DOI: 10.1002/cbic.

201800629 Minireviews

Delivery of CRISPR/Cas9 by Novel Strategies for Gene

Therapy
Le Wang,[a, b] Wenfu Zheng,*[b] Shaoqin Liu,[a] Bing Li,[b, c] and Xingyu Jiang*[a, b, d]

Precise editing of the genome of a living body is a goal pur- hindering wide and deep application. In this review, recently
sued by scientists in many fields. In recent years, CRISPR (clus- developed delivery strategies for various CRISPR/Cas9 formula-
tered regularly interspaced short palindromic repeat)/Cas tions and their applications in treating gene-related diseases
(CRISPR-associated) genome-editing systems have become a are briefly summarized. This review should inspire others to
revolutionary toolbox for gene editing across various species. explore more efficient strategies for CRISPR system delivery
However, the low transfection efficiency of the CRISPR/Cas9 and gene therapy.
system to mammalian cells in vitro and in vivo is a big obstacle

1. Introduction

According to a report from the National Institutes of Health bioinformatics, a CRISPR/Cas system based on a RNA-guided
(NIH), over hundreds of thousands of human diseases are nuclease was identified to induce genome editing in mamma-
caused by genetic alterations in the genome, and only a very lian cells (Figure 1).[2, 5] Before CRISPR/Cas was used for gene
small portion of these diseases can be cured;[1] thus, the emer- editing, numerous studies showed that zinc finger nucleases
gence of a powerful genome-editing toolbox is highly antici- (ZFNs)[6] and transcription activator-like effector nucleases
pated. In recent years, the CRISPR (clustered regularly inter-
spaced short palindromic repeat)/Cas(CRISPR-associated)
system was developed to manipulate mammalian genomes
and to provide opportunities for treating gene-related diseas-
es. The CRISPR/Cas system is an acquired species that has
evolved in bacteria and archaea to defend against attack by
viruses and plasmids.[2–4] Although the CRISPR sequence was
discovered as early as 1987, genome editing made a huge leap
forward with the report that the CRISPR/Cas system could be
repurposed for genome editing in mammalian cells in early
2013. With the development of sequencing technology and

Figure 1. Key studies characterizing and engineering CRISPR systems.

[a] L. Wang, Prof. S. Liu, Prof. X. Jiang
School of Life Science and Technology, Harbin Institute of Technology
2 Yikuang Road, Nangang District, Harbin 150001 (China)
E-mail: xingyujiang@nanoctr.cn (TALENs)[7] could target and edit specific DNA sequences. How-
[b] L. Wang, Prof. W. Zheng, B. Li, Prof. X. Jiang ever, both ZFN- and TALEN-mediated gene editing rely on pro-
Beijing Engineering Research Center for BioNanotechnology
tein–DNA recognition; thus, each experiment requires the con-
CAS Key Lab for Biological Effects of Nanomaterials and Nanosafety
CAS Center of Excellence for Nanoscience struction of different proteins with different targeting specifici-
National Center for NanoScience and Technology ties. On the contrary, in the CRISPR/Cas system, the specificity
Beijing, 100190 (China) of the genome recognition is determined by gRNA, the design
E-mail: zhengwf@nanoctr.cn
and preparation of which are vastly simpler and easier than
[c] B. Li
those of either ZFNs or TALENs, which simplifies the operation
Biomedical Engineering Institute, Jinan University
No.601, West Huangpu Avenue, TianHe District process and provides technical conditions for its application in
Guangzhou, 510632 (China) various research fields.
[d] Prof. X. Jiang On the basis of the core element content and sequences,
Department of Biomedical Engineering CRISPR/Cas systems can be categorized into three main types:
Southern University of Science and Technology
type I, type II, and type III, and the type II CRISPR/Cas system
No. 1088 Xueyuan Road, Nanshan District, Shenzhen
Guangdong 518055 (China) (i.e., CRISPR/Cas9 system) has been widely applied in biomedi-
The ORCID identification numbers for the authors of this article can be cal applications owing to its simplicity, versatility, high specifici-
found under https://doi.org/10.1002/cbic.201800629. ty, and efficiency. The mechanism of CRISPR/Cas9 was elucidat-

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ed in 2012.[8] The CRISPR/Cas9 system contains three key com- DNA, mRNA, or protein, usually has a large size that is difficult
ponents: CRISPR RNA (crRNA), transacting CRISPR RNA (tracr- for loading onto delivery vehicles. In addition, if the CRISPR/
RNA), and Cas9 endonuclease. A portion of the crRNA se- Cas9 system is administrated in vivo, it is expected to accumu-
quence can be paired with tracrRNA to form a chimeric RNA late in the desired organs or tissues and be internalized by the
(tracrRNA/crRNA), and the other portion can be base paired cells of interest, subsequently functioning as a gene-editing
with the target DNA site. By modifying tracrRNA/crRNA, a tool in the cell nucleus. This targeted transportation process
guide RNA (gRNA) with a guiding role can be constructed. requires that the stability and functionality of the CRISPR/Cas9
Within the gRNA-targeted DNA region, each protospacer must system be maintained, so that degradation and elimination
always be accompanied by a protospacer-adjacent motif during circulation in the body can be avoided. Hence, develop-
(PAM), which varies according to the specific CRISPR system. ing safe and effective delivery vehicles for the CRISPR/Cas9
Under the guide of gRNA, Cas9 nucleases target specific DNA system is crucial for its clinical applications, and this has at-
sequences and induce DNA double-strand breaks (DSBs). DSB tracted increasing attention from researchers and has achieved
can be repaired by two endogenous repair mechanisms, error- rapid development in recent years. Here, we summarize recent
prone nonhomologous end joining (NHEJ) and homology-di- advances in the field of delivery vehicles for the CRISPR/Cas9
rected repair (HDR), to achieve gene editing (Figure 2). system, including viral delivery, physical delivery, nanomateri-
The advanced CRISPR/Cas9 technology provides a powerful als, and biochemical modifications. The features of all the de-
toolbox for gene correction to cure diseases. In recent years, livery vehicles are discussed below (Table 1).

