Key considerations in designing CRISPR/Cas9-carrying nanoparticles for therapeutic genome editing

Yunxue Xu ab, Renfa Liu a and Zhifei Dai *a
aDepartment of Biomedical Engineering, College of Engineering, Peking University, Beijing 100871, China. E-mail: zhifei.dai@pku.edu.cn
bAcademy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China

Received 22nd July 2020 , Accepted 1st October 2020

First published on 5th October 2020


Abstract

CRISPR-Cas9, the breakthrough genome-editing technology, has emerged as a promising tool to prevent and cure various diseases. The efficient genome editing technology strongly relies on the specific and effective delivery of CRISPR/Cas9 cargos. However, the lack of a safe, specific, and efficient non-viral delivery system for in vivo genome editing remains a major limit for its clinical translation. In this review, we will first briefly introduce the working mechanism of CRISPR/Cas9 and the patterns of CRISPR/Cas9 delivery. Furthermore, the physiological obstacles for the delivery process in vivo are elaborated. Finally, the key considerations will be deeply discussed in designing non-viral nanovectors for therapeutic CRISPR/Cas9 delivery in vivo, including the effective encapsulation of large-size macromolecules, targeting specific tissues and cells, efficient endosomal escape and safety concerns of the vector systems, in the hope of inviting more comprehensive studies on the development of safe, specific, and efficient non-viral nanovectors for delivering a CRISPR/Cas9 system.


1 Introduction

Clustered regularly interspaced short palindromic repeats (CRISPR), a sequence of repeated segments of DNA, in collaboration with Cas (CRISPR-associated) proteins, are originally a kind of immune defense function in bacteria or archaea to prevent invading genetic elements.1 A milestone event happened in 2013 when Cong et al. reported that the CRISPR/Cas9 system could be exploited to edit the genome in eukaryotic cells.2 The genome-editing tool CRISPR/Cas9 is composed of single guide RNA (sgRNA) and Cas9 endonuclease. Cas9 is the most widely used Cas protein owing to its simplicity and high efficiency. With targeted recognition of the protospacer adjacent motif (PAM) via base pairing, sgRNA guides the Cas9 protein to cleave specific DNA sites.3,4 With the aim of improving the editing efficiency and facilitating the delivery process, novel feasible CRISPR systems, such as CRISPR/Cas12a, CRISPR/Cas12b and CRISPR/Casx, have been reported.5–8

Compared with traditional biomolecular drugs, such as siRNA, therapeutic mRNA or protein, CRISPR/Cas9 can introduce stable insertions and deletions in the genome with high specificity in a relatively simple way. In the past few years, the application of CRSPR/Cas9 for gene therapy has achieved exciting results both in preclinical and clinical trials.9–11 Recently, it has been proven that using the CRSPR/Cas9 system is safe and feasible in several clinical trials, such as CRISPR/Cas9-edited T cells in patients with refractory cancers and stem cells in patients with acute lymphocytic leukemia.12–14 In addition, the first in vivo CRISPR/Cas9 genome-editing medicine (EDIT-101) was administered to patients with leber congenital amaurosis 10 (LCA10) via sub-retinal injection (ClinicalTrials.gov Identifier: NCT03872479).

Despite the promising prospects, the efficient and safe systemic delivery of CRISPR/Cas9 elements in vivo remains challenging, and it is the perquisite for widely translating the genome-editing CRISPR/Cas9 system into clinic. To date, the most popular approaches for CRISPR/Cas9 delivery include viral vectors and non-viral vectors.15 Viral vectors, mainly comprising lentivirus, adenovirus, and adeno-associated virus (AAVs), are widely utilized to deliver the CRISPR/Cas9 system owing to its high efficiency. Nevertheless, the undesired viral-genome integration, immunogenetic responses, limited cargo size and high cost in production are existing obstacles for the clinical application of viral-based vectors for in vivo delivery of CRISPR/Cas9 components.16,17 In contrast, non-viral vectors (including liposomes, polymer-based nanoparticles and cell-penetrating peptides) have advantages over viral-based vectors owing to their lower immune response and flexible cargo size.18–20

Herein, we first briefly describe the working mechanism of CRISPR/Cas9 and the delivery patterns of CRISPR/Cas9. Then, the complicated physiological barriers to the in vivo delivery are elaborated. Finally, we highlight the key considerations in the design of non-viral nanovectors for CRISPR/Cas9 delivery in vivo by summarizing some delivery strategies and providing potential solutions to address the existing limitations (Scheme 1).


image file: d0nr05452f-s1.tif
Scheme 1 Key considerations in designing CRISPR/Cas9-carrying nanoparticles for therapeutic genome editing.

2 Genome-editing mechanism and delivery patterns of CRISPR/Cas9

2.1 Working mechanism of CRISPR/Cas9

The type II CRISPR/Cas9 is the most popularly applied gene-editing format among multiple types of CRISPR systems. CRISPR/Cas9 consists of two components: sgRNA and Cas9 endonuclease. sgRNA is made up of CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA). The ∼20 nucleotides at the 5′ end of crRNA is designed to combine the target DNA sequence via base-pairing. The 5′ end of tracrRNA is base-paired with the 3′ end of crRNA, and this duplex structure binds the Cas9 protein. The Cas9 protein is a kind of endonuclease containing the RuvC and HNH domains. The HNH domain is responsible for cutting the complementary strand of the targeted DNA sequence, whereas the RuvC domain cleaves the other strand.21 In addition, the protospacer adjacent motif (PAM), with a sequence of “NGG”, is required for Cas9 binding and should be at the 3′ end downstream of the target DNA sequence. Hence, sgRNA acts as a positioning bullet to recognize the specific site in the genome, and Cas9 works as scissors to cut the sequence.

Once Cas9 cleaves the target DNA sequence, double strand breaks (DSB) form and there exists two genetic repair modes.22 (i) In the absence of the donor DNA template, the non-homologous end-joining mode (NHEJ) will be applied and insertion/deletion mutations (indels) will appear, leading to gene knockout. (ii) With the DNA template, the homology-directed repair (HDR) will be conducted and the DSB site will be repaired using the donor DNA template.23,24 In general, generating a gene knockout is much easier than a knock-in. This is because NHEJ is much more effective than the HDR mode, which only occurs in dividing cells.

In recent years, in order to improve the specificity and efficacy of CRISPR-based gene editing, tremendous efforts have been made to optimize the existing CRISPR/Cas9 system and explore an alternative CRISPR system. Kocak et al. engineered sgRNA secondary structures by adding a hairpin structure in the spacer region, which contributed to an increase of several orders of magnitude in the editing specificity.25 Cas12a was proven to cause less off-target effects than Cas9.26 In addition, since Cas12a has a smaller genetic size and only requires crRNA to recognize the target DNA sequence, it is easier to encapsulate and deliver the Cas12a/crRNA system with non-viral vectors in vivo.8 Cas13a was found to target the RNA sequence and mediate the RNA knockdown with the guidance of crRNA. Compared with genome editing, therapeutic RNA editing induced by the Cas13a/crRNA system holds less risk owing to its reversibility.27

2.2 Delivery patterns of CRISPR/Cas9 elements

At present, there are mainly three delivery patterns of the CRISPR/Cas9 components (Fig. 1). The first delivery pattern is a plasmid, such as pX260 and pX335, which encodes both sgRNA and Cas9. The second delivery pattern is a mixture of sgRNA and Cas9 mRNA. The third delivery pattern is Cas9/sgRNA ribonucleoprotein (RNP).28,29 Various advantages and shortcomings lie in each delivery pattern for clinical translation.
image file: d0nr05452f-f1.tif
Fig. 1 Delivery patterns of CRISPR/Cas9 components. The first delivery pattern is a plasmid encoding both sgRNA and Cas9, which would go through transcription and translation to form the Cas9/sgRNA complex and exert the genome-editing function. The second delivery pattern is a mixture of sgRNA and Cas9 mRNA. The third pattern is to directly deliver Cas9/sgRNA RNP.

The plasmid-based delivery pattern is the most convenient and stable among the three patterns. Compared with Cas9/sgRNA RNP, the plasmid is easier to prepare and more cost-effective. However, the DNA sequence expressing Cas9 is more than 4.5 kb and the large size of the total plasmid coding both Cas9 and CRISPR is about 7 kb–9 kb, which will significantly decrease the delivery efficiency.30,31 In addition, the plasmid needs to go through the transcription and translation process to produce the Cas9 protein, which takes a much longer time to edit the genome. Furthermore, the uncontrolled integration of the plasmid sequence into the host genome is a potential risk, leading to an uncontrolled intracellular concentration of Cas9 and more off-target effects.32

The second delivery pattern is the mixture of Cas9 mRNA/sgRNA.33,34 The Cas9 mRNA can be translated into Cas9 endonuclease in the cytoplasm, and then bind sgRNA to conduct genome-editing. Compared with the delivery of the plasmid into nucleus, it is much easier to deliver the mixture of Cas9 mRNA/sgRNA into the cytoplasm. Besides, the Cas9 mRNA could just induce the transient expression of Cas9 endonuclease, contributing to a lower off-target risk.35 Despite the appealing advantages, efficiently delivering the Cas9 mRNA is still challenging owing to the instability of mRNA.

