Rational designs of in vivo CRISPR-Cas delivery systems

Journal Pre-proof Rational designs of in vivo CRISPR-Cas delivery systems

Cong-Fei Xu, Guo-Jun Chen, Ying-Li Luo, Yue Zhang, Gui Zhao, Zi-Dong Lu, Anna Czarna, Zhen Gu, Jun Wang PII:

S0169-409X(19)30208-X

DOI:

https://doi.org/10.1016/j.addr.2019.11.005

Reference:

ADR 13517

To appear in:

Advanced Drug Delivery Reviews

Received date:

14 October 2019

Revised date:

9 November 2019

Accepted date:

19 November 2019

Please cite this article as: C.-F. Xu, G.-J. Chen, Y.-L. Luo, et al., Rational designs of in vivo CRISPR-Cas delivery systems, Advanced Drug Delivery Reviews (2019), https://doi.org/ 10.1016/j.addr.2019.11.005

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2019 Published by Elsevier.

Journal Pre-proof

Rational designs of in vivo CRISPR-Cas delivery systems Cong-Fei Xu1, 4, †, Guo-Jun Chen2, 3 †, Ying-Li Luo8, Yue Zhang1, 5, Gui Zhao1, 6, ZiDong Lu8, Anna Czarna8, Zhen Gu2, 3, *, Jun Wang1, 4, 5, 7 * 1

Guangzhou First People’s Hospital, School of Biomedical Sciences and Engineering,

Guangzhou International Campus, South China University of Technology, Guangzhou 510006, P. R. China Department of Bioengineering, California Nanosystems Institute, University of

of

2

California, Los Angeles 90095, USA

Jonsson Comprehensive Cancer Center, California NanoSystems Institute, and Center

ro

3

-p

for Minimally Invasive Therapeutics, University of California, Los Angeles 90095, USA

National Engineering Research Center for Tissue Restoration and Reconstruction,

re

4

South China University of Technology, Guangzhou 510006, P. R. China Key Laboratory of Biomedical Engineering of Guangdong Province, and Innovation

lP

5

Center for Tissue Restoration and Reconstruction, South China University of 6

na

Technology, Guangzhou 510006, P. R. China Key Laboratory of Biomedical Materials and Engineering of the Ministry of Education,

7

Jo ur

South China University of Technology, Guangzhou 510006, P. R. China Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou

510005, P. R. China 8

Institutes for Life Sciences, School of Medicine, South China University of

Technology, Guangzhou 510006, P. R. China *

Corresponding Authors:

Jun Wang, E-mail: [email protected] Zhen Gu, E-mail: [email protected] †

Cong-Fei Xu and Guo-Jun Chen contributed equally to this work.

Journal Pre-proof Abstract The CRISPR-Cas system initiated a revolution in genome editing when it was, for the first time, demonstrated success in the mammalian cells. Today, scientists are able to readily edit the genome, regulate gene transcription, engineer posttranscriptional events, and image nucleic acids using CRISPR-Cas-based tools. However, to efficiently transport CRISPR-Cas into target tissues/cells remains challenging due to many extraand intra-cellular barriers, therefore largely limiting the applications of CRISPR-based therapeutics in vivo. In this review, we summarize the features of plasmid-, RNA- and ribonucleoprotein (RNP)-based CRISPR-Cas therapeutics. Then, we survey the current

of

in vivo delivery systems. Finally, we specify the requirements for efficient in vivo

ro

delivery in clinical settings, and highlight both efficiency and safety for different

-p

CRISPR-Cas tools.

Jo ur

na

lP

re

Keywords: CRISPR-Cas; Drug delivery; Genome editing; Nanomedicine

Journal Pre-proof

Introduction

2

Features of CRISPR-Cas

5

2.2

Plasmid-based CRISPR-Cas

2.3

RNA-based CRISPR-Cas

2.4

RNP-based CRISPR-Cas

2.5

Other forms of CRISPR-Cas

Current delivery systems of CRISPR-Cas

Delivery systems of plasmid-based CRISPR-Cas

3.2

Delivery systems of RNA-based CRISPR-Cas

3.3

Delivery systems of RNP-based CRISPR-Cas

3.4

Delivery systems of other forms of CRISPR-Cas

lP

re

-p

3.1

Rational design of CRISPR-Cas delivery systems for clinical use

na

4

Brief history of CRISPR-Cas

4.1

The clinical requirements of in vivo genome editing

4.2

Selection of CRISPR-Cas tools

4.3

Rational design of in vivo delivery systems

Jo ur

3

2.1

ro

1

of

Contents

Conclusions

Acknowledgments Additional information References

Journal Pre-proof 1.