2.1. Viral vectors

In recent years, viral vehicles have been widely utilized for the
delivery of the CRISPR/Cas9 system in vitro and in vivo owing
to its high delivery efficiency and stable expression of the
transgene. The commonly used viral vehicles include adenovi-
rus, adeno-associated virus (AAV), and lentivirus. Among these
vectors, AAVs are the most frequently used carriers for the
delivery of the CRISPR/Cas9 system. For instance, recombinant
AAVs can deliver the CRISPR/Cas9 system into mouse embryos
to form transgenic mice, which provides an in vivo strategy to
generate genetically modified mice and eliminates the need to
isolate mouse embryos and re-implant the treated zygotes
into pseudopregnant females.[20] AAV vectors can also deliver
muscle-targeting Cas9/gRNA expression cassettes and success-
fully induce HDR-mediated Dmd gene correction in vivo.[21] Al-
though viral vectors show high transfection efficiency, there
are still some concerns with their clinical applications. There
Figure 2. Schematic of Cas9/gRNA-mediated genome editing. are challenges associated with their high risks of mutagenesis
and carcinogenesis. In addition, the limited loading capacity of
viruses (e.g., AAVs,  4.7 kb) also restricts the efficient delivery
many gene-related diseases such as immunodeficiency,[9] obesi- of the sequence encoding Cas9/gRNA, which is about 9.3 kb.
ty,[10] granulomatous disease,[11] muscular dystrophy,[12] Hunting- Thus, the development of novel nonviral delivery approaches
ton’s disease,[13] hemophilia,[14, 15] hereditary tyrosinemia,[16] car- is essential for CRISPR-mediated therapeutics.
diac syndrome,[17] and metabolic liver disease[18] have been
treated by the CRISPR/Cas9 system. However, owing to its
2.2. Physical delivery
large size (Cas9 expression plasmids usually exceed 9.3 kb[19]),
the low transfection efficiency of CRISPR/Cas9 is still a huge Physical delivery, a strategy for intended delivery by temporari-
obstacle hindering its wider and deeper application. Thus, the ly disrupting the physical barriers of tissues without the sup-
development of novel delivery approaches is essential for port of the delivery vehicles, has been used for transporting
CRISPR-mediated therapeutics. the CRISPR/Cas9 system. This strategy can generate physical
force upon the cell membranes or nuclear membrane to
momentarily produce membrane pores, which can facilitate
2. Delivery of CRISPR/Cas9
the delivery of cargos. Electroporation, microinjection, hydro-
Although the CRISPR/Cas9 system has great potential in gene dynamic injection, and membrane deformation are representa-
editing and regulation, targeted delivery remains a bottleneck tive physical approaches for delivering CRISPR/Cas9.
for its therapeutic application. Several critical obstacles Electroporation is an approach that directly applies an elec-
emerged in the delivery of the CRISPR/Cas9 system. As far as tric field on the cell membrane to generate pores and, thus,
we know, the CRISPR/Cas9 system, whether in the form of facilitates the delivery of large molecules. In vitro, this strategy

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Table 1. Strategies to deliver the CRISPR/Cas9 system: examples of viral delivery, physical delivery, nanomaterials, and biochemical modification.

Delivery vector Target gene Model system Research objective Ref.

adeno-associated virus Tyr mouse embryos and mouse in vivo model generate genetically modified mice [20]
electroporation CXCR4PD-1 CD4 + T cells functional gene knock-out [22]
functional gene knock-in
microinjection EGFP frog embryos functional gene knock-in [23]
physical
hydrodynamic injection Fah mouse model of hereditary tyrosinemia correction of genetic mutation [24]
delivery
P38 MAPKs MDA-MB-231 cells, SUM-159 cells, and functional gene knock-out [26]
membrane deformation primary human T cells
PD-1 CD4 + T cells functional gene knock-in [26]
nanomaterials: hybrid nanoparticles Runx2hCTNNB1 mesenchymal stem cells functional gene knock-out [30]
Plk-1 A375 cells and mouse in vivo model functional gene knock-out [31]
mdx mdx mice model hdr-mediated repair of genetic mutation [32]
nanomaterials: lipid-based nanoparticles Plk-1 A375 cells and mouse in vivo model functional gene knock-out [33]
Ntn1 T2D mice model functional gene knock-out [35]
nanomaterials: polymer-based nanoparticles GFPPlk-1 U87 cells functional gene knock-out [37]
EGFP EGFP transfected CHO cells functional gene knock-down [40]
nanomaterials: gold nanoparticles AAVS1PTEN HeLa cells functional gene knock-out [41]
GFP GFP-B16F10 cells functional gene knock-out [42]
biochemical modification CCR5 HEK293T, HeLa, and NCCIT cells lines functional gene knock-out [44]
EMX1 HEPG2 functional gene knock-out [46]