Cas9/sgRNA RNP is the most straightforward delivery pattern. Once delivered into the cells, Cas9/sgRNA RNP can be immediately utilized to perform gene-editing without the transcription and translation process, leading to the high efficiency of gene-editing.36 On the other hand, it is reported that the Cas9 protein can be degraded gradually in the cells, reducing the potential off-target effects.37 Nevertheless, the big size of the Cas9 protein (160 kDa) increases the difficulty of the delivery process.38 Moreover, the high cost and complicated process of the Cas9 protein purification is another main limitation for the broad application of Cas9/sgRNA RNP in clinic.39

Apart from the above three dominant delivery patterns, there exist many other combinations or modifications of CRISPR/Cas9 components. Owing to the challenges of packaging all of the elements in a single nanocarrier, it is an alternative to load Cas9 protein/mRNA and sgRNA in a separated delivery system. For example, Yin et al. combined lipid nanoparticles carrying Cas9 mRNA and adeno-associated viruses encoding sgRNA and a DNA donor to repair a liver disease gene in mice.40 It is hard for a single positively-charged Cas9 protein to form a complex with cationic carriers, which inspired researchers to conjugate it with cationic carriers, such as Cell-penetrating peptide (CPP) and polymer bPEI.41,42 In addition, Rotello et al. modified the N-terminus of the Cas9 protein with an oligo glutamic acid tag (E-tag) to form Cas9En, which carries a net negative charge, leading to its effective self-assembly with the cationic arginine gold nanoparticles. The Cas9En and sgRNA were co-assembled with cationic gold nanoparticles to form a single vector, which could directly transport Cas9En and sgRNA into the cytoplasm and nucleus in cultured cells.43

3 Physiologic obstacles for in vivo delivery

Successful genome editing requires efficient delivery of the CRISPR/Cas9 system into target organs, tissues and cells. To deliver the gene-editing tool into the desired site, the non-viral based nanocarriers need to get over various physiological obstacles after systemic or local administration.44 For systemic delivery, the cargo-loaded nanoparticles would be first exposed to blood circulation, where lots of compounds or enzymes may cause clearance or degradation of the cargo. Then, the nanoparticles need to get through the endothelial cells of the blood vessel and the interstitial area to make contact with the target cells. At last, the cell membrane, endosome and nuclear membrane are the final intracellular obstacles that nanocarriers must overcome.

In blood circulation, there are all kinds of compounds, such as albumin and opsonin, which could bind with the surface of the nanoparticles to form a protein corona.45 The size, surface charge and composition of the nanocarrier make a great difference in the formation of various protein coronas.46–50 Typically, the opsonin-based protein corona will probably be cleared from the blood circulation by a mononuclear phagocyte system. To increase the circulation time of the nanoparticles, polyethylene glycol (PEG) was proven to greatly prevent opsonizing.51–55 In addition, there exist the risks that the Cas9 protein could be degraded by multiple proteases, Cas9 mRNA and sgRNA degraded by RNase, and plasmid degraded by DNase.

Before the CRISPR/Cas9-loaded nanocarrier makes contact with the targeted cells, encountering the endothelial cells of the blood vessel and the interstitial area is inevitable. In particular, the leaky vessels (which are common in tumor and inflammation tissues) facilitate the extravasation of the nanocarriers from blood vessel.56–58 In contrast, the cerebral vascular endothelial cells, which are connected tightly with each other, combined with the astrocyte and basal lamina to form a blood brain barrier, severely hinder the delivery of CRISPR/Cas9 to the brain.59–61 In addition, the positively charged nanocarriers are prone to being stuck in the interstitial area, as the general extracellular matrix is negatively charged.62,63 Typically, the interstitial hypertension in solid tumors, resulting from the dysfunction of the lymphatic system and enhanced permeability effect, would greatly impede the transport of CRISPR/Cas9-loaded nanoparticles.64–66

The intracellular obstacles mainly include the cytomembrane, endosome and nuclear membrane for delivering plasmid or Cas9/sgRNA RNP. For delivering the mixture of Cas9 mRNA/sgRNA, the intracellular barriers mainly contain the cytomembrane and endosome entrapment because the Cas9 mRNA translated into the Cas9 protein in the cytoplasm do not need to get into the nucleus. The internalization of nanoparticles or macrobiomolecules mainly relies on endocytosis or phagocytosis.67,68 The efficiency of the internalization process can be improved by modifying the nanoparticles with a specific ligand, resulting in the receptor-mediated cellular uptake of the cargo.69,70 After internalization, the CRISPR/Cas9-loaded nanoparticles might be enveloped in the endosome or lysosome, where the acidic fluid (pH = ∼5) and various enzymes contribute to the degradation of the nanoparticles, as well as the cargo. Escape from the endosome is the prerequisite for any delivery patterns of the CRISPR/Cas9 system.71,72 Finally, the plasmid-based components and Cas9/sgRNA RNP need to get into the nucleus. Modifying the Cas9 protein of Cas9/sgRNA RNP with nuclear localization sequences (NLS) will facilitate its nuclear entry. Similarly, designing the plasmid and Cas9 mRNA with sequences encoding NLS is helpful for the nuclear entry of their translational product Cas9 protein. Going across the nuclear membrane of the dividing cells would be much easier, as cracks appear in the nuclear membrane during mitosis.73

In some cases, treatment of some diseases in the eye, brain and muscle take advantage of the local injection of the CRISPR/Cas9 machinery (ClinicalTrials.gov Identifier: NCT03872479).74,75 For local delivery, the delivery nanocarriers are generally injected into the interstitial area of the target organ/tissue, bypassing some obstacles in blood circulation, such as opsonin, multiple DNase, RNase or protease, and circumventing the vascular endothelium. However, the dense extracellular matrix can still interfere with the transporting process of the nanocarrier by preventing its homogeneous dispersion around the injection region. Hence, for local administration, the CRISPR/Cas9 system might be mainly restricted in the point of injection and distributed heterogeneously in the target organ/tissue.

4 Critical considerations in designing non-viral nanocarriers for CRISPR/Cas9

As a revolutionary gene-editing tool, the CRISPR/Cas9 system has attracted quite a lot of attention from researchers in various fields, and greatly promoted the progress of gene therapy. Despite the remarkable gene-editing potential of CRISPR/Cas9, the development of safe and efficient nanocarriers for in vivo genome-editing remains challenging for its clinical application. Unlike the traditional biomolecular drug, such as siRNA, therapeutic mRNA or protein, the CRISPR/Cas9 cargo (whether in the pattern of plasmid (DNA), mixture of Cas9 mRNA/sgRNA, or Cas9/sgRNA RNP) has a large genetic size, which is difficult for vehicles to effectively envelope and transport. Moreover, when systemically administrated, gene-editing is expected to perform in the desired organs, tissues and specific cells, with the least off-target effect. In addition, after cellular internalization, it is necessary for the CRISPR/Cas9 system to enter the nucleus for genome editing. This part discusses in depth the critical considerations in designing novel CRISPR/Cas9 vehicles in the hope of introducing more valuable remarks and inviting more comprehensive studies.

4.1 Efficient packaging of the large cargo

The genetic size of the Cas9 protein is ∼160 kDa, Cas9 mRNA is ∼4500 nt and the plasmid encoding Cas9 is 7–9 kb.16 The large size of the plasmid, mRNA and Cas9 protein makes it challenging to effectively load multiple elements in a single nanocarrier. Therefore, it can be much easier to package the macromolecules with separated delivery carriers.40 For instance, Ramakrishna et al. conjugated the Cas9 protein with a CPP, and complexed sgRNA with another cationic arginine-based CPP. The results demonstrated that the mixture of the CPP/sgRNA complex and CPP/Cas9 conjugate led to a mutation frequency of 2.3%–16% in the culture cells.41

Compared with the separated delivery systems, encapsulating both elements of sgRNA and Cas9 into a single nanovehicle could ensure that they would be simultaneously delivered to the same cell. Owing to the sufficient negative charge of the plasmid, mRNA/sgRNA or Cas9/sgRNA RNP, the most common technique to condense the large size of the anionic plasmid, Cas9 mRNA/sgRNA or Cas9/sgRNA RNP is complexing with cationic compounds via electrostatic interaction. Positively charged materials (including cationic lipids, polymers, polypeptides and gold nanoclusters) were widely used to condense the negatively charged cargo to form the compact delivery system. For example, Lee constructed thiolated DNA-coated gold nanoparticles (GNPs), which were hybridized with donor DNA and subsequently coated with Cas9/sgRNA RNP by electrostatic forces, effectively condensing various macromolecules (Fig. 2A). Then, the complex of GNPs, donor DNA and RNP was encapsulated with negatively charged silica. Finally, the positively charged endosomal disruptive polymer PAsp(DET) formed CRISRP-Gold. After intramuscular injection of the mdx mice with Duchenne muscular dystrophy (DMD), CRISRP-Gold corrected 5.4% of the dystrophin gene, which is much higher than treatment with naked RNP and donor DNA.75


image file: d0nr05452f-f2.tif
Fig. 2 Non-viral nanovehicles effectively packaging the CRSPR/Cas9 system for local in vivo and ex vivo delivery. (A) Schematic depiction of a thiolated DNA-coated gold nanoparticle (CRISPR-gold) to simultaneously load Cas9/sgRNA RNP and donor DNA by electrostatic forces (Adapted with permission from ref. 75. Copyright 2017 Springer Nature). (B) Rational design of DNA nanoclew (NC)-based nanocarriers to envelop Cas9/sgRNA RNP by DNA–RNA base pairing (Reproduced with permission from ref. 76. Copyright 2015 Wiley-VCH).