Introduction

Currently, CRISPR-Cas, clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9, is one of the easiest genome editing machinery [1-3]. In this system, a Cas nuclease is guided by a single guide RNA (gRNA) to cleave a target DNA sequence following the base pairing of gRNA and genomic DNA [4]. It only requires the design of gRNA to target the genome of interest, which makes CRISPRCas available to the researchers who are not specialists in molecular biology. In addition, the genome editing efficacy and specificity are fairly high due to the high nuclease activity of Cas and the recognition mechanism of gRNA [3]. These advantages broaden

of

the usage of CRISPR-Cas to various fields, including life science, biotechnology,

ro

medicine and agriculture [5].

-p

Except for CRISPR-Cas9 from Streptococcus pyogenes (SpCas9), many other Cas9like nucleases with different sizes, target requirements and substrate preferences were

re

subsequently discovered [3]. These CRISPR-Cas nucleases provide an abundant toolbox of “molecular scissors” for editing almost all loci in eukaryotic genomes [6].

lP

The mechanism of CRISPR-Cas-mediated genome editing is creating site-specific double-strand breaks (DSB) at gRNA-targeting locations, and the damaged genome can

na

be repaired by either nonhomologous end joining (NHEJ) or homology-directed repair (HDR) [4]. While NHEJ results in random insertions/deletions (indels) for gene

Jo ur

knockout, HDR can be utilized for precise gene repair or insertion with a DNA donor. To date, CRISPR-Cas-based genome editing has been applied for genome-wide screens to probe basic biological functions and identify potential drug targets in complex diseases [7]. Moreover, multiple therapeutic strategies based on CRISPR-Cas have demonstrated feasibility for treating hereditary diseases (e.g., Duchenne muscular dystrophy (DMD), blood disorders, severe combined immunodeficiency (SCID)) [810], viral infections (hepatitis B virus (HBV) and human immunodeficiency virus (HIV)) [11], inflammatory diseases (autoimmune diseases and inflammatory bowel diseases (IBD)) [12], and cancers [13] and for cell engineering (e.g., generating chimeric antigen receptor (CAR) T cells [14]). Apart from gene editing, researchers have also re-engineered CRISPR-Cas nucleases with other functionalities [3]. They mutated the catalytic domains (HNH and RuvC) to create catalytically inactive (dead) Cas9 (dCas9) [15]. By fusing dCas9 with a

Journal Pre-proof deaminase, the single-base editors were created for catalyzing transitions of nucleic acid bases, which are able to correct point mutations without inducing DSBs [16]. By fusing dCas9 with transcription activators or repressors, researchers developed CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi), respectively, which enable gene regulation at the transcriptional level [15]. Furthermore, dCas9 can be fused with a green fluorescent protein (GFP) or a red fluorescent protein (RFP) to visualize nucleic acids [17]. Although CRISPR-Cas systems hold great promise in medicine and various aspects, delivery systems that are able to efficiently and safely deliver CRISPR-Cas components

of

into target cells remain limited [18]. Several previous reviews have covered the material

ro

solutions of CRISPR-Cas delivery [19, 20], the nonviral systems of CRISPR-Cas for establishing disease models and developing therapies [21-24], and in vivo delivery

-p

strategies for CRISPR-Cas [25-30]. This review is focal on designing optimal carriers

re

to deliver plasmid-, RNA- and ribonucleoprotein (RNP)-based CRISPR-Cas into target cells in vivo (Fig. 1). First, we introduce the major features of CRISPR-Cas, summarize

lP

the current nonviral delivery systems. Then, we highlight the efficiency and the safety when designing CRISPR-Cas-based medicine. Finally, we discuss how to rationally

Jo ur

research.

na

design in vivo delivery systems for CRISPR-Cas and provide prospects for future

lP

re

-p

ro

of

Journal Pre-proof

na

Fig. 1. Delivery of CRISPR-Cas for genome editing. (A) Rational designs of in vivo delivery systems of plasmid-, RNA- and RNP-based CRISPR-Cas. (B) Representative

2.

