle85 (S85), were non-covalently combined with AsCas12a RNP, which improved the gene editing of NK cells with achieved indels of 25%, 23%, and 26%, respectively²⁸⁵. In addition, AuNP-based CRISPR nanoformulation (AuNP/CRISPR) was used for targeted homology-directed repair of blood stem and progenitor cells without cytotoxicity²⁸⁶. This AuNP/CRISPR was synthesized by conjugation of Cas12a RNP on the surface of AuNP, briefly, an 18-nucleotide oligo ethylene glycol spacer and a terminal thiol linker linked crRNA, which was attached to AuNP by semi covalent gold–thiol interaction.

Cas12a has a much shorter crRNA (about 41 nt) for gene editing as compared with crRNA-tracrRNA chimera (sgRNA, about 100 nt) of Cas9, which is available for chemical modification of the crRNA^6,287. It was reported that chemical modification of the extended 5′ end of the crRNA resulted in enhanced serum stability. The extension of the length of crRNA at the 5′ end by 59 nucleotides could enhance the delivery efficiency of Cas12a RNP both in vitro and in vivo by cationic delivery vehicles, including Lipofectamine 2000 or cationic polymer pAsp (DET) complexation²⁸⁸. To improve the delivery efficiency of CRISPR-Cas12a, Sun et al.⁵⁷ developed a DNA nanoclews (NCs) for systemic delivery of Cas12a RNP through base-complementation between the DNA NCs and the crRNA. Addition of a charge reversal polymer layer condensed the DNA NCs and stabled the DNA-template core in blood circulation and facilitated the endosomal escape. The mentioned effects might occur due to the charge reversal polymer, which exhibited a negative charge under physiological pH but reverted to positive charge under an acidic environment and was composed of the targeting ligand galactose, polyetherimide and anionic dimethylmaleic anhydride. The results in vivo showed that the knock out efficiency of the targeted gene PCSK9 was about 48%, with obvious serum cholesterol reduction (∼45%). Nevertheless, the emerging Cas12a genome editing system exists several disadvantages, which are summarized as follows: i, the gene editing efficiency is generally lower than Cas9 nuclease; ii, the highly active ssDNA nuclease after binding to target DNA triggers non-specific ssDNA degradation, which makes it unsuitable for HDR applications.

5.2. CRISPR-Cas13a

Another new type of CRISPR nuclease, CRISPR-Cas13a (previous known as C2c2), is defined as an RNA-guided RNA targeting CRISPR effector^289,290. In other word, the general RNase activity is activated by Cas13a/crRNA complexes binding to the target RNA, which leads to non-specific cleavage of cellular RNAs. Based on the programmed cell death or dormancy induced by CRISPR-Cas13a, Zhang et al.²⁹¹ designed a dual-locking nanoparticle (DLNP) that triggered by pH and H₂O₂ concentration in the TME, which could restrict the activation of CRISPR-Cas13a in tumor regions so as to avoid undesired activation in normal tissues. In this study, the DLNP was designed using a core–shell structure in which plasmid DNA encoding the CRISPR-Cas13a system was encapsulated inside the core with a dual-responsive polymer network layer. Once the DLNP reached to a TME, the polymer layer was degraded into a cationic polymer and the CRISPR-Cas13a system was released by low pH and high H₂O₂ concentration of TME, which enhanced the cellular internalization and gene editing efficiency of CRISPR-Cas13a, thus resulting in significantly increased antitumor effect and improved survival rate.