Apart from electrostatic interaction, single strand DNA–RNA base pairing is an alternative for condensing CRISPR/Cas9 components. Sun et al. constructed a DNA nanoclew (NC) to compact Cas9/sgRNA RNP via DNA–RNA base-pairing (Fig. 2B). Driven by encoded palindromic sequences, the self-assembled yarn-like NC was fabricated by rolling circle amplification (RCA). Sequences complementary to sgRNA in NC were designed to bind Cas9/sgRNA RNP. The RNP-loaded NC was subsequently encapsulated with cationic endosomal disruptive polyetherimide (PEI) to self-assemble into uniform Cas9/sgRNA/NC-12/PEI particles. The significant difference in the EGFR mutation frequencies between Cas9/sgRNA/NC-12/PEI (28%) and Cas9/sgRNA/PEI (1.5%) in vitro confirmed the effective packaging of Cas9/sgEGFP RNP by NC.76

As mentioned above, condensing the CRISPR/Cas9 components with nanocarriers could facilitate their in vitro delivery or local delivery in vivo. However, for systemic administration in vivo, additional modifications are needed to protect them from degradation and increase the circulation time. The core–shell multilayer nanoparticles can ensure the stability of the CRISPR/Cas9 elements by utilizing the inner layer to condense the large cargo. In addition, the outer layer can be applied to protect them from degradation and increase the circulation time, which make it a promising platform for effective systemic delivery in vivo.18 Modifying the shells with PEG or applying anionic polymers as the outer shells are proven to greatly decrease the nonspecific uptake, undesired clearance and degradation of the CRISPR/Cas9 components. Recently, Liu et al. constructed charge-conversional core–shell polymeric nanoparticles (MDNP) to deliver the CRISPR/dCas9 plasmid (pDNA) with an anionic polymer 2,3-dimethylmaleic anhydride (DMMA)-conjugated poly (ethylene glycol)-polylysine (mPEG113-b-PLys100/DMMA) as the shell (Fig. 3). The anionic mPEG113-b-PLys100/DMMA could greatly enhance the circulation stability of MDNP, and convert into a positively charged polymer when exposed to the acidic microenvironment of the tumor. The core–shell MDNP remained intact and stable in the blood circulation, whereas it would decompose in the target tumor, owing to the reversal charge of mPEG113-b-PLys100/DMMA (from anionic to cationic). Then, the cationic core would effectively accumulate in the targeted tumor, transfect the tumor cell and perform gene-editing. It is demonstrated that MDNP/dCas9-miR-524 was a feasible approach to activate the miR-524 gene, and significantly inhibit the tumor progress.77 Similarly, Sun et al. improved the above-mentioned DNA nanoclew (NC) for systemic delivery by coating it with a charge-reversal polymer, which was composed of the targeting ligand galactose (Gal), polyetherimide (PEI) and anionic dimethylmaleic anhydride (DM). The charge-reversal polymer Gal-PEI-DM exhibited a negative charge under physiological pH to stabilize the cationic compact core in blood circulation, and converted to being positively charged in the acidic endosome to facilitate endosomal escape. The in vivo results revealed that the targeted gene PCSK9 was knocked out by 48%, which effectively induced the decrease of serum cholesterol by 45%.78


image file: d0nr05452f-f3.tif
Fig. 3 Schematic diagram of charge-conversional core–shell polymeric nanoparticles (termed MDNP) for delivering the CRISPR/dCas9 plasmid (pDNA) with an anionic polymer 2,3-dimethylmaleic anhydride (DMMA)-conjugated poly (ethylene glycol)-polylysine (mPEG113-b-PLys100/DMMA) as the shell. MDNP was transferred in the blood circulation, while the core–shell structure would decompose in acidic environment, as a result of the charge conversion of mPEG113-b-PLys100/DMMA (from anionic to cationic) (Adapted with permission from ref. 77. Copyright 2018 Wiley-VCH).

4.2 Targeting specificity for in vivo delivery

One of the most important prerequisites for efficient genome-editing is the target delivery of the CRISPR/Cas9 tool in the desired organs, tissues and cells. Notably, the physicochemical properties, such as the size and surface charge of nanocarriers, greatly influence their biodistribution and excretion.79–81 Typically, particles larger than 2 μm are usually caught by pulmonary capillary vessels, while those with diameters of less than 5 nm are easily cleared via renal system. It had been proven that nanoparticles with a diameter of 10–200 nm could substantially accumulate in the tumor, inflammatory tissue or hepatic sinusoid, owing to the high vascular permeability of these tissues.82–86 In general, a large proportion of the systemically administered nanovehicles would be captured by the reticuloendothelial systems of the liver and spleen.86,87 The surface charge is another important factor that greatly affects the fate of the administered nanoparticle in vivo. Compared with the negatively charged or neutral nanocarriers, the positively charged particles are prone to be taken up by the liver and easier to be internalized by cells.88,89 However, the differential fates of the nanoparticle surface charge are not always constant in biological systems, as the formation of the protein corona may cause a shift of the surface charge.90,91 Hence, the differential effects of the physicochemical properties on the biodistribution should be kept in mind for the design of organ-specific CRISPR/Cas9-carrying nanocarriers.

Cationic lipid nanoparticles (LNPs) containing ionizable cationic lipid have been extensively applied for the delivery of various macromolecules, such as siRNA, mRNA and therapeutic proteins.92–95 In general, most LNP-based delivery systems are prone to associate with various serum proteins, like apolipoprotein E, which might account for the sufficient uptake of LNP by hepatocyte endocytosis.96 The tropism of LNP to the liver makes it an efficient liver-specific delivery system for therapeutic gene editing in the hepatocyte. Finn et al. developed an ionizable cationic lipid (LP-01)-based LNPs (termed LNP-INT01) for the liver-specific delivery of the Cas9/sgRNA system, which mediated the effective gene editing of transthyretin (Ttr) in the mouse and rat liver. LP-01 with a pKa of 6.1 exhibited an effective condensation of the anionic Cas9 mRNA/sgRNA complex at low pH conditions, which contributed to the strong electrostatic interaction between the lipids and the Cas9 mRNA/sgRNA complex by converting the tertiary amine group of LP-01 to the positively charged quaternary amine groups. With a single administration, LNP-INT01 induced a 70% gene-knockout of transthyretin in the liver, leading to a sharp decrease of the serum transthyretin by 97%, which lasted for more than 12 months.97

As a promising nanomaterial, it is of importance to sensibly optimize LNP for the multiple organ-specific delivery of the gene-editing tool. Recently, based on the speculation that the internal or external charge plays a key role in determining the tissue tropism, researchers reported that the rational design of LNP with different lipid formulations or compositions allowed for the tissue-specific delivery of the CRISPR/Cas9 tool. The reported selective organ targeting (SORP) strategy could realize the controllable delivery of Cas9 mRNA/sgRNA or Cas9/sgRNA RNP into the liver, spleen or lung via tuning the adding percentage of the supplemental lipid molecule (SORT molecule) in traditional LNPs (Fig. 4A). The results demonstrated that by tuning the internal charge with various SORT molecules (including DOTAP), the SORT nanoparticles specifically delivered the CRISPR/Cas9 components into the liver. This led to about 60% mutation at the sequence of PCSK9, which reduced the serum level of PCSK9 by 100%.98 This work brings us some sort of order to rationally improve the existing nanovectors for organ-targeting delivery.


image file: d0nr05452f-f4.tif
Fig. 4 Strategies for the targeted delivery of the CRISPR/Cas9 system. (A) Tuning the adding percentage of supplemental lipid molecule (SORT molecule) in traditional lipid nanoparticles (LPNs) allows for the organ-specific delivery of CRISPR/Cas9 components (Adapted with permission from ref. 98. Copyright 2020 Springer Nature). (B) Schematic depiction of the preparation and cell-type-specific internalization of biomimetic MCF-7 cell membrane-coated zeolitic imidazolate frameworks (C3-ZIFMCF) (Reproduced with permission from ref. 108. Copyright 2020 American Chemical Society).