Jo ur

genome editing route by three forms of CRISPR-Cas.

Features of CRISPR-Cas

2.1 Brief history of CRISPR-Cas 2.1.1

The history of CRISPR-Cas9

CRISPR-Cas9 was built on several decades of tracing the fundamental mechanisms of the acquired immune systems of archaea and bacteria [31]. The first CRISPR repeat clusters were observed in Escherichia coli (E. coli) by the Nakata’s group when they analyzed the alkaline phosphatase gene and found that an unique repetitive DNA sequence was appeared in distinct DNA spacer sequences [32]. In the following decade, similar CRISPR repeat clusters were found in other archaea and bacteria [33]. However, the function and origin of these CRISPR sequences were still unknown. Until the genomes of many bacteriophages, archaeal viruses, and plasmids were sequenced,

Journal Pre-proof researchers discovered that the spacer sequences of CRISPR were derived from the foreign viruses or plasmids [34-36]. In addition, several well-conserved genes encoding DNA repair proteins were identified to be adjacent to CRISPR and were called cas genes [37, 38]. These comparative genomic analyses implied that CRISPR sequences and cas genes worked together as the acquired immune systems of archaea and bacteria. In 2007, Horvath Group reported evidence that CRISPR-Cas provided prokaryotes acquired immunity against viruses [39]. They found that removal of particular spacers abolished the phage-resistance ability of bacteria. Shortly after this discovery, the groups of van der Oost, Sontheimer and Moineau demonstrated that Cas nuclease was

of

assisted by a CRISPR RNA (crRNA) transcribed from the spacer sequences to cut the

ro

invading viruses and plasmids [40-42].

In 2012, Charpentier’s and Doudna’s groups collaboratively reported that Cas9

-p

nuclease-mediated DNA cleavage is directed by a two-RNA structure, which is formed

re

by base-pairing crRNA and trans-activating crRNA (tracrRNA) [43]. Furthermore, they combined tracrRNA and crRNA into a single gRNA, which greatly simplified the

lP

preparation of CRISPR-Cas9. This work is the landmark of developing CRISPR-Cas9 into a genome editing tool. In 2013, Zhang and Church demonstrated genome editing

na

in mammalian cells with a CRISPR-Cas9 system [2, 44]. Their findings attracted attentions of genome editing into life science, biotechnology, medicine and agriculture.

Jo ur

Henceforward, researchers are able to readily design reliable and effective tools for editing the genomes of all species by only changing the base-pairing sequence of gRNA.

2.1.2

Newly developed CRISPR-Cas tools after CRISPR-Cas9

Although SpCas9 is the pioneering genome editing machinery, its large size (1368 amino acids) is not suitable for viral delivery [45, 46]. As mentioned above, many other Cas9-like nucleases have been subsequently discovered (Table 1). For example, the smallest Cas9 nuclease is from Campylobacter jejuni (CjCas9), which has only 984 amino acids [47]. These smaller nucleases enable researchers to use virus vectors for genome editing. In 2017, Zhang Group screened Cas13 orthologues, which can target RNA for interference and imaging, providing tools for RNA study in cells [48]. Table 1. Features of Cas9 and other Cas9-like nucleases. Adapted with permission from [3]. Copyright (2018) Springer Nature.