5.3. CRISPR/nuclease-inactivated Cas9 (dCas9)

CRISPR-Cas9 system uses a nuclease-deficient Cas9 (dCas9) protein, called CRISPR-dCas9, which retains the ability to bind the guide RNA but lost its endonuclease activity292, 293, 294, 295, 296. Various effector domains fuse dCas9 enables the sequence-specific recruitment of transcription regulators for gene regulation, fluorescent proteins for genome imaging, and epigenetic modifiers for epigenetic modification²⁹⁷. For activated transcription, the VP64-p65-Rta (VPR) activator that was developed by modifying the conventional activator virion protein 64 (VP64)²⁹⁸ following the addition of the transcription factors p65 and Rta to the C terminus of dCas9-Vp64²⁹⁹ was bound to the gRNA RNPs complex. Lee et al.³⁰⁰ synthesized a magnetic peptide-imprinted chitosan nanostructure and combined this nanocomposite to the dCas9-VPR: gRNA RNPs with various gRNAs, which was used to activate OSKM genes in HEK-293T cells. In the system, four pairs of sgRNA were employed for the binding sites recognition and targeted the same gene to enhance gene expression. It was shown that the activated expression of OSKM genes is up to three-fold higher than that of the other gene. dCas9 was also used to enhance the expression of CT45 and to improve the efficacy of cisplatin in the cisplatin grafted PEG-PEI glutathione reductase sensitive delivery system³⁰¹.

DNA methylation has a crucial effect on biological processes, including the determination of cell-fate, genomic imprinting, the advanced architecture of chromatin and the manipulation of gene expression³⁰². To manipulate the methylation status of specific genes, a CRISPR Cas9-based near-infrared upconversion-activated DNA methylation editing system (CNAMS) was designed³⁰³. The dCas9 was fused to CIBN and the catalytic domain of DNMT3A or TET1 was fused to the CRY2PHR. The CNAMS could remotely erasure the installation of DNA 5-cytosine methylation editing in HEK-293T cells and inhibit tumor growth in vivo by regulating gene expression.

The CRISPR-dCas9 system is also an intelligent tool for nuclear targeting of anticancer drugs to avoid uncontrolled cytoplasmic release. Hence, a CRISPR-dCas9-guided and telomerase-responsive nano system was used for nuclear targeting and smart release of anticancer drugs²¹. To be specific, mesoporous silica nanoparticles (MSNs) was employed to deliver the CRISPR-dCas9 into nuclei so as to achieve precise targeting and tumor-specific release of anticancer drugs, such as DOX. The dCas9 protein was fused with a short lipoic acid ligase (LplA) acceptor peptide (LAPtag) for trans-cyclooctene (TCO2) decoration and labeled with a tetrazine-conjugated MSN by Diels–Alder cycloaddition. A specifically designed wrapping DNA was used for the release of telomerase-responsive drug, which encapsulated DOX to MSNs. The wrapping DNA, extended and formed a rigid hairpin-like structure in the presence of telomerase, was translocated from the surface of MSNs to release DOX.

The development of CRISPR-dCas9-based anti-cancer therapy remains challenging due to the conflicting requirements for the design of the delivery system. The classical cationic and membrane-binding surface nanocarriers facilitate the tumor accumulation and cellular uptake of CRISPR-dCas9 system, but hinder the circulating stability in vivo. Based on the mentioned above, Liu et al.⁵⁶ designed a multistage delivery nanoparticle (MDNP) that could achieve tumor-targeted delivery of CRISPR-dCas9 systems and restored endogenous miRNA expression in vivo. The MDNP was a core–shell structure in which the shell was made of a responsive polymer that endowed MDNP with the capability to present different surface properties in response to its surrounding microenvironment, allowing the MNDP overcoming multiple physiological barriers and delivering the payload to tumor tissues with an optimal efficiency. It was found that systemic administration of MDNP/dCas9-miR-524 to tumor-bearing mice achieved effective upregulation of miR-524 in tumors, which led to the simultaneous interferences of multiple signal pathways related to cancer cell proliferation and showed remarkable tumor growth retardation.