has been widely used in the transfection of various biological Hydrodynamic injection is another physical delivery strategy
molecules, including hard-to-transfect cell types such as neu- for large molecules, and it involves the rapid injection of a
rons. By electroporation, researchers have successfully deliv- high-volume solution into organisms through a vein. The high
ered Cas9 ribonucleoproteins (Cas9 protein in complex with pressure caused by a sudden increase in volume induces the
gRNA, Cas9 RNP) into CD4 + T cells, which efficiently induced formation of temporary pores on the vascular wall or cell
DSB of the target gene CXCR4 and down-regulated the expres- membrane, and this facilitates effective delivery of cargos to
sion of CXCR4 by NHEJ repair. They also achieved efficient the desired tissues. CRISPR/Cas9 delivered by this means accu-
genetic knock-in with HDR to induce CXCR4 or PD-1(PDCD1) mulates significantly in the liver, so it has been widely used in
ablation. For editing CXCR4, the CXCR4 HDR template was gene editing for liver-related diseases. For example, hydrody-
designed to replace 12 nucleotides from a human reference namic injection of a plasmid-based CRISPR/Cas9 system and a
genome, including the PAM sequence required for CRISPR- HDR repair template was shown to correct a Fah mutation in
mediated DNA cleavage, and to introduce a HindIII restriction hepatocytes in a hereditary tyrosinemia mouse model. The de-
enzyme cleavage site. For PD-1 editing, the PD-1 HDR template livery efficiency of hydrodynamic injection was 1/250 liver cells,
was designed to generate a frameshift mutation and a knock- which is sufficient to rescue some functions of the liver, but it
in HindIII restriction enzyme cleavage site in the first exon of has much lower editing efficiency than other delivery strat-
PD-1. Cas9 RNPs and CXCR4 or PD-1 HDR templates were co- egies.[24] In addition, its impact on large animals is still unclear.
electroporated into primary CD4 + T cells and realized im- Membrane deformation can promote the diffusion of macro-
proved efficacy of CXCR4 or PD-1 ablation.[22] T cells with muta- molecules in the surrounding medium into the cytosol by
tions on the HIV coreceptor CXCR4 can gain resistance to HIV using physical constriction to generate a transient membrane
infection, whereas T cells with deletion of the genes encoding disruption or holes on the deformed cells. Recently, researchers
key immune checkpoint PD-1 can recognize and kill cancer designed membrane-deformation-based microfluidic devices
cells. The knock-out or knock-in of PD-1 or CXCR4 demon- with constrictions smaller than the cell diameter, imposing
strates the robustness of the CRISPR/Cas9 system in creating shear and compressive forces on cells to form a rapid mechani-
genome mutations at will. Nevertheless, although electropora- cal deformation for intracellular delivery. Researchers have opti-
tion shows high transfection efficiency, the drawbacks of a low mized the specifications of the delivery chip, including con-
targeting capability, requirement for specialized equipment, striction dimensions, fluid flow rates, and duration of cell pas-
and irreversible damage to cells limit its application. sage through the chip, to realize safe and efficient delivery for
Microinjection can directly inject the exogenous biomolecule different cell types containing hard-to-transfect cells, such as
into cells by using a micron-scale needle, which provides a immune cells and stem cells. This intracellular delivery method
good strategy for the delivery of CRISPR/Cas9 with high effi- is not restricted by the types of target cells or exogenous mac-
ciency, controllable dosage, and precise delivery. Microinjecting romolecules of interest, and thus, it achieves high-throughput
the components of CRISPR/Cas9 into the nucleus is the most delivery of almost all macromolecules into almost any cell type
direct approach to achieve gene editing.[23] However, this with high efficiency and a low cell-death rate.[25] Based on the
method requires professional microoperating equipment and broad applicability of these membrane-deformation-based
professionals to operate it. Moreover, this technique has low microfluidic devices, Cas9 RNP delivery has been shown to effi-
throughput, which is still considered its biggest limitation. ciently modify the genome in human primary T cells; hence,