Journal Pre-proof Size Source (amino acids)

St3Cas9

CjCas9

AsCpf1

LbCpf1

aureus Neisseria meningitides Streptococcus thermophilus Streptococcus thermophilus Campylobacter jejuni Acidaminococcus sp Lachnospiraceae bacterium

Multiple sources

NGG

20

1628

190.3

NGG

20

1052

123.8

NNGR RT

21

1081

124.2

NNNNG ATT

24

1122

129.5

NNAGA AW

20

1409

164.9

983

114.8

1307

1228

Multiple

orthologs

NGGNG

20

NNNNACAC

22

TTTV

24

143.7

TTTV

24

Variable

RNA targeting

28

151.2

na

Cas13

Staphylococcus

sequence (bp)

158.3

of

St1Cas9

novicida

Length of guiding

1368

ro

NmCas9

Francisella

PAM sequence

-p

SaCas9

pyogenes

re

FnCas9

Streptococcus

weight (kDa)

lP

SpCas9

Molecular

Starting from a genome editing technology, CRISPR-Cas9 has been re-engineered with

Jo ur

many other functionalities, including gene regulation, base editing and imaging (Fig. 2) [3]. In 2013, Lim group repurposed dCas9/gRNA to CRISPRi, which repressed gene expression by interfering with the binding of RNA polymerase [49]. Several months later, Qi and colleagues reported different kinds of CRISPRi whose dCas9 was fused with transcriptional repressors, such as dCas9-KRAB, to achieve stronger gene silencing effects [50]. In 2013, the CRISPRa systems were also reported by two groups, who fused dCas9 with the transcriptional activator VP64 to increase gene expression [51, 52]. A CRISPRa system with higher efficiency was soon created using tripartite transcriptional activators (VP64-p65-Rta, VPR) [53]. In another strategy called SunTag, dCas9 was fused with a protein scaffold to recruit an antibody-transcriptional activator fusion protein [54]. In addition, the gRNA scaffold was engineered to contain an MS2 aptamer for recruiting bacteriophage MS2-coat protein (MCP). When MCP was fused with VP64 or the P65-HSF1 complex, these synergistic activation mediators (SAM) can be recruited for transcriptional activation [55, 56]. In 2016 and 2017, Liu reported

Journal Pre-proof the CRISPR base editors for directly mutating one target base into another without introducing DSBs [57, 58]. They fused a Cas9 protein with a cytidine or adenosine deaminase enzyme, which allowed all four transition mutations (C•G to T•A or A•T to G•C substitutions). Many groups further developed CRISPR base editors to introduce stop codons for gene knockout or repair stop codons for gene recovery [59-61]. Similarly, epigenetic modifiers (e.g., DNA methyltransferases, histone demethylases or deacetylases), protein dimers (e.g., ABI1 and PYL1 from the abscisic acid pathway) or fluorescent proteins were combined with dCas9/gRNA to create tools for epigenome

na

lP

re

-p

ro

of

editing, chromatin structure manipulation and chromatin imaging [62-67].

Jo ur

Fig. 2. Summary of CRISPR-Cas-based technologies. The original Cas9 induces gene editing with guidance of gRNA. dCas9 can be engineered with trans-effectors, epigenetic modifiers, fluorescent proteins (FP), uracil glycosylase inhibitor (UGI) and protein dimers for gene regulation, epigenome editing, chromatin imaging, base editing and chromatin topology. The Cas13 orthologs are for RNA targeting. Reprinted with permission from [3]. Copyright (2018) Springer Nature.

2.2 Plasmid-based CRISPR-Cas The primary CRISPR-Cas9 for eukaryotic cells was a two-in-one plasmid (pX330, Addgene plasmid #42230) from Zhang Group [2]. The plasmid has two expression cassettes: one cassette uses a U6 promoter to drive gRNA expression, and the other cassette uses a chicken β-actin promoter to drive SpCas9 expression [4]. Many other CRISPR-Cas plasmids (pCas) have also been constructed. For example, the pX458