The cell-specific uptake of CRISPR/Cas9-loaded nanocarriers can be further improved by modifying the nanoparticles with specific ligands, which contributes to the receptor-mediated enhanced cellular uptake of ligand-conjugated nanocarriers.99–102 For instance, in order to realize the effective hepatocyte-targeting delivery of Cas9/sgPCSK9, Tang et al. modified the TAT peptides-conjugated gold nanocluster with the 4-aminophenyl β-D-galactopyranoside (Gal) modified polyethylene glycol phospholipid (Gal-PEG-DSPE) rather than DSPE-PEG, for the reason that Gal could contribute to the asialoglycoprotein receptor (ASGPR)-mediated hepatocytic uptake of Cas9/sgPCSK9-loaded Gal-LGCP. The in vivo results showed that Gal-LGCP effectively knocked down the PCSK9 expression in the hepatocyte, down-regulating the serum LDLC level by 30%.103

Besides surface modification with ligands, a novel strategy that utilizing the biomimetic cell membrane coating nanotechnology to realize the cell-specific delivery of therapeutic agents has attracted a lot of attention.104–106 Compared with the functionalizing ligand, the biomimetic cell membrane has the advantages of a facile preparation, effective escape of RES and minimal immune responses.107 Recently, a biomimetic cancer cell membrane encapsulated metal organic framework for the active targeting and cell-specific delivery of CRISPR/Cas9 components was reported (Fig. 4B). After being incubated with multiple cell lines, the MCF-7 cell membrane-coated zeolitic imidazolate frameworks (C3-ZIFMCF) revealed the highest internalization by MCF-7 cells. This was due to the intrinsic homotypic binding effect of the cancer cells. The Cas9/sgEGFP-loaded C3-ZIFMCF exhibited an effective down-regulation of EGFP by 3-fold in MCF-7, while the Cas9/sgEGFP-loaded C3-ZIFHELA just repressed the expression of EGFP by 1-fold in MCF-7. This confirmed the efficient MCF-7 cell-specific delivery of C3-ZIFMCF.108

4.3 Cellular internalization

Up to now, most synthetic non-viral vectors loaded with drugs or therapeutic biomacromolecules (such as mRNA, siRNA or CRISPR/Cas9 components) enter the targeted cells via endocytosis.109–112 Following the endocytosis, the nanoparticles are entrapped in a vesicle termed endosome, which contains an acidic environment (pH = ∼5) and multiple digestive enzymes contributing to the degradation of the therapeutic agents. Hence, it is critical to overcome this obstacle for the successful delivery of the CRISPR/Cas9 components. The best solution is bypassing the endosome, that is, the nanocarriers directly enter the cytoplasm after internalization by cells. Alternatively, another strategy is disrupting the endosomal membrane to escape from the endosome.
4.3.1 Bypassing the endosome. Inducing pore formation of the cell membrane is an effective strategy for delivery vehicles to directly enter the cytoplasm after cellular internalization (Fig. 5). For instance, Wang et al. reported on poly(γ-4-((2-(piperidin-1-yl)-ethyl)-aminomethyl)-benzyl-L-glutamate) (PPABLG)-based PEGylated nanoparticles (termed P-HNP) for delivering Cas9 plasmid/sgRNA (Fig. 6). The cationic polypeptide PPABLG has an α-helical structure, which contributes to the evasion of the endosome and induces a direct intracellular internalization via pore formation of the cytomembrane. With the outstanding cell-penetrating capability of PPABLG, P-HNP performed at higher transfection efficiency than commercial Lipofectamine 3000 in multiple cell lines, and 1.9- to 4.9-fold higher than that of PEI/px458 treatment. The in vivo anticancer results revealed that the P-HNP-targeting Plk1 gene induced 35% gene mutation of Plk1 after intratumoral injection, leading to more than 71% inhibition of tumor growth.113
image file: d0nr05452f-f5.tif
Fig. 5 Cellular internalization and endosomal escape of the CRISPR/Cas9-loaded nanoparticles. After internalization via endocytosis, disrupting endosomal membrane subsequently by proton sponge effect or non-bilayer Hexagonal HII conformation (cationic lipids) leads to effective endosomal escape. Alternatively, some CRISPR/Cas9-loaded nanoparticles circumvent endosomal entrapment and directly enter the cytoplasm via pore formation of the cytomembrane or receptor-mediated cytomembrane fusion.

image file: d0nr05452f-f6.tif
Fig. 6 The α-helical polypeptide PPABLG-based PEGylated nanoparticles (P-HNP) transporting Cas9 plasmid/sgRNA circumvent the endosomal entrapment and directly enter the cytoplasm via pore formation of the cytomembrane (Adapted with permission from ref. 113. Copyright 2018 National Academy of Sciences).

Apart from the pore formation of the cytomembrane, receptor-mediated cytomembrane fusion is an alternative to circumvent endosomal encapsulation. Based on the fact that the membrane fusion of the liposome and cell is determined by the elasticity of the nanocarrier, a kind of non-cationic nanolipogel (tNLG) could directly enter cell plasma by membrane fusion mainly because of its low elasticity.101 In contrast, most other conventional CRISPR-carrying nanoparticles has high elasticity (>0.76 GPa), which inhibited the fusion pathway.114 This work provided a promising platform for effectively evading endosomal entrapment.

4.3.2 Disrupting the endosomal membrane.
4.3.2.1 Proton sponge effect. The most common approach to achieving endosomal escape for delivering the CRISPR/Cas9 system is disrupting the endosomal membrane via the pH-buffering effect. The pH-buffering effect (also termed proton sponge effect) is usually induced by protonated agents that cause a sufficient inflow of water, H+ and Cl+ into the endosome, leading to osmotic swelling and fragmentation of the endosome membrane. Sufficient tertiary amine groups and amine groups are known to show a considerable buffering effect once protonated.115 Specifically, some cationic polymers (such as polyethyleneimine (PEI), poly(amidoamine) (PAMAM) and chitosan) containing the tertiary amine group or amine groups are extensively utilized, and believed to facilitate endosomal escape for the delivery of the CRISPR/Cas9 components.116–119

It was reported that the cationic polymer, poly[N-(N-(2-aminoethyl)-2-aminoethyl) aspartamide] (PAsp(DET)), could display high transfection efficacy for siRNA, plasmid DNA and Cas9/sgRNA RNP with minimal cytotoxicity.74,75,120,121 The outstanding transfection efficacy and negligible cytotoxicity were proven to be due to the effective pH-dependent endosome-destabilizing of PAsp(DET), which exhibited weak membrane destabilization at neutral pH, while it can induce complete disruption of the endosomal membrane via the proton sponge effect at acidic endosomal pH (pH = ∼5), owing to the conversion of PAsp(DET) from the monoprotonated form to the diprotonated form under an acidic environment. The above-mentioned CRISRP-Gold took advantage of the cationic PAsp(DET) to facilitate cellular internalization, and exhibited outstanding endosomal escape to promote intracellular delivery. The results of inducing 3%–4% gene mutation in cells and 5.4% mutation frequency for local delivery in vivo demonstrated that PAsp(DET) is a promising endosomal disruptive polymer.75


4.3.2.2 Cationic lipids induced hexagonal HII conformation. Cationic lipids are known to effectively induce the inverted non-bilayer conformation termed Hexagonal HII once combined with an anionic lipid, disrupting the endosomal membrane.96,122 When cationic LNPs are internalized via endocytosis, they were entrapped in the endosome. The positively charged lipids of the nanovectors will adhere to the negatively charged lipids of the endosomal membrane owing to the electrostatic interaction, contributing to the membrane fusion of LNPs and endosome. Sufficient cationic-anionic ion pairs finally lead to the HII phase formation of the entire endosomal membrane, accelerating the release of macromolecular cargo. Compared with the conventional cationic lipid, which contains permanently charged quaternary amine groups, the ionizable cationic lipid composed of the tertiary amine exhibited a neutral or weakly cationic surface charge at physiological pH (7.4), which could reduce the non-specific adsorption with the serum protein, contributing to a longer circulation time and more effective delivery.123 Once exposed to the acidic environment of the endosome, the tertiary amine of the ionizable cationic lipid would convert to the positively charged quaternary amine, inducing the HII phase formation and accelerating the cargo release from the endosome.

4.4 Safety concerns of the cargo-vector system

4.4.1 Biocompatibility of the nanomaterials. The safety of the CRISPR/Cas9 system is one of the critical considerations for designing CRSPR-carrying nanoparticles. As for the delivery materials, it is advisable to exploit FDA-approved pharmaceutical adjuvants for the reason that their metabolic pathway, metabolic products or adverse reactions have been well investigated, ensuring higher biocompatibility. On the other hand, the well-studied pharmaceutical adjuvants could shorten the development duration to promote clinical translation.

The cationic lipid nanoparticle is one of the most widely used delivery vectors in clinic for siRNA and mRNA. Up to now, the majority of ionizable cationic lipids applied in clinic are non-biodegradable, resulting in the unintended retention in the liver and potential long-term toxicity.124,125 In order to maximize the delivery efficacy and simultaneously minimize the potential safety risks of LNPs, substantial improvements have been made. For instance, the combinatorial chemistry-based synthesis, together with the structure–activity relationship-based screening from the diverse library, allowed to identify the novel lipids that could achieve the efficient delivery of therapeutic macromolecules with extremely low administering doses, effectively reducing the potential toxicity of the undesired retention of nanomaterials.96,124 On the other hand, rationally designing the biodegradable ionizable cationic lipid by integrating a labile ester linkage or bioreducible disulfide bond is an alternative to minimizing the toxicity of unintended retention.95,97 Wang et al. designed one such biodegradable ionizable cationic lipid by inserting disulfide bonds in hydrophobic chains, which could respond to reductive glutathione in the cytoplasm (Fig. 7A). Meanwhile, reduction of the lipid was proven to greatly facilitate the endosomal release of the Cas9/sgRNA complex, improving the delivery efficiency.95


image file: d0nr05452f-f7.tif
Fig. 7 Biocompatibility of the nanovectors. (A) Sensible design of a biodegradable ionizable cationic lipid for delivering the Cas9/sgRNA complex (Reproduced with permission from ref. 95. Copyright 2016 National Academy of Sciences). (B) Schematic diagram of the core–shell non-cationic nanolipogel (tNLG) for the simultaneous delivery of 3 plasmids and the deformation of tNLG during extravasation from blood vessels (Reproduced with permission from ref. 101. Copyright 2019 National Academy of Sciences).