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this technique shows great promise for engineering T cells to realize gene mutation correction by homology-directed repair,
treat autoimmunity and infections. Furthermore, the trium- the simultaneous delivery of Cas9, sgRNA, and donor DNA is
phant genome editing of PD-1 might benefit cancer immuno- challenging. A nanocarrier (named CRISPR-Gold) consisting of
therapies.[26] A combination of the advantages of high- gold nanoparticles, Cas9 RNP, donor DNA, and endosomal dis-
throughput microfluidic chips and efficient CRISPR/Cas9 gene ruptive polymer (poly{N-[N-(2-aminoethyl)-2-aminoethyl]aspart-
editing can also realize rapid gene function screening and, amide}) was recently reported. CRISPR-Gold enters cells by
thus, provides a powerful method to facilitate systematic ge- endocytosis and releases the cargo into the cytoplasm by dis-
netic analyses.[27] rupting the endosome membrane. DNA is released from the
gold core of CRISPR-Gold by cytosolic glutathione and effec-
tively edits the target gene. In vivo experiments demonstrate
2.3. Nanomaterials
that CRISPR-Gold corrects dystrophin mutation in mdx mice
Currently, nanomaterials have emerged as prevailing delivery with minimal off-target damage and improves animal strength
systems for gene-regulation tools.[28, 29] Nanomaterials for (Figure 3 B).[32] CRISPR-Gold offers a new therapeutic strategy
CRISPR/Cas9 delivery can protect these easily degradable for treating diseases caused by point mutations and small de-
cargos from clearance from the body by wrapping them tight- letions.
ly. The physicochemical properties of nanomaterials can be Lipid-based nanoparticles are prevalent in the delivery of
tuned to achieve effective and targeted transportation. All of the CRISPR/Cas9 system. In lipid-based delivery systems, cat-
these merits provide the nanomaterials with the ability to de- ionic lipids usually encapsulate anionic Cas9 DNA, mRNA, or
liver the CRISPR/Cas9 system in vivo. protein through electrostatic interactions, and this precludes
Hybrid nanoparticles are burgeoning carriers that have degradation of the CRISPR/Cas9 system and the immunological
attracted much attention in the field of CRISPR/Cas9 delivery. response of the organism. Recently, our group reported a poly-
Combining the excellent properties of different materials ethylene glycol (PEG)-modified cationic lipid-based nanoparti-
makes hybrid nanoparticles perform splendidly. Researchers cle that condenses the protamine/Cas9-gPlk-1 plasmid DNA/
have developed exosome–liposome hybrid nanoparticles to chondroitin sulfate ternary complex to form a core–shell struc-
deliver the CRISPR/Cas9 system into mesenchymal stem cells. ture with about 47.4 % transfection efficiency in A375 cells in
The platform solves the challenges associated with a low load- vitro and 67 % suppression of tumor growth in vivo.[33] Other
ing capacity of the exosomes and difficulties in the transfection work has identified lipids from a library of synthetic cationic
of mesenchymal stem cells with liposomes at the same time.[30] lipids that effectively deliver the Cas9 protein. This screening
Recently, on the basis of our work in the preparation of lipid- has shed light on the structure–activity relationship between
based nanoparticles and gold nanoparticles, we developed a lipid structure and delivery efficacy.[34] A library of PEG-block-
lipid/gold nanocluster delivery system that could successfully poly(d,l-lactic-co-glycolic acid) (PEG-b-PLGA)-based cationic
deliver Cas9 proteins and gPlk-1 plasmids for gene therapy. In lipid-assisted nanoparticles (CLANs) that carry macrophage-
this delivery system, besides effective encapsulation of the specific promotor (CD68)-driving Cas9 expression plasmid was
lipid shell, TAT-modified gold nanoclusters also condense the recently reported. With the guidance of the sgRNA-targeting
complex of Cas9 proteins and gRNA plasmids to decrease the Ntn1 gene, the CLANs specifically edit the Ntn1 gene only in
packing volume and promote nuclear transport. After that, we macrophages and reduce the expression of netrin1 and im-
created another photothermal-activatable lipid/gold nanoparti- prove type II diabetes symptoms in vivo (Figure 3 C).[35]
cle (AuNP) platform for the delivery of Cas9-gPlk-1 plasmids A large number of polymer-based nanoparticles are useful
that could be thermally triggered to release TAT peptides from for the delivery of gene-editing toolboxes. Polyethyleneimine
the AuNPs by cleavage of Au S bonds under l = 514 nm laser (PEI) is a popular polymer for CRISPR/Cas9 delivery. For in-
irradiation. Moreover, Cas9/gPlk-1 plasmids that condense with stance, a graphene oxide–polyethylene glycol–polyethylene-
TAT peptides through electrostatic interactions are dissociative imine nanocarrier can load Cas9/gRNA through physical ab-
in the cytosol and can subsequently enter the nucleus under sorption and p-stacking interactions. This method achieves effi-
the guidance of penetrating peptides. The multifunctional cient gene editing in AGS cells.[36] In another report, liposome-
lipid/gold nanoparticles combine the functions of multiple ma- templated hydrogel nanoparticles (LHNPs) composed of a PEI
terials. In more detail, the lipid shell maintains the stability of hydrogel are capable of encapsulating Cas9 protein and cat-
the plasmid and also promotes the uptake of hybrid nanoparti- ionic lipids for efficient delivery of gene-editing tools. After
cles; the AuNPs function as both plasmid carriers and photo- loading Cas9/gPlk-1, the RGD-modified LHNPs effectively sup-
thermal reagents; the TAT peptides mediate the binding of press the growth of U87 tumor.[37] In addition to PEI, there are
plasmids and guide them to the nucleus (Figure 3 A). This still other kinds of polymers that can be used as delivery vehi-
AuNP-condensed, lipid-encapsulated, and laser-controlled de- cles for CRISPR/Cas9. Quaternary ammonium terminated poly-
livery platform combines the individual functions of two excel- (propylene oxide) (PPO-NMe3) and amphiphilic Pluronic F127
lent platforms for gene delivery, lipid formulation and AuNPs, form a lower charge density, self-assembled micelle as the de-
that act in a collaborative way to realize high delivery efficien- livery platform for the plasmid-based CRISPR/Cas9 system, and
cy and targeted gene therapy in vivo. Our strategy shows it efficiently delivers Cas9 plasmid and disrupts the HPV18-E7
great promise in high-efficiency CRISPR/Cas9 delivery and gene to suppress cancer progression.[38] Other interesting re-
gene therapy for treating a wide spectrum of diseases.[31] To search reports the self-assembly of nucleic acids to construct a