Journal Pre-proof plasmid (Addgene plasmid #48138) co-expresses SpCas9 with enhanced GFP (EGFP), which helps to easily detect the expression of SpCas9. The most actively used adenine base editor (ABE) plasmid (pCMV-ABE7.10, Addgene plasmid # 102919) was developed by Liu Laboratory and expresses ABE7.10 which is a fusion protein of the TadA-evolved TadA*(7.10) mutant and Cas9 (D10A) nickase [58]. Due to the large size of Cas proteins, the length of pCas is usually larger than 8,000 base pairs (bp), which requires delivery systems capable of a high payload. For clarity, we are using the popular pX330 plasmid as an example to analyze the features of plasmid-based CRISPR-Cas (Fig. 3). pX330 is a circular plasmid that has

of

8,963 bp with a molecular weight of approximately 5.55×103 kDa. The radius of

ro

uncondensed pX330 is estimated to be 233.58 nm according to the radius of the pUC18 plasmid [68]. In addition, one pX330 has 1.79×104 phosphate groups, and each

-p

phosphate group exhibits one negative charge [69]. Thus, one pX330 intrinsically

re

carries 1.79×104 negative charges. Based on the high negative charge of pX330, cationic nanoparticles or liposomes can be used as delivery systems. For example, the

lP

commercial Lipofectamine 2000 was used for transfecting pX330 into a variety of cells [4, 70]. Typically, 1 μL Lipofectamine 2000 can adsorb 1 μg pX330 to its surface via

na

electrostatic interactions, and the formed complex can be endocytosed by cells. Because pX330 carries high negative charges, the delivery systems should have sufficient

Jo ur

positive charges for encapsulation. Usually, cationic lipids or polymers with abundant amino groups are good choices for preparing delivery systems. However, a high positive charge may also cause cytotoxicity; thus, designing delivery systems with the least number of amino groups is an important factor to be considered.

Fig. 3. The map and features of pX330. Generated by SnapGene Viewer software. Enhancing the loading capacity of delivery systems is just one of the requirements for

Journal Pre-proof in vivo genome editing. From injection to editing, pX330 with carriers must go through a series of delivery barriers. First, the serum and body fluids are rich in nucleases, where pX330 can be easily degraded. Although the double-strand structure of pX330 is relatively stable, delivery systems to protect pX330 from degradation are still necessary. In addition, innate immune cells that highly express Toll-like receptors are able to recognize DNA and cause inflammation [71]. Thus, delivery systems should be able to reduce the immunogenicity of pX330. Second, pX330 needs to be selectively delivered into target cells. Since the genome of different tissue cells is identical, pX330 can unselectively edit the genome of each cell as soon as it is expressed into Cas9 nuclease

of

and gRNA. Thus, delivery systems with cell targeting ability can minimize the off-

ro

target effects and potential safety risks. Alternatively, the promoter of Cas9 in the plasmid can be replaced by a cell-specific promoter to selectively express Cas9 in target

-p

cells [70]. Third, pX330 needs to be transcribed in the nucleus, which contains enzymes, especially RNA polymerases. For dividing cells, the nuclear envelope is dissociated,

re

and pX330 can easily diffuse into the nucleus [72]. However, for nondividing cells, an extra nuclear localization signal (NLS) is required for transporting pX330 into the

lP

nucleus. For example, Smith and colleagues utilized the SV40 NLS for enhancing the transfection efficacy by opening the nuclear pore [73]. Cationic cell penetrating

na

peptides (CPPs), which can not only bind to pX330, but also disrupt the nuclear envelope, can function as NLSs [74, 75]. Fourth, the transcribed Cas9 mRNA needs to

Jo ur

be transferred into the cytoplasm for translating. To conclude, the plasmid-based CRISPR-Cas system is large in size and has a highly negative charge. Delivery systems need to bind and condense CRISPR-Cas systems into small sizes or encapsulate them. In addition, delivery systems that can overcome in vivo barriers, including nuclease degradation, immunogenicity, selective cell targeting and nuclear envelope obstacles, can greatly enhance genome editing efficiency and reduce off-target risks. Because additional single strand DNA (ssDNA) or double-strand DNA (dsDNA) donors are required for gene repair or insertion, delivery systems that can simultaneously carry ssDNA or dsDNA can broaden the capability of genome editing.