To date, cationic materials are prevalent delivery carriers for the CRISPR/Cas9 system, as the cationic nanocarriers could easily compact the large cargo and increase the transfection frequency by contact with the negatively charged cellular membrane. However, the excess cationic components of the carrier usually disrupt and destabilize the cellular membrane, leading to severe cytotoxicity. To address this problem, the non-cationic nanolipogel (tNLG) was developed to deliver CRISPR/Cas9 components for the knockout of the oncogene Lcn2 in triple negative breast cancer (TNBC) (Fig. 7B). The core–shell structure of tNLG comprised CRISPR plasmids-embedded alginate hydrogel (core) and non-cationic lipid layer made up of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and DSPE-PEG2000-COOH. Furthermore, tNLG was modified with ligand ICAM to specifically target TNBC and directly enter the cell plasma by receptor-mediated membrane fusion. As a consequence, it is demonstrated that the systemic injection of tNLG suppressed the expression of Lcn2 by 81%, and significantly inhibited the tumor growth by 77%.101

4.4.2 Systemic dissemination of off-target gene-editing. The off-target effect is an undesired gene editing in untargeted cells or targeted cells, which might cause tumorigenic or various genetic disorders, and is another important problem related with the safety of the CRISPR/Cas9 system. In particular, developing a nanovehicle to selectively realize the activatable gene-editing of CRISPR/Cas9 could minimize the risk of systemic dissemination of off-target gene-editing.

Recently, based on the light-thermal conversion capability of gold nanoparticles (Au NPs), Wang et al. developed a lipid-encapsulated Au NPs (LACP) to realize light-triggered Cas9/CRISPR release and genome-editing. When exposed to light irradiation, the production of hot electrons would cleave Au-S bonds between Au NPs and the TAT peptide, leading to the release of the Cas9/sgPLK-1 plasmid that was electrostatically adhered to TAT. Following Cas9/sgPLK-1 plasmid release, it can enter the nucleus and further edit the gene. As a result, with the activation of light, LACP effectively down-regulated the expression of PLK-1 (65%) and inhibited tumor growth (85%).126

For in vivo application, a light trigger was limited by the poor penetration depth in the tissue. Magnetic materials were reported to regulate some cellular processes with a magnetic field, which had no obvious side effects and could penetrate deeply without attenuation.127,128 Zhu et al. developed a hybrid viral-magnetic nanoparticle complex delivery system to realize the magnetically-activated gene-editing of CRISPR/Cas9. The complex was made up of an engineered baculovirus (BV) and iron oxide nanoparticles (MNP-BV). Notably, the baculoviral could effectively transfect multiple somatic cells, whereas it could not replicate or integrate into the somatic cells, providing transient gene-editing and reducing genotoxicity. When exposed to the serum, bare BV was inactive, owing to the complement system where some proteins might adhere to the surface of BV to form a protein corona. With a local magnetic field, the magnetic iron nanoparticles of MNP-BV could effectively activate BV to perform genome-editing, minimizing off-target effects. Applying the on–off switch, MNP-BV induced transient and liver-specific gene modification by 50%.129

5 Future perspectives

Until now, most of the ongoing CRISPR-related clinical trials for therapeutic genome-editing are based on the ex vivo approach, which utilizes the patient-isolated cells whose genome was edited with the CRISPR/Cas9 system in vitro. Then, the isolated cells with the correct gene sequence were subsequently delivered back into the patients. For ex vivo gene editing, the delivery of the CRISPR/Cas9 components mainly relies on viral-vectors and physical methods, such as electroporation, microinjection and hydrodynamic injection. Despite the recent exciting results in the CRISPR-based ex vivo clinical trials for treating refractory cancer and acute lymphocytic leukemia, many types of genetic disorders, such as Alzheimer's disease (AD), cystic fibrosis and spinal muscular atrophy, require the direct delivery of the CRISPR/Cas9 system to the related organs or tissues of patients.

In particular, the lack of safe and efficiently targeted delivery systems is the major challenge for using CRISPR/Cas9 in clinic. To improve the delivery specificity of CRISPR/Cas9, the non-viral carrier can be modified with cell-specific targeting ligands to facilitate its interaction with host cells. However, in some cases, modifying ligands on the surface of the delivery vehicles can make the fabrication process complex, and may induce stronger immune responses.130–132 Alternatively, efficient tissue-targeted delivery can be achieved by utilizing the tropism of specific materials for corresponding organs, tissues and cells. For instance, intravenously administered LNPs-based nanomaterials are prone to be sufficiently taken up by the hepatocyte, which makes it an efficient liver-specific delivery system for gene editing in the hepatocyte. In addition, another solution is coating the cargo with a biomimetic membrane, which enables the cell-specific delivery of CRISPR/Cas9 due to the intrinsic homotypic binding effect.

The safety concern is crucial for the successful clinical translation of CRISPR/Cas9. To improve safety, one possible solution is developing a conditional expressing Cas9 system that contains a specific promoter that is only expressed in the targeted organs or an inducible promoter, which could prevent the systemic dissemination of the off-target effect.133 Similarly, designing sgRNA based on tissue-specific DNA alterations could greatly contribute to the targeted cellular genome-editing, minimizing the off-target effect.134,135 Besides, the delivery of CRISPR/Cas9 in the pattern of RNP, which would be gradually degraded in the cells and has a short exposure time, can reduce the off-target effect compared to the plasmid-based delivery form.136 On the other hand, novel biocompatible, biodegradable and non-immunogenic materials are desirable to minimize the risk of long-term toxicity and local inflammatory responses. Furthermore, system dissemination of CRISPR/Cas9 can be reduced by designing delivery carriers with an “on–off” switch that can be activated by a specific microenvironment, such as enzymatic activity and pH, or that respond to external stimulation, such as thermal, optical or a magnetic field.126,129 The rational design of safe and efficiently-targeted delivery systems is an undoubtedly promising platform to achieve clinical transform of CRISPR/Cas9. Meanwhile, we must take into consideration that some accompanied complications, such as the dilemma in scale-up, complex quality control and high cost of such delivery systems, might hinder their clinical translation.

Non-viral vector-based therapeutic genome editing with CRISPR/Cas9 in vivo has started a new era in the application of molecular therapy for treating various genetic diseases. Although most of the existing CRISPR-carrying nanovectors cannot fully meet the requirements for clinical translation, we believe that emerging delivery systems will solve the issues of delivery efficiency, biosafety and scale-up production in the near future.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This contribution was financially supported by the National Key Research and Development Program of China (No. 2016YFA0201400), State Key Program of National Natural Science of China (No. 81930047), Projects of International Cooperation and Exchanges NSFC-PSF (No. 31961143003), National Project for Research and Development of Major Scientific Instruments (No. 81727803), Beijing Natural Science Foundation, Haidian, Original Innovation Joint Fund (No. 17L20170), and the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (No. 81421004).