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Figure 3. Nanomaterials for CRISPR/Cas9 delivery. A) Schematic for the preparation of a lipid/gold nanoparticles platform and laser-enhanced knock-out of the
Plk-1 gene in A375 cells. ACP = AuNPs/Cas9-gPlk-1 plasmids, DSPE = 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, LACP = lipid-encapsulated ACP. Repro-
duced from ref. [31] with permission. Copyright: 2018, Wiley-VCH Verlag GmbH & Co. KGaA. B) Ingenious design of CRISPR-Gold for delivering Cas9 RNPs and
donor DNA to correct the gene mutation of Duchenne muscular dystrophy in mice. Reproduced from ref. [32] with permission. Copyright: 2017, Nature Pub-
lishing Group. C) Illustration of the strategy for specific gene editing in macrophages in vivo with the CRISPR/Cas9 system. Reproduced from ref. [35] with per-
mission. Copyright: 2018, American Chemical Society. D) Design of arginine nanoparticles for the delivery of Cas9 or Cas9-RNP into the cytoplasm by mem-
brane fusing. Reproduced from ref. [41] with permission. Copyright: 2017, American Chemical Society.

vector to deliver the CRISPR/Cas9 system.[39] However, consid- Biodegradable carriers are ideal tools for in vivo delivery, as
ering the essential drawback of nucleic acids, this strategy these kinds of carriers are safer than nondegradable materials.
needs to combine other approaches to avoid their degradation Recently, a biodegradable two-dimensional black phosphorus
and enhance their intracellular delivery. Lately, nanoscale zeolit- nanosheet based nanocarrier for delivering CRISPR/Cas9 RNP
ic imidazole frameworks (ZIFs) have been utilized to encapsu- was reported. The RNP was engineered with three nuclear locali-
late CRISPR RNPs and to enhance the endosomal escape of zation signals at the C terminus. The nanocarrier enters the
payloads.[40] target cell through the endocytosis pathway, and the nano-
Gold nanomaterials are ideal vectors for the delivery of the sheets degrade and release RNP through endosomal escape. In
CRISPR/Cas9 system. Recently, a nanocarrier composed of cat- vitro and in vivo experiments demonstrated successful genome
ionic arginine gold nanoparticles (ArgNPs) and engineered Cas9 editing and gene silencing. This simple and versatile cytosolic
proteins with a glutamate peptide tag at the N terminus and a delivery platform is a convenient tool for delivering not only
nuclear localization sequence (NLS) at the C terminus were re- CRISPR/Cas9 but also other bioactive macromolecules.[43]
ported to deliver RNPs directly into the cytosol with high effica-
cy (  90 %). The nanocarrier enters the nucleus of HeLa cells
2.4. Biochemical modification
with the guidance of the NLS, which results in a gene editing
(AAVS1 gene) efficiency of about 30 % (Figure 3 D).[41] Ultra- Biochemical modification of the components of the CRISPR/
sound-propelled gold nanowires (AuNWs) that bind Cas9/gRNA Cas9 system can significantly improve their delivery efficiency.
through reversible disulfide linkages effect rapid and efficient in- Modifying proteins and nucleic acids with the cell-penetrating
tracellular delivery of the CRISPR/Cas9 system. This strategy can peptide (CPP) is a common approach to enhance their intracel-
effectively knock-out the target gene at a low concentration lular and nuclear-targeted delivery. CPPs are short peptides
(0.6 nm) extracellular of the Cas9/gRNA complex.[42] with positive charges and excellent cell-penetrating ability.

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Conjugation of CPPs to the Cas9 protein and/or gRNA results
Table 2. Characteristics of different CRISPR/Cas9 genome-editing sys-
in improved delivery of the CRISPR/Cas9 system. Recently, CPP- tems.
conjugated Cas9 protein and CPP-complexed gRNA were re-
ported to induce efficient CCR5 gene disruption with reduced Cas9 Vehicle Advantages Shortcomings Ref.
delivery
off-target mutations in human cells, including dermal fibro-
method
blasts, HeLa, HEK239T, embryonic stem cells, and embryonic
carcinoma cells.[44] Modifying the NLS to the proteins and
nucleic acids of the CRISPR system is an alternative approach
low efficiency,
to improve delivery efficacy, because only if the endonuclease low cost,
DNA delayed onset, [47]
great stability
Cas9 and gRNA reach the nucleus can the gene-editing func- integration risk
tion of the CRISPR/Cas9 system be realized. In recent work,
Cas9 protein modified with multiple SV40 NLSs exhibits a ten-
fold increase in the efficiency of in vivo neuronal editing.[45] transient
The successful application of CPPs or NLSs requires that they expression,
be combined with other strategies, because although CPP mRNA quick onset, poor stability [51]–[53]
modification or NLS modification of Cas9 proteins can promote low off-target
effects
intracellular delivery and subsequent nuclear transport, Cas9
proteins simply modified by a CPP or NLS are not stable in the
presence of proteases and are weak in cell-type-specific target- transient
protein– duration, high cost,
ing, which are still challenges for effective in vivo gene editing. RNA swift onset, endotoxin [54]
Recently, Cas9 RNP was modified with an asialoglycoprotein re- complex low off-target contamination
ceptor and a NLS that can be recognized and internalized by effects
liver cells. This biochemical-modified RNP complex selectively
gathers in the liver and conducts cell-selective gene editing.[46]