2.3 RNA-based CRISPR-Cas Compared to plasmids that hold the risk of genome integration, Cas mRNA and gRNA

Journal Pre-proof are safer for clinical therapy [76]. Cas mRNA has a short half-life, which can be degraded within 24 h, therefore reducing the risks of immunogenicity and off-target effects. Cas mRNA and gRNA are usually produced by in vitro transcription using dsDNA templates, whose sequences are derived from the pCas and driven by the T7 promoter for transcription. All plasmid-based CRISPR-Cas can be transcribed into RNA for delivery. Due to the various types of Cas proteins and their modifiers, the length of Cas mRNA ranges from 3,000 nucleotides (nt) to over 5,000 nt. Using SpCas9 as an example (Fig. 4), SpCas9 mRNA is a linearized RNA with a singlestranded structure. The length of SpCas9 mRNA without a poly A tail is approximately

of

4,300 nt. The molecular weight is approximately 1.38×103 kDa. It contains

ro

approximately 4,300 negative charges. The average length of SpCas9 mRNA is approximately 600~700 nm, which is larger than the plasmid-based systems due to the

-p

linear structure of mRNA. Hence, it is necessary to pack Cas mRNA into a small size

re

using the cationic lipids or polymers. Because the gRNA has only approximately 100 nt and the Cas mRNA and gRNA need to be delivered together into target cells,

lP

desirable carriers need to encapsulate long and short RNAs at the same time. In addition, Cas mRNA does not require transcription and can be readily translated into the Cas

na

protein after entering the cytoplasm. The translation elongation speed of mRNA is approximately 5 amino acids per second [77]. For the Cas9 nuclease with 1,367 amino

Jo ur

acids, the whole translation takes less than 5 min. The translated Cas9 nuclease can form Cas9 RNP with gRNA and then localize into the nucleus.

Fig. 4. Structure and features of SpCas9 mRNA and gRNA. The biggest challenge for the delivery of Cas mRNA and gRNA is that single strand

Journal Pre-proof RNA (ssRNA) is typically unstable. Various methods have been developed for improving their stability. For Cas mRNA, different cap analogs were used for cotranscriptional capping, such as the anti-reverse cap analog (ARCA) [78]. In addition, pseudouridine, N6-methyladenosine, or inosine can be used for mRNA modifications to enhance its resistance to RNases [78, 79]. For gRNA, chemical modifications comprising 2′-O-methyl, 2′-O-methyl 3′phosphorothioate, or 2′-O-methyl 3′thioPACE can significantly enhance the genome editing efficiency [80]. In addition, these chemical modifications can further reduce the immunogenicity of Cas9 mRNA and gRNA [78]. Apart from chemical modifications, delivery systems can also improve

of

their stability. The best design is to encapsulate Cas9 mRNA and gRNA inside

ro

nanoparticles, which prevents their exposure to RNases and the immune system in vivo. In addition, unlike the plasmid-based CRISPR-Cas, which can rely on specific

-p

promoters to drive Cas expression to enhance specificity, Cas mRNA can nonspecifically edit every kind of cell after translation into Cas nuclease. Thus, delivery

re

systems that can selectively enter target cells are essential for accurate therapeutic genome editing. After endocytosis by target cells, the nanoparticles should be able to

lP

remain intact in the endosomes that are full of nucleases and escape into the cytoplasm by crossing the endosome membrane. Moreover, as soon as in the cytoplasm, RNAs

na

need to be released rapidly to function due to their short half-lives.

Jo ur

2.4 RNP-based CRISPR-Cas

Delivering the RNP complex of the Cas protein and gRNA is the most straight-forward method for genome editing. The RNP-based CRISPR-Cas system can directly edit the genome after entering the nucleus. It is also supposed to cause the least off-target effects because the amount of RNPs delivered into target cells is limited and can be degraded quickly after entering the cell [81]. The Cas protein and gRNA are available from commercial vendors, but they are costly and not customized. Thus, using the pET-28b plasmid to express the Cas protein is a good alternative. For example, the pET-28bCas9-His plasmid can be utilized to express His-tagged Cas9 in E. coli. [82]. gRNAs can be prepared by in vitro transcription. Then, the RNP complex can be generated by in vitro sample incubation of the Cas protein and the gRNA [83]. The size of the RNP complex is about 10 nm. In addition, the RNP complex consists of both a positively charged Cas protein and a negatively charged gRNA.