References

  1. R. Barrangou, C. Fremaux, H. Deveau, M. Richards, P. Boyaval, S. Moineau, D. A. Romero and P. Horvath, Science, 2007, 315, 1709–1712 CrossRef CAS.
  2. L. Cong, F. A. Ran, D. D. Cox, S. Lin, R. P. J. Barretto, N. Habib, P. Hsu, X. Wu, W. Jiang and L. A. Marraffini, Science, 2013, 339, 819–823 CrossRef CAS.
  3. B. Wiedenheft, S. H. Sternberg and J. A. Doudna, Nature, 2012, 482, 331–338 CrossRef CAS.
  4. J. V. Der Oost, E. R. Westra, R. N. Jackson and B. Wiedenheft, Nat. Rev. Microbiol., 2014, 12, 479–492 CrossRef.
  5. J. Strecker, S. Jones, B. Koopal, J. Schmid-Burgk, B. Zetsche, L. Gao, K. S. Makarova, E. V. Koonin and F. Zhang, Nat. Commun., 2019, 10, 212 CrossRef CAS.
  6. F. Teng, T. Cui, G. Feng, L. Guo, K. Xu, Q. Gao, T. Li, J. Li, Q. Zhou and W. Li, Cell Discovery, 2018, 4, 63 CrossRef.
  7. J.-J. Liu, N. Orlova, B. L. Oakes, E. Ma, H. B. Spinner, K. L. M. Baney, J. Chuck, D. Tan, G. J. Knott and L. B. Harrington, Nature, 2019, 566, 218–223 CrossRef CAS.
  8. D. C. Swarts and M. Jinek, Wiley Interdiscip. Rev.: RNA, 2018, e1481 CrossRef.
  9. E. Beyret, H.-K. Liao, M. Yamamoto, R. Hernandez-Benitez, Y. Fu, G. Erikson, P. Reddy and J. C. Izpisua Belmonte, Nat. Med., 2019, 25, 419–422 CrossRef CAS.
  10. O. Santiago-Fernández, F. G. Osorio, V. Quesada, F. Rodríguez, S. Basso, D. Maeso, L. Rolas, A. Barkaway, S. Nourshargh and A. R. Folgueras, Nat. Med., 2019, 25, 423–426 CrossRef.
  11. Q. Ding, A. Strong, K. M. Patel, S.-L. Ng, B. S. Gosis, S. N. Regan, C. A. Cowan, D. J. Rader and K. Musunuru, Circ. Res., 2014, 115, 488–492 CrossRef CAS.
  12. Y. Lu, J. Xue, T. Deng, X. Zhou, K. Yu, L. Deng, M. Huang, X. Yi, M. Liang, Y. Wang, H. Shen, R. Tong, W. Wang, L. Li, J. Song, J. Li, X. Su, Z. Ding, Y. Gong, J. Zhu, Y. Wang, B. Zou, Y. Zhang, Y. Li, L. Zhou, Y. Liu, M. Yu, Y. Wang, X. Zhang, L. Yin, X. Xia, Y. Zeng, Q. Zhou, B. Ying, C. Chen, Y. Wei, W. Li and T. Mok, Nat. Med., 2020, 26, 732–740 CrossRef CAS.
  13. L. Xu, J. Wang, Y. Liu, L. Xie and H. Chen, N. Engl. J. Med., 2019, 381, 1240–1247 CrossRef CAS.
  14. E. A. Stadtmauer, J. A. Fraietta, M. M. Davis, A. D. Cohen, K. L. Weber, E. Lancaster, P. A. Mangan, I. Kulikovskaya, M. Gupta, F. Chen, L. Tian, V. E. Gonzalez, J. Xu, I.-Y. Jung, J. J. Melenhorst, G. Plesa, J. Shea, T. Matlawski, A. Cervini, A. L. Gaymon, S. Desjardins, A. Lamontagne, J. Salas-Mckee, A. Fesnak, D. L. Siegel, B. L. Levine, J. K. Jadlowsky, R. M. Young, A. Chew, W.-T. Hwang, E. O. Hexner, B. M. Carreno, C. L. Nobles, F. D. Bushman, K. R. Parker, Y. Qi, A. T. Satpathy, H. Y. Chang, Y. Zhao, S. F. Lacey and C. H. June, Science, 2020, 367, eaba7365 CrossRef CAS.
  15. C. Liu, L. Zhang, H. Liu and K. Cheng, J. Controlled Release, 2017, S0168365917308453 Search PubMed.
  16. S. Tong, B. Moyo, C. Lee, K. W. Leong and G. Bao, Nat. Rev. Mater., 2019, 4, 726–737 CrossRef CAS.
  17. H. Deng, W. Huang and Z. Zhang, Nano Res., 2019, 12, 10 Search PubMed.
  18. H. Tang, X. Zhao and X. Jiang, Adv. Drug Delivery Rev., 2020 DOI:10.1016/j.addr.2020.03.001.
  19. J. A. Doudna, Nature, 2020, 578, 229–236 CrossRef CAS.
  20. P. K. Jain, J. H. Lo, S. Rananaware, M. Downing, A. Panda, M. Tai, S. Raghavan, H. E. Fleming and S. N. Bhatia, Nanoscale, 2019, 11, 21317–21323 RSC.
  21. L. A. Marraffini and E. J. Sontheimer, Science, 2008, 322, 1843–1845 CrossRef CAS.
  22. M. Jinek, K. Chylinski, I. Fonfara, M. Hauer, J. A. Doudna and E. Charpentier, Science, 2012, 337, 816–821 CrossRef CAS.
  23. L. Krejci, V. Altmannova, M. Spirek and X. Zhao, Nucleic Acids Res., 2012, 40, 5795–5818 CrossRef CAS.
  24. X. Xu, T. Wan, H. Xin, D. Li, H. Pan, J. Wu and Y. Ping, J. Gene Med., 2019, 21, 7 CrossRef.
  25. D. D. Kocak, E. A. Josephs, V. Bhandarkar, S. S. Adkar, J. B. Kwon and C. A. Gersbach, Nat. Biotechnol., 2019, 37, 657–666 CrossRef CAS.
  26. Y. Li, X. Teng, K. Zhang, R. Deng and J. Li, Anal. Chem., 2019, 91, 3989–3996 CrossRef CAS.
  27. O. O. Abudayyeh, J. S. Gootenberg, P. Essletzbichler, S. Han, J. Joung, J. J. Belanto, V. Verdine, D. B. T. Cox, M. J. Kellner, A. Regev, E. S. Lander, D. F. Voytas, A. Y. Ting and F. Zhang, Nature, 2017, 550, 280–284 CrossRef.
  28. C. Liu, L. Zhang, H. Liu and K. Cheng, J. Controlled Release, 2017, 266, 17–26 CrossRef CAS.
  29. N. Chang, C. Sun, L. Gao, D. Zhu, X. Xu, X. Zhu, J. Xiong and J. J. Xi, Cell Res., 2013, 23, 465–472 CrossRef CAS.
  30. S. Chakraborty, H. Ji, A. M. Kabadi, C. A. Gersbach, N. Christoforou and K. W. Leong, Stem Cell Rep., 2014, 3, 940–947 CrossRef CAS.
  31. P. Mali, L. Yang, K. M. Esvelt, J. Aach, M. Guell, J. E. Dicarlo, J. E. Norville and G. M. Church, Science, 2013, 339, 823–826 CrossRef CAS.
  32. Y. Fu, J. A. Foden, C. Khayter, M. L. Maeder, D. Reyon, J. K. Joung and J. D. Sander, Nat. Biotechnol., 2013, 31, 822–826 CrossRef CAS.
  33. B. Shen, W. Zhang, J. Zhang, J. Zhou, J. Wang, L. Chen, L. Wang, A. Hodgkins, V. Iyer and X. Huang, Nat. Methods, 2014, 11, 399–402 CrossRef CAS.
  34. Y. Niu, B. Shen, Y. Cui, Y. Chen, J. Wang, L. Wang, Y. Kang, X. Zhao, W. Si, W. Li, A. P. Xiang, J. Zhou, X. Guo, Y. Bi, C. Si, B. Hu, G. Dong, H. Wang, Z. Zhou, T. Li, T. Tan, X. Pu, F. Wang, S. Ji, Q. Zhou, X. Huang, W. Ji and J. Sha, Cell, 2014, 156, 836–843 CrossRef CAS.
  35. B. Shen, J. Zhang, H. Wu, J. Wang, K. Ma, Z. Li, X. Zhang, P. Zhang and X. Huang, Cell Res., 2013, 23, 720–723 CrossRef CAS.
  36. S. Kim, D. Kim, S. Cho, J. Kim and J. Kim, Genome Res., 2014, 24, 1012–1019 CrossRef CAS.
  37. A. Hendel, R. O. Bak, J. T. Clark, A. Kennedy, D. E. Ryan, S. Roy, I. Steinfeld, B. D. Lunstad, R. J. Kaiser and A. B. Wilkens, Nat. Biotechnol., 2015, 33, 985–989 CrossRef CAS.
  38. J. W. Woo, J. Kim, S. I. Kwon, C. Corvalan, S. W. Cho, H. Kim, S. Kim, S. Kim, S. Choe and J. Kim, Nat. Biotechnol., 2015, 33, 1162–1164 CrossRef CAS.
  39. L. Li, S. Hu and X. Chen, Biomaterials, 2018, 171, 207–218 CrossRef CAS.
  40. H. Yin, C.-Q. Song, J. R. Dorkin, L. J. Zhu, Y. Li, Q. Wu, A. Park, J. Yang, S. Suresh, A. Bizhanova, A. Gupta, M. F. Bolukbasi, S. Walsh, R. L. Bogorad, G. Gao, Z. Weng, Y. Dong, V. Koteliansky, S. A. Wolfe, R. Langer, W. Xue and D. G. Anderson, Nat. Biotechnol., 2016, 34, 328–333 CrossRef CAS.
  41. S. Ramakrishna, A. K. Dad, J. Beloor, R. Gopalappa, S. Lee and H. Kim, Genome Res., 2014, 24, 1020–1027 CrossRef CAS.
  42. Y. K. Kang, K. Kwon, J. S. Ryu, H. N. Lee, C. Park and H. J. Chung, Bioconjugate Chem., 2017, 28, 3936–3936 CrossRef.