The second method is to deliver Cas9 mRNA and gRNA. The

3. Modes of CRISPR/Cas9 Delivery
mRNA encoding the Cas9 protein can be expressed by ribo-
The CRISPR/Cas9 gene-editing system provides an efficient somes in the cytosol of cells. Cas9 mRNA can induce faster
approach for specific, complete, and permanent knock-out of gene editing during cell culture than a plasmid delivery
gene expression, and this makes it a potent research tool for system, because the transcription process is not required.[50]
determining key players in specific biological pathways. Cas9 mRNA delivery leads to transient protein expression, which can
cassettes can be introduced into CRISPR systems and delivered more directly control the duration and dose of Cas9 protein in
in three modes: DNA, mRNA, and protein (Table 2). Each the cell and limit the occurrence of off-target editing events
method has overall effectiveness but still has shortcomings in during gene editing. However, the shorter expression time
facing unique challenges. On the basis of the cell type and the might cause inefficiency of gene editing. In addition, mRNA is
applications, the most effective delivery format can be chosen vulnerable to RNases both in vitro and in vivo. Therefore, ap-
and then paired with optimal cell-culture reagents and analysis propriate delivery methods are needed to protect mRNA from
tools. enzyme-induced degradation. Chemical modification to im-
The first method is to deliver a plasmid encoding the Cas9 prove the stability of the mRNA molecules and to control their
gene and gRNA genes. At the laboratory level, plasmid-based expression in cells was recently reported.[51] As Cas9 mRNA and
DNA has many advantages, such as high stability, simple oper- gRNA are all single-strand RNA molecules, both of them have
ation, and low cost, all of which make plasmid-encoded Cas9 a the same delivery vehicle. However, the delivery time has
common delivery method.[47] Cas9 or gRNA expression cas- become a new issue.[52] Specifically, for genome editing, the
settes and HDR templates can be packaged in the same plas- complete gRNA and the functional Cas9 protein must be pres-
mid, which is more stable than mRNA or protein. However, the ent in the cell at the same time, but the delivered Cas9 mRNA
large size of the Cas9 gene (4 to 7 kb) and total plasmid molecule must first be translated into Cas9 protein in situ. To
(  9.3 kb) pose great difficulty in the delivery or expression of avoid this time issue completely, a hybrid approach has been
the CRISPR/Cas9 system.[48] DNA-encoded delivery to eukaryot- developed, and the mRNA encoding the Cas9 protein is code-
ic cells needs penetration of the cell membrane and a nuclear livered with the viral genome that constitutively expresses
membrane, because DNA needs to be transcribed within the gRNA.[53] In addition, gRNA could begin to degrade before
nucleus. Moreover, DNA-encoded delivery shows reduced Cas9 mRNA is translated into protein. Thus, the delivery of
genome editing efficiency and delayed therapeutic effects, gRNA about 6 h after mRNA increases the editing efficiency.[52]
during which Cas9 protein experiences a longer active period, The third CRISPR system delivery method is to codeliver
and this leads to possible higher off-target effects than those Cas9 protein and gRNA (Cas9/gRNA ribonucleoprotein com-
found for transient formats such as protein delivery.[49] More- plex, RNP); this is the most direct way to deliver the CRISPR
over, the integration of plasmids into the host genome has system for the fastest genome editing, because the transcrip-
potential risks. tion and/or translation processes are not required. Further-