Journal Pre-proof Typically, as shown in Fig. 5, the Cas9 protein has two lobes: an α-helical recognition (REC) lobe and a nuclease (NUC) lobe [84]. The NUC lobe consists of the HNH and RuvC domains for cleaving DNA, and the PAM-interacting domain, respectively. The REC lobe is responsible for sensing nucleic acids, regulating the conformational transition and site locking of the HNH domain [85]. Wright et al. have split Cas9 into two lobes, and they demonstrated that a gRNA could recruit them into complex which recapitulates the similar cleavage activity of wild-type Cas9 [86]. The REC and NUC lobes are positively charged, and the negatively charged gRNA is loaded into the groove [87]. The Cas9 protein has 22 positive charges, and the gRNA has ~100 negative

of

charges; the total charge of the RNP complex is negative. Therefore, Zuris et al. have

ro

used cationic liposomes to deliver RNP both in vitro and in vivo, which significantly disrupted GFP in U2OS reporter cells and mouse inner ear cells [88]. Other than the

-p

use of electrostatic interactions for RNP delivery, the active groups on the amino acids of the Cas9 protein or the base-pairing ability of gRNA can also be harnessed for the

re

design of delivery systems. For example, Wang and colleagues immobilized the RNP complex onto the surface of a gold nanowire (AuNW) through a disulfide linkage

lP

between the AuNWs and the Cas9 protein [89]. Murthy and coworkers coated a donor DNA on gold nanoparticles (AuNPs) where the RNP complex adsorbed via the specific

na

base pairing between the gRNA and the donor DNA [90]. Hence, although the RNP complex is not as simple as a plasmid or RNA, the specific features of macromolecules

Jo ur

still provide multiple ways for the RNP complex delivery. The most important consideration in addition to loading efficiency is to maintain the nuclease activity of Cas9 and the integrity of the gRNA.

Fig. 5. Structure and features of SpCas9 RNP. Adapted with permission from [84]. Copyright (2014) Elsevier. Unlike plasmids and RNAs, which mainly induce innate immune responses, the RNP

Journal Pre-proof complex can cause adaptive immune responses. IgG antibodies against SaCas9 were detected in 78% of human adults, and 58% of human adults carried IgG antibodies against SpCas9 [91]. The cellular immunity of anti-SpCas9 T cells was detected in 67% human donors [91]. In other words, anti-Cas9 antibodies and T cells are pre-exist in humans due to infections. The antibodies can directly bind to the Cas9 RNP, and the anti-Cas9 T cells can attack the target cells when the Cas9 RNP is presented on their surfaces [92]. Therefore, delivery systems need to prevent the RNP complex from being recognized by the antibodies and T cells before transporting them inside the target cells. In addition, delivery systems also need to protect the RNP complex against proteases

of

and nucleases in the serum and body fluids. Once inside the target cells, the delivery

ro

systems should help RNP rapidly escape into the cytoplasm from the endosomes. Then, the RNP complex can enter the nucleus facilitated by the NLS at the end of the Cas

-p

protein. Additionally, penetrating peptides or the NLS from the delivery systems can

lP

2.5 Other forms of CRISPR-Cas

re

also guide RNP into the nucleus for subsequent genome editing.

As aforementioned, CRISPR-Cas systems are highly flexible and have been expanded

na

into many other forms. On the one hand, Cas nucleases can be re-engineered into other functional proteins by mutating their nuclease activity sites or fusing trans-effectors,

Jo ur

epigenetic modifiers and fluorescent proteins to the Cas protein. The gRNA can also be re-designed with functional motifs for recruiting transcription factors or other effectors. On the other hand, mixed component systems of plasmids, RNA, proteins and linear DNA can be delivered. For genome editing by NHEJ, delivery of only CRISPR-Cas in a single component is sufficient. For genome editing by HDR, additional dsDNA or ssDNA templates are needed. Furthermore, pX330 can only express one gRNA. Multiple gRNAs targeting other genome sites can be delivered together with pX330, which makes it possible to edit several targets at the same time. To further enhance the efficiency of genome editing, small molecule inhibitors (for example, Scr7, which is the DNA ligase IV inhibitor) or HDR enhancers (for example, RS-1 and L755507) can be simultaneously delivered [93-95]. Therefore, versatile delivery systems that are able to deliver plasmids, RNA, RNPs, linear dsDNA, ssDNA, small molecule compounds or their mixtures are needed.