  43. R. Mout, M. Ray, G. Y. Tonga, Y. Lee, T. Tay, K. Sasaki and V. M. Rotello, ACS Nano, 2017, 11, 2452–2458 CrossRef CAS.
  44. E. Blanco, H. Shen and M. Ferrari, Nat. Biotechnol., 2015, 33, 941–951 CrossRef CAS.
  45. V. Mirshafiee, R. Kim, S. Park, M. Mahmoudi and M. L. Kraft, Biomaterials, 2016, 75, 295–304 CrossRef CAS.
  46. R. M. Visalakshan, M. N. Macgregor, S. Sasidharan, A. Ghazaryan, A. Mierczynskavasilev, S. Morsbach, V. Mailander, K. Landfester, J. D. Hayball and K. Vasilev, ACS Appl. Mater. Interfaces, 2019, 11, 27615–27623 CrossRef CAS.
  47. A. K. Srivastav, N. Dhiman, H. Khan, A. K. Srivastav, S. K. Yadav, J. Prakash, N. Arjaria, D. Singh, S. Yadav and S. Patnaik, J. Pharm. Sci., 2019, 108, 1872–1889 CrossRef CAS.
  48. R. Liu, J. Tang, Y. Xu and Z. Dai, ACS Nano, 2019, 13, 5124–5132 CrossRef CAS.
  49. C. Gao, P. Bhattarai, M. Chen, N. Zhang, S. Hameed, X. Yue and Z. Dai, Bioconjugate Chem., 2018, 29, 3967–3981 CrossRef CAS.
  50. M. Chen, X. Liang, C. Gao, R. Zhao, N. Zhang, S. Wang, W. Chen, B. Zhao, J. Wang and Z. Dai, ACS Nano, 2018, 12, 7312–7326 CrossRef CAS.
  51. A. C. Anselmo, C. L. Moderypawlowski, S. Menegatti, S. Kumar, D. R. Vogus, L. L. Tian, M. Chen, T. M. Squires, A. S. Gupta and S. Mitragotri, ACS Nano, 2014, 8, 11243–11253 CrossRef CAS.
  52. F. S. Mozar and E. H. Chowdhury, J. Pharm. Sci., 2018, 107, 2497–2508 CrossRef CAS.
  53. L. Rao, Q. Meng, L. Bu, B. Cai, Q. Q. Huang, Z. Sun, W. Zhang, A. Li, S. Guo and W. Liu, ACS Appl. Mater. Interfaces, 2017, 9, 2159–2168 CrossRef CAS.
  54. Z. Gong, M. Chen, Q. Ren, X. Yue and Z. Dai, Signal Transduction Targeted Ther., 2020, 5, 12 CrossRef.
  55. S. Mo, X. Zhang, S. Hameed, Y. Zhou and Z. Dai, Theranostics, 2020, 10, 2130–2140 CrossRef CAS.
  56. H. F. Dvorak, J. A. Nagy, J. T. Dvorak and A. M. Dvorak, Am. J. Pathol., 1988, 133, 95–109 CAS.
  57. X. Zhang, R. Liu and Z. Dai, Nanoscale, 2018, 10, 20347–20353 RSC.
  58. S. Hameed, P. Bhattarai, X. Liang, N. Zhang, Y. Xu, M. Chen and Z. Dai, Theranostics, 2018, 8, 5501–5518 CrossRef CAS.
  59. D. Mcmahon, C. Poon and K. Hynynen, Expert Opin. Drug Delivery, 2019, 16, 129–142 CrossRef.
  60. B. D. Gastfriend, S. P. Palecek and E. V. Shusta, Curr. Opin. Biomed. Eng., 2018, 5, 6–12 CrossRef.
  61. N. Zhang, F. Yan, X. Liang, M. Wu, Y. Shen, M. Chen, Y. Xu, G. Zou, P. Jiang, C. Tang, H. Zheng and Z. Dai, Theranostics, 2018, 8, 2264–2277 CrossRef CAS.
  62. H.-X. Wang, Z.-Q. Zuo, J.-Z. Du, Y.-C. Wang, R. Sun, Z.-T. Cao, X.-D. Ye, J.-L. Wang, K. W. Leong and J. Wang, Nano Today, 2016, 11, 133–144 CrossRef CAS.
  63. W. F. Richter and B. Jacobsen, Drug Metab. Dispos., 2014, 42, 1881–1889 CrossRef.
  64. Y. Boucher and R. K. Jain, Cancer Res., 1992, 52, 5110–5114 CAS.
  65. Y. Boucher, L. T. Baxter and R. K. Jain, Cancer Res., 1990, 50, 4478–4484 CAS.
  66. S. Hameed, H. Chen, M. Irfan, S. Z. Bajwa, W. S. Khan, S. M. Baig and Z. Dai, Bioconjugate Chem., 2019, 30, 13–28 CrossRef CAS.
  67. D. A. Kuhn, D. Vanhecke, B. Michen, F. Blank, P. Gehr, A. Petri-Fink and B. Rothen-Rutishauser, Beilstein J. Nanotechnol., 2014, 5, 1625–1636 CrossRef.
  68. T.-G. Iversen, T. Skotland and K. Sandvig, Nano Today, 2011, 6, 176–185 CrossRef CAS.
  69. S. Xu, B. Z. Olenyuk, C. T. Okamoto and S. F. Hamm-Alvarez, Adv. Drug Delivery Rev., 2013, 65, 121–138 CrossRef CAS.
  70. H. Tang, H. Zhang, H. Ye and Y. Zheng, J. Phys. Chem. B, 2018, 122, 171–180 CrossRef CAS.
  71. R. J. Lee, S. Wang and P. S. Low, Biochim. Biophys. Acta, Mol. Cell Res., 1996, 1312, 237–242 CrossRef.
  72. S. A. Smith, L. I. Selby, A. P. R. Johnston and G. K. Such, Bioconjugate Chem., 2019, 30, 263–272 CrossRef CAS.
  73. U. Kutay and M. W. Hetzer, Curr. Opin. Cell Biol., 2008, 20, 669–677 CrossRef CAS.
  74. B. Lee, K. Lee, S. Panda, R. Gonzalesrojas, A. T. Chong, V. Bugay, H. M. Park, R. Brenner, N. Murthy and H. Y. Lee, Nat. Biomed. Eng., 2018, 2, 497–507 CrossRef CAS.
  75. K. Lee, M. J. Conboy, H. M. Park, F. Jiang, H. J. Kim, M. A. Dewitt, V. A. Mackley, K. Chang, A. Rao and C. Skinner, Nat. Biomed. Eng., 2017, 1, 889–901 CrossRef CAS.
  76. W. Sun, W. Ji, J. M. Hall, Q. Hu, C. Wang, C. L. Beisel and Z. Gu, Angew. Chem., Int. Ed., 2015, 54, 12029–12033 CrossRef CAS.
  77. Q. Liu, K. Zhao, C. Wang, Z. Zhang, C. Zheng, Y. Zhao, Y. Zheng, C. Liu, Y. An and L. Shi, Adv. Sci., 2019, 6, 1801423 CrossRef.
  78. W. Sun, J. Wang, Q. Hu, X. Zhou, A. Khademhosseini and Z. Gu, Sci. Adv., 2020, 6, eaba2983 CrossRef.
  79. D. H. Jo, J. H. Kim, T. G. Lee and J. H. Kim, Nanomedicine, 2015, 11, 1603–1611 CrossRef CAS.
  80. C. He, Y. Hu, L. Yin, C. Tang and C. Yin, Biomaterials, 2010, 31, 3657–3666 CrossRef CAS.
  81. C. Schleh, M. Semmlerbehnke, J. Lipka, A. Wenk, S. Hirn, M. Schaffler, G. Schmid, U. Simon and W. G. Kreyling, Nanotoxicology, 2012, 6, 36–46 CrossRef CAS.
  82. M. Durymanov, T. Kamaletdinova, S. E. Lehmann and J. Reineke, J. Controlled Release, 2017, 261, 10–22 CrossRef CAS.
  83. J. Fang, H. Nakamura and H. Maeda, Adv. Drug Delivery Rev., 2011, 63, 136–151 CrossRef CAS.
  84. E. Fitzpatrick, R. R. Mitry and A. Dhawan, J. Intern. Med., 2009, 266, 339–357 CrossRef CAS.
  85. K. Yuan, C. Lai, L. Wei, T. Feng, Q. Yang, T. Zhang, T. Lan, Y. Yao, G. Xiang and X. Huang, Stem Cells Int., 2019, 2019, 5310202 Search PubMed.
  86. X. Duan and Y. Li, Small, 2013, 9, 1521–1532 CrossRef CAS.
  87. S. M. Moghimi, A. C. Hunter and J. C. Murray, Pharmacol. Rev., 2001, 53, 283–318 CAS.
  88. Z. Yue, W. Wei, P. Lv, H. Yue, L. Wang, Z. Su and G. Ma, Biomacromolecules, 2011, 12, 2440–2446 CrossRef CAS.
  89. K. Xiao, Y. Li, J. Luo, J. Lee, W. Xiao, A. M. Gonik, R. G. Agarwal and K. S. Lam, Biomaterials, 2011, 32, 3435–3446 CrossRef CAS.
  90. S. Tenzer, D. Docter, J. Kuharev, A. Musyanovych, V. Fetz, R. Hecht, F. Schlenk, D. Fischer, K. Kiouptsi and C. Reinhardt, Nat. Nanotechnol., 2013, 8, 772–781 CrossRef CAS.
  91. A. S. Zahr, C. A. Davis and M. V. Pishko, Langmuir, 2006, 22, 8178–8185 CrossRef CAS.
  92. P. R. Cullis and M. J. Hope, Mol. Ther., 2017, 25, 1467–1475 CrossRef CAS.
  93. J. C. Kraft, J. P. Freeling, Z. Wang and R. J. Y. Ho, J. Pharm. Sci., 2014, 103, 29–52 CrossRef CAS.
  94. K. J. Kauffman, J. R. Dorkin, J. H. Yang, M. W. Heartlein, F. DeRosa, F. F. Mir, O. S. Fenton and D. G. Anderson, Nano Lett., 2015, 15, 7300–7306 CrossRef CAS.