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more, this method, in theory, can reduce off-target effects and relevant to human disease.[56] Other work involves the use of
cytotoxicity. However, this method faces some challenges. For base editors targeting the Dmd or Tyr gene in mouse zygotes,
example, it is not easy to pack the large volume of Cas9 as this induces nonsense mutations with allelic frequencies of
protein (  160 kDa) and gRNA (  31 kDa) into the vehicles, up to 100 % in mice.[57] Base editing improves precise genome
although there are successful cases both in vitro and in editing to a single-base level with very little indel formation,
vivo.[31, 54] RNP provides the most transient expression time. At and it, thus, shows great potential in the treatment of point-
the same time, it is necessary to obtain a highly pure, active mutation diseases.
Cas9 protein and to consider the issues of bacterial endotoxin It is inconvenient to manipulate and measure RNA, even
contamination. Most commonly, Cas9 protein and gRNA are though it plays significant and diverse roles in biology. Usually,
produced in vitro and are then combined into an RNP complex RNAs can be knocked down efficiently by RNA interference.
and delivered as a single unit. Owing to the presence of gRNA However, off-target effects cannot be avoided, and exogenous
in the RNP, this method should, in particular, protect the pay- tags need to be introduced to visualize the RNAs. To tackle
load in any degradable pathway. It is more difficult to obtain a this problem, researchers have employed Cas13a8 to bind and
pure protein than to obtain a plasmid or mRNA, and thus, the knock-down mammalian cell RNA. Cas13a from Leptotrichia
economic viability of this method might be compromised. wadei (LwaCas13a) controllably knocks down either reporter or
Finally, if dose levels are not carefully monitored, the sudden endogenous transcripts as RNA interference. This assay demon-
introduction of an unnaturally occurring bacterial protein into strates that CRISPR/Cas13a can be used as a flexible strategy
a mammal might trigger an immune response or cause toxici- to investigate theranostics concerning RNAs. In other work, the
ty. Cas13b orthologue from Prevotella sp. P5-125 (PspCas13b) has
been identified to be the most efficient and specific for editing
mammalian cells. The ADAR2 deaminase domain (ADAR2DD)
4. CRISPR/Cas9 for Therapeutics
and catalytically inactive PspCas13b are fused to cause RNA
The CRISPR/Cas9 system has great potential for administrating editing for A to I (G) replacement of reporter, endogenous
gene-related diseases. By simply designing gRNA sequences, transcripts, and disease-related mutations.
large-scale genomic interference can be achieved by Cas9. Fur-
thermore, by appropriate modification, Cas9 can have more
4.2. The applications of CRISPR/Cas9
functions for elucidating pathogenic genetic variations or for
exploring more genome functions. For example, wild-type 4.2.1. Cancer therapy: Cancer is still a big challenge to clinical
Cas9 nuclease can be converted into a deactivated form treatment and leads to high mortality. The pathogenesis of
(dCas9) by inactivating the catalytic domain for positioning cancer is associated with genetic changes, such as mutation or
specific gene sites. A variety of proteins and RNAs can be aberrant methylation of genes. Gene therapy shows great po-
linked to Cas9 or gRNAs to monitor or even to influence the tential in cancer therapy. Approaches such as gene silencing
transcription status of specific genomic sites.[55] and gene expression are useful in this field. However, these
approaches also suffer from problems, such as complicated op-
eration, low efficiency in gene regulation, and serious off-
4.1. DNA and RNA editing
target effects. CRISPR/Cas9 shows great potential to resolve
A great number of point mutations can lead to genetic diseas- the above problems. Considering that the major obstacle hin-
es. At present, the correction of point mutations is usually dering the CRISPR/Cas9 system is the large size of Cas9 plas-
based on traditional gene-editing technology, which induces mids, we have proposed a strategy to deliver Cas9 protein and
random insertions and deletions at the targeted sites. The in- gRNA plasmid by a nanocarrier comprising lipid-shell-encapsu-
efficiency of the current approaches limits the application of lated gold nanoclusters. TAT-peptide-decorated gold nanoclus-
gene editing in the correction of point mutations. “Base edit- ters improve the delivery of the Cas9/gRNA system to cell
ing” is a new approach to genome editing that has been nuclei (Figure 4 A). These nanoparticles lead to effective down-
developed to enable the direct, irreversible conversion of one regulation of Plk1 and subsequent tumor cell viability both in
target DNA base into another in a programmable manner vitro and in vivo.[58] Understanding drug resistance and devel-
without requiring cleavage of the double-stranded DNA back- oping new therapeutics will be useful in screening novel drugs
bone or a donor template. This approach does not cause and drug combinations for multidrug-resistant cancer therapy.
double-strand breaks and does not require a donor template, We previously reported drug-resistant cancer model cell lines
but it can transform the base from cytidine into uridine direct- by using the CRISPR/Cas9 system that accelerates the estab-
ly. The resulting “base editors” convert cytidines within a lishment of cancer-cell-line-based, inheritable drug-resistant
window of approximately five nucleotides and efficiently cor- models by specific knock-out of the MED12 gene. The gene-
rect a variety of point mutations relevant to human disease. A edited cells are fully established within 3 weeks, which is much
recently reported approach involves fusing CRISPR/Cas9 with a less than the time required for the construction of animal
cytidine deaminase enzyme that realizes “base editing” by models and resistant cell lines prepared by gradient-dosage in-
transforming the base from cytidine into uridine directly, and duction or patient derivation. Evaluation of the anticancer
this leads to single-base-pair substitutions in eukaryotic cells. drugs and their combinations has shown that certain combina-
This approach efficiently corrects a variety of point mutations tions of BRAF inhibitors and TGF-b receptor (TGF-b R) inhibi-

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Figure 4. Nonviral vectors for in vivo CRISPR/Cas9 delivery. A) Schematic of the synthesis process of the nanocarrier of lipid-shell-encapsulated gold nanoclus-
ters for delivery of CPISPR/Cas9. GN = gold nanocluster, GCP = TAT-GNs/Cas9 protein/sgPlk1 plasmid, LGCP = lipid-coated GCP. Reproduced from ref. [58] with
permission. Copyright: 2017, Wiley-VCH Verlag GmbH & Co. KGaA. B) Model of CRISPR/Cas9-directed intracellular defense against lentiviral infection. Repro-
duced from ref. [63] with permission. Copyright: 2015, Nature Publishing Group.