Journal Pre-proof 3.

Current delivery systems of CRISPR-Cas

Although Intellia Therapeutics plans to file an investigational new drug (IND) for treating transthyretin amyloidosis using lipid nanoparticles (LNP)-carried CRISPR/Cas, electroporation and viral vectors are still the main strategies for clinical tests of CRISPR-Cas (Fig. 6) [96]. For example, Yang Group designed a nontoxic nanopore electroporation (NanoEP) for the transfection of CRISPR-Cas, which achieved 80% delivery efficiency [97]. Researchers also utilized electroporation to locally deliver the CRISPR-Cas RNP or plasmid into the skin stem cells or pancreatic cells of mice for in vivo genome editing [98, 99]. Qin Group designed microfluidics-based membrane

of

deformation for delivering CRISPR-Cas9 into lymphoma cells or embryonic stem cells

ro

[100]. Adeno-associated viruses (AAVs), which have been approved for clinical use [101], can also efficiently deliver CRISPR-Cas into multiple tissues, including eye,

-p

liver and muscle tissues [102]. Nevertheless, local electroporation and AVVs are limited

re

to a few target tissues. In addition, AAVs can induce strong immunity, and they usually express CRISPR-Cas nuclease constitutively, which may cause serious safety risks. To

Jo ur

na

lP

this end, many nonviral delivery systems have been designed for CRISPR-Cas.

Fig. 6. Current strategies for clinical tests of CRISPR-Cas therapy.

3.1 Delivery systems of plasmid-based CRISPR-Cas Because the two major features of plasmid-based CRISPR-Cas are a high negative charge and large size, most reported delivery systems utilized electrostatic interactions to condense these plasmids into smaller size complexes and loaded them by coating on the surface or encapsulating in the core of nanoparticles. The positively charged materials of these delivery systems mainly are cationic lipids, PEI-based polymers, cationic polypeptides, protamine sulfate and quaternary ammonium-terminated polymers. Two exceptions were using cell-derived exosomes to encapsulate CRISPR-

Journal Pre-proof Cas9 plasmids (pCas9) or assemble pCas9 and histones into chromatin for delivery.

3.1.1

Cationic lipid-based delivery systems

Wang and coauthors have developed a versatile platform, named cationic lipid-assisted polymeric nanoparticles (CLANs), to deliver pCas9 into chronic myeloid leukemia (CML) cells, neutrophils, B cells and macrophages for in vivo gene editing (Fig. 7A) [103]. CLANs were first designed for siRNA delivery using a cationic lipid N,N-bis(2hydroxyethyl)-N-methyl-N-(2-cholesteryloxycarbonyl

aminoethyl)

ammonium

of

bromide (BHEM-Chol) to drive siRNAs encapsulated in poly(ethylene glycol)-bpolylactide (PEG-b-PLA) matrices [104]. Although CLANs were originally designed

ro

for siRNA, they have been proved to be able to deliver various nucleic acids, including siRNAs, miRNAs, CpG oligodeoxynucleotides, mRNAs and large plasmids [103].

-p

CLANs were fabricated using a double emulsion method. During the primary

re

emulsification step, nucleic acids and the cationic lipids assemble into tight complexes at the water-oil interface. Then, the complexes were encapsulated into the inner aqueous

lP

core of PEG-b-PLA or poly(ethylene glycol)-b-poly(lactide-co-glycolide) (PEG-bPLGA) nanoparticles in the second emulsification. Because nucleic acids are

na

encapsulated in the core, the PEG surface can prevent nucleic acids from being degraded by nucleases or recognized by the immune system. The PEG sheath can also

Jo ur

stabilize CLANs by preventing aggregation and protein adsorption [105]. To adapt CLANs for pCas9 delivery, Liu et al. modulated the formulations for preparing CLANs by changing the feeding weight of pCas9 