  95. M. Wang, J. A. Zuris, F. Meng, H. A. Rees, S. Sun, P. Deng, Y. Han, X. Gao, D. Pouli and Q. Wu, Proc. Natl. Acad. Sci. U. S. A., 2016, 113, 2868–2873 CrossRef CAS.
  96. S. C. Semple, A. Akinc, J. Chen, A. P. Sandhu, B. L. Mui, C. K. Cho, D. W. Y. Sah, D. Stebbing, E. J. Crosley and E. Yaworski, Nat. Biotechnol., 2010, 28, 172–176 CrossRef CAS.
  97. J. D. Finn, A. R. Smith, M. C. Patel, L. Shaw, M. R. Youniss, J. van Heteren, T. Dirstine, C. Ciullo, R. Lescarbeau, J. Seitzer, R. R. Shah, A. Shah, D. Ling, J. Growe, M. Pink, E. Rohde, K. M. Wood, W. E. Salomon, W. F. Harrington, C. Dombrowski, W. R. Strapps, Y. Chang and D. V. Morrissey, Cell Rep., 2018, 22, 2227–2235 CrossRef CAS.
  98. Q. Cheng, T. Wei, L. Farbiak, L. T. Johnson, S. A. Dilliard and D. J. Siegwart, Nat. Nanotechnol., 2020, 15, 313–320 CrossRef CAS.
  99. G. Chen, A. A. Abdeen, Y. Wang, P. K. Shahi, S. Robertson, R. Xie, M. Suzuki, B. R. Pattnaik, K. Saha and S. Gong, Nat. Nanotechnol., 2019, 14, 974–980 CrossRef CAS.
  100. C. Liang, F. Li, L. Wang, Z. Zhang, C. Wang, B. He, J. Li, Z. Chen, A. B. Shaikh and J. Liu, Biomaterials, 2017, 147, 68–85 CrossRef CAS.
  101. P. Guo, J. Yang, J. Huang, D. T. Auguste and M. A. Moses, Proc. Natl. Acad. Sci. U. S. A., 2019, 116, 18295–18303 CrossRef CAS.
  102. B.-Y. Liu, X.-Y. He, R.-X. Zhuo and S.-X. Cheng, Nanoscale, 2018, 10, 21209–21218 RSC.
  103. L. Zhang, L. Wang, Y. Xie, P. Wang, S. Deng, A. Qin, J. Zhang, X. Yu, W. Zheng and X. Jiang, Angew. Chem., Int. Ed., 2019, 58, 12404–12408 CrossRef CAS.
  104. R. H. Fang, A. V. Kroll, W. Gao and L. Zhang, Adv. Mater., 2018, 30, 1706759 CrossRef.
  105. A. Parodi, N. Quattrocchi, A. L. V. De Ven, C. Chiappini, M. Evangelopoulos, J. O. Martinez, B. S. Brown, S. Z. Khaled, I. K. Yazdi and M. V. Enzo, Nat. Nanotechnol., 2013, 8, 61–68 CrossRef CAS.
  106. Z. Chen, P. Zhao, Z. Luo, M. Zheng, H. Tian, P. Gong, G. Gao, H. Pan, L. Liu and A. Ma, ACS Nano, 2016, 10, 10049–10057 CrossRef CAS.
  107. H. Wang, Y. Liu, R. He, D. Xu, J. Zang, N. Weeranoppanant, H. Dong and Y. Li, Biomater. Sci., 2020, 8, 552–568 RSC.
  108. M. Z. Alyami, S. Alsaiari, Y. Li, S. S. Qutub, F. A. Aleisa, R. Sougrat, J. S. Merzaban and N. M. Khashab, J. Am. Chem. Soc., 2020, 142, 1715–1720 CrossRef CAS.
  109. Z. Kaźmierczak, K. Szostak-Paluch, M. Przybyło, M. Langner, W. Witkiewicz, N. Jędruchniewicz and K. Dąbrowska, Bioorg. Med. Chem., 2020, 28, 115556 CrossRef.
  110. I. M. S. Degors, C. Wang, Z. U. Rehman and I. S. Zuhorn, Acc. Chem. Res., 2019, 52, 1750–1760 CrossRef CAS.
  111. P. N. Yaron, B. D. Holt, P. A. Short, M. Lösche, M. F. Islam and K. N. Dahl, J. Nanobiotechnol., 2011, 9, 45 CrossRef CAS.
  112. R. Rouet, B. A. Thuma, M. D. Roy, N. G. Lintner, D. M. Rubitski, J. E. Finley, H. M. Wisniewska, R. Mendonsa, A. Hirsh, L. de Oñate, J. Compte Barrón, T. J. McLellan, J. Bellenger, X. Feng, A. Varghese, B. A. Chrunyk, K. Borzilleri, K. D. Hesp, K. Zhou, N. Ma, M. Tu, R. Dullea, K. F. McClure, R. C. Wilson, S. Liras, V. Mascitti and J. A. Doudna, J. Am. Chem. Soc., 2018, 140, 6596–6603 CrossRef CAS.
  113. H. Wang, Z. Song, Y. Lao, X. Xu, J. Gong, D. Cheng, S. Chakraborty, J. S. Park, M. Li and D. Huang, Proc. Natl. Acad. Sci. U. S. A., 2018, 115, 4903–4908 CrossRef CAS.
  114. P. Guo, D. Liu, K. Subramanyam, B. Wang, J. Yang, J. Huang, D. T. Auguste and M. A. Moses, Nat. Commun., 2018, 9, 130–130 CrossRef.
  115. A. K. Varkouhi, M. Scholte, G. Storm and H. J. Haisma, J. Controlled Release, 2011, 151, 220–228 CrossRef CAS.
  116. S. S. Rohiwal, N. Dvorakova, J. Klima, M. Vaskovicova, F. Senigl, M. Slouf, E. Pavlova, P. Stepanek, D. Babuka, H. Benes, Z. Ellederova and K. Stieger, Sci. Rep., 2020, 10, 4619 CrossRef CAS.
  117. N. Ryu, M. A. Kim, D. Park, B. Lee, Y. R. Kim, K. H. Kim, J. I. Baek, W. J. Kim, K. Y. Lee and U. K. Kim, Nanomedicine, 2018, 14, 2095–2102 CrossRef CAS.
  118. J. A. Kretzmann, D. Ho, C. W. Evans, J. H. C. Planilam, B. Garciabloj, A. E. Mohamed, M. L. Omara, E. Ford, D. Tan and R. Lister, Chem. Sci., 2017, 8, 2923–2930 RSC.
  119. B.-Y. Liu, X.-Y. He, C. Xu, L. Xu, S.-L. Ai, S.-X. Cheng and R.-X. Zhuo, Biomacromolecules, 2018, 19, 2957–2968 CrossRef CAS.
  120. K. Miyata, M. Oba, M. Nakanishi, S. Fukushima, Y. Yamasaki, H. Koyama, N. Nishiyama and K. Kataoka, J. Am. Chem. Soc., 2008, 130, 16287–16294 CrossRef CAS.
  121. H. J. Kim, A. Ishii, K. Miyata, Y. Lee, S. Wu, M. Oba, N. Nishiyama and K. Kataoka, J. Controlled Release, 2010, 145, 141–148 CrossRef CAS.
  122. I. Hafez, N. Maurer and P. R. Cullis, Gene Ther., 2001, 8, 1188–1196 CrossRef CAS.
  123. J. A. Kulkarni, P. R. Cullis and R. V. Der Meel, Nucleic Acid Ther., 2018, 28, 146–157 CrossRef CAS.
  124. M. Jayaraman, S. M. Ansell, B. L. Mui, Y. K. Tam, J. Chen, X. Du, D. Butler, L. Eltepu, S. Matsuda and J. K. Narayanannair, Angew. Chem., Int. Ed., 2012, 51, 8529–8533 CrossRef CAS.
  125. E. P. Thi, C. E. Mire, A. Lee, J. B. Geisbert, R. Ursicbedoya, K. N. Agans, M. Robbins, D. J. Deer, R. W. Cross and A. S. Kondratowicz, J. Clin. Invest., 2017, 127, 4437–4448 CrossRef.
  126. P. Wang, L. Zhang, W. Zheng, L. Cong, Z. Guo, Y. Xie, L. Wang, R. Tang, Q. Feng and Y. Hamada, Angew. Chem., Int. Ed., 2018, 57, 1491–1496 CrossRef CAS.
  127. M. A. Wheeler, C. J. Smith, M. Ottolini, B. S. Barker, A. M. Purohit, R. M. Grippo, R. P. A. Gaykema, A. J. Spano, M. P. Beenhakker and S. Kucenas, Nat. Neurosci., 2016, 19, 756–761 CrossRef CAS.
  128. Y. Qiu, S. Tong, L. Zhang, Y. Sakurai, D. R. Myers, L. Hong, W. A. Lam and G. Bao, Nat. Commun., 2017, 8, 15594–15594 CrossRef CAS.
  129. H. Zhu, L. Zhang, S. Tong, C. M. Lee, H. Deshmukh and G. Bao, Nat. Biomed. Eng., 2019, 3, 126–136 CrossRef CAS.
  130. J. Guan, Q. Shen, Z. Zhang, Z. Jiang, Y. Yang, M. Lou, J. Qian, W. Lu and C. Zhan, Nat. Commun., 2018, 9, 2982 CrossRef.
  131. M. Hadjidemetriou and K. Kostarelos, Nat. Nanotechnol., 2017, 12, 288–290 CrossRef CAS.
  132. G. Caracciolo, Nanomedicine, 2015, 11, 543–557 CrossRef CAS.
  133. W. Zhou and A. Deiters, Angew. Chem., Int. Ed., 2016, 55, 5394–5399 CrossRef CAS.
  134. K. Zhang, R. Deng, H. Gao, X. Teng and J. Li, Chem. Soc. Rev., 2020, 49, 1932–1954 RSC.
  135. K. Zhang, R. Deng, X. Teng, Y. Li, Y. Sun, X. Ren and J. Li, J. Am. Chem. Soc., 2018, 140, 11293–11301 CrossRef CAS.
  136. B. Suresh, S. Ramakrishna and H. Kim, Methods Mol. Biol., 2017, 1507, 81–94 CrossRef CAS.

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