tors are active in suppressing the growth of MED12KO A375 viral reactivation in infected cells seems to be effective. How-
cells, and a few combinations of EGFR inhibitors and TGF-b R ever, this temporary inhibition is also not an ideal treatment.
inhibitors are active in suppressing the growth of MED12KO Permanent inhibition of the HIV genome by damaging the in-
PC9 cells.[59] The efficiency and specificity of the CRISPR/Cas9 tegrated proviral DNA is a new approach to treat HIV. The pre-
system remain unsatisfactory. Recently, researchers have pro- cise gene-editing system CRISPR/Cas9 raises the possibility for
posed a self-assembled micelle composed of quaternary am- this therapy. Researchers have identified highly specific targets
monium terminated poly(propylene oxide) (PPO-NMe3) and within the HIV-1 LTR U3 region and have transfected HIV-1-in-
amphiphilic Pluronic F127 that enables effective delivery of the fected cells with Cas9/gRNA. Successful gene editing in the tar-
Cas9 plasmid targeting the human papillomavirus (HPV) E7 geted sites excises a large fragment of integrated proviral DNA
oncogene and inhibits cancerous activity both in vitro and in completely, and this results in inactivation of viral gene expres-
vivo. These micelles represent an efficient delivery system for sion and replication in latently infected microglias, promono-
nonviral gene editing and add to the armamentarium of gene- cytes, and T cells. Furthermore, the gene-editing system is not
editing tools to advance safe and effective precision medicine- genotoxic and does not have any off-target effect on the host
based therapeutics.[60] cells. These results indicate that Cas9/gRNA has great potential
in the treatment of AIDS specifically and efficiently.[62] In other
4.2.2. Neurodegenerative diseases: Neurodegenerative diseases
work, intracellular defense against viruses on a HIV-1 infection
involve mutations of the genome, and they can be corrected
model by using the CRISPR/Cas9 system has been evaluated.
by the CRISPR/Cas9 system. Huntington’s disease derives from
The CRISPR/Cas9 system disrupts integrated HIV-1 genome in
a gain-of-function cytosine-adenine-guanine (CAG) expansion
cells (Figure 4 B). Besides, the cells build a long-term adaptive
mutation. Recently, researchers have proposed a strategy to in-
response defense against new viral infection. Furthermore, the
activate the mutant allele by haplotype-specific CRISPR/Cas9 to
researchers also construct human-induced pluripotent stem
selectively excise the transcription start site and the CAG ex-
cells expressing HIV-targeted CRISPR/Cas9 that differentiate
pansion mutation. This strategy leads to complete inactivation
into HIV reservoir cells that are resistant to HIV-1 infection.
of the mutant allele without impacting the normal one. This
These studies illustrate the great potential of the CRISPR/Cas9
highly selective excision on the target allele is promising for
system as a new therapeutic strategy against viral infections.[63]
broader applicability in disorders with diverse disease haplo-
types.[61]
4.2.3. Acquired immune deficiency syndrome (AIDS): AIDS results
5. Challenges and Opportunities of CRISPR/
from infection with human immunodeficiency virus (HIV) and
Cas9 Delivery
is still incurable and affects public health worldwide. Antiretro-
viral therapies can inhibit the life cycle of HIV and reduce viral The CRISPR/Cas9 system, which is simple but multifunctional,
replication greatly. However, these approaches cannot clear not only is a powerful tool for biological research but also pro-
the virus completely, and HIV can replicate in infected cells motes the development of gene regulation and gene therapy.
and tissues. A strategy to suppress viral protein expression and However, despite the great potential of the CRISPR/Cas9

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system in genome editing, there are still some tough challeng- 5.5. Targeted delivery
es that need to be addressed.
The successful delivery of CRISPR/Cas9 involves three steps:
cellular uptake, cytoplasmic mobility, and nuclear import. To
5.1. Encapsulation
boost cellular uptake, targeting ligands such as antibodies are
The large size and different charges of Cas9 protein, mRNA, required.[69] However, modification of the targeting molecule
and plasmid make it difficult to package all the components increases the difficulty with which a delivery vector can incor-
into a single vector. For example, with a weight of 160 KD, porate additional elements inside. Furthermore, CRISPR/Cas9
Cas9 protein is larger than most proteins. Moreover, the sizes plasmids need to enter the nucleus for transcription and go
of mRNA and DNA used in the CRISPR/Cas9 system are also out to the cytosol for translation and return back to the nu-
large, which makes it more difficult to develop suitable vectors. cleus for gene editing. Thus, nucleus-targeting ligands such as
Additionally, the charge of the native Cas9 protein is positive, the NLS are needed not only for the vectors but also for the
whereas traditional vectors are designed to be positively sequences of the plasmid.
charged; thus, they cannot encapsulate the Cas9 protein Although there are many challenges associated with CRISPR/
through electrostatic interactions.[64] All of these disadvantages Cas9 delivery, we believe that with the rapid development of
should be taken into consideration for designing nonviral vec- materials science and nanotechnology more robust delivery
tors. methods will be created to overcome these challenges to push
forward the clinical application of “magic scissors”.
5.2. Stability
Preservation of the stability of the CRISPR/Cas9 system under
Acknowledgements
physiological conditions is also a big challenge that needs to
We appreciate support of the Minister of Science and Technology
overcome. First, plasmids, proteins, and RNA are easily degrad-
of China (2017YFA0205901), the Chinese Academy of Sciences
ed by proteases and nucleases in the body. For example,
(121D11KYSB20170026), and National Nature Science Foundation
gRNA, which plays a vital role in the positioning process, can
of China (31470911, 81673039, 21535001, 81730051,
be degraded by ribonucleases in the serum and tissues.[65]
21761142006, 81671784, 21505027).
Second, the CRISPR/Cas9 system might interact with plasma
proteins and be recognized and cleared by the reticuloendo-
thelial system (RES). Therefore, vectors that can maintain the Conflict of Interest
stability of the CRISPR/Cas9 system are needed.
The authors declare no conflict of interest.
5.3. Immune responses
Keywords: carriers · delivery systems · nanomaterials · gene
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technology · gene therapy
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lo, ACS Nano 2017, 11, 2452 – 2458. Version of record online: && &&, 0000

& ChemBioChem 2018, 19, 1 – 11 www.chembiochem.org 10  2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

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MINIREVIEWS
L. Wang, W. Zheng,* S. Liu, B. Li, X. Jiang*
&& – &&
Delivery of CRISPR/Cas9 by Novel
Strategies for Gene Therapy

In the genes: In this review, recently eases are briefly summarized. This
developed delivery strategies for various review should inspire others to explore
CRISPR/Cas9 formulations and their ap- more efficient strategies for CRISPR
plications in treating gene-related dis- system delivery and gene therapy.

ChemBioChem 2018, 19, 1 – 11 www.chembiochem.org 11  2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim &

These are not the final page numbers! ÞÞ