5.4. CRISPR-mediated base editors

CRISPR-mediated base editing (BE) system, as a new generation of genome editing tool, enables precise base changes without inducing a high level of double-strand breaks (DSBs) and the need of homology-directed repair (HDR) donors for gene repair^20,304. To date, there are two classes of CRISPR-BE system have been developed, i.e., cytosine CRISPR-BE and adenine CRISPR-BE^305,306. For cytosine CRISPR-BE, it is composed of cytosine deaminases, Cas9 nickases, and uracil glycosylase inhibitor, which introduces C·G to T·A base changes and has been applied to successfully introduce a stop codon or correct T·A to C·G mutations in prokaryotes, fungi, plants, insects, amphibians, fish, and mammals^304,307, 308, 309, 310, 311. For adenine CRISPR BE, it includes an evolved Escherichia coli TadA (ecTadA∗, a tRNA adenosine deaminase evolved to accept DNA substrate) and Cas9 nickase, which caused A·T to G·C substitutions^306,312. Recently, Koblan et al.³¹² reported the use of CRISPR-mediated adenine base editor (ABE) to correct the Hutchinson–Gilford progeria syndrome (HGPS)-causing mutations, which attenuated the symptoms and extended the lifespan of mice.

To improve the base-editing efficiency of CRISPR-ABE, a lipid-mediated delivery of chemical modified sgRNA and mRNA of a codon-optimized BEs was designed, which possessed a higher capacity for generation of Fah⁺ hepatocytes than the standard CRISPR-ABE³¹³. Following correction by engineered CRISPR-ABE, sufficient Fah⁺ hepatocytes were generated and the phenotypic weight loss of Fah mutant mice was ameliorated. Additionally, Musunuru et al.³¹⁴ reported the highly efficient in vivo delivery of a CRISPR-ABE using lipid nanomaterials in cynomolgus monkeys to introduce a precise single-nucleotide PCSK9 loss-of-function mutation, which resulted in reduction of PCSK9 and LDL cholesterol for at least 8 months. Although the phenotypes using lipid NPs to delivery CRISPR-ABE have shown advantages both in vitro and in vivo. Some challenges still need to be solved: i. As an initial delivery vehicle, the delivery efficiency of lipid NPs is limited; ii. The delivery or translation of ABE mRNA is less efficient than Cas9 mRNA because ABE mRNA is longer than Cas9 mRNA (5.2 kb vs. 4.1 kb); iii. Further evaluation of the risks of BE in vivo is warranted. Hence, it is needed to develop methods to improve delivery and enhance mRNA stability/translation so as to broaden the therapeutic application of BEs^313,314.

6. Conclusions and perspectives

The field of gene editing is booming and continuous innovations in CRISPR-Cas technology have brought great hope on biomedical applications in the future. To achieve progress in translational medicine, it is urgent to develop therapeutic strategies to increase the safety and effectiveness for CRISPR delivery vectors. Customized single stimulus-responsive nanocarriers have been developed, which possess site specificity and spatiotemporal tunability, thereby greatly improving the therapeutic efficacy and reducing the side effects of CRISPR-based genome editing.

Although stimuli-responsive nanocarrier delivery systems have great advantages in controllable and enhanced activation of CRISPR system at specific disease sites. However, it is still difficult for nanocarriers to realize clinical applications due to the complex ingredients for catering the complicated biological environment. Additionally, since single stimulus only achieves a single function with either temporal or spatial controllability, it is risky to maintain a good balance of the entire system to meet the overall demands. Therefore, an ideal nanocarrier should possess high drug-loading efficiency, stability during circulation, specificity to lesions and intelligent response to specific stimuli. Overall, this multimodal stimulus responsive platforms of CRISPR delivery with high safety and precision, which can possess synergistic treatment effects, i.e., 1 + 1 > 2, and might be a new trend in nanotherapeutic carrier design in the future.

Acknowledgments

This study was funded by National Natural Science Foundation of China (No. 31901010); Jiangsu Specially Appointed Professorship Foundation; the Priority Academic Program Development of Jiangsu Higher Education Institutions (Integration of Chinese and Western Medicine).

Author contributions

Xin Han and Xiaoxiang Guan conceived and designed this review. Chao Chen and Wu Zhong wrote the original manuscript. Shiyu Du, Yayao Li, Yunfei Zeng, and Kunguo Liu collected the references. Chao Chen, Wu Zhong, and Jingjing Yang made the figures and tables. Xin Han and Chao Chen revised the manuscript. All authors have read and approved the final manuscript.

Conflicts of interest

The authors declare no conflicts of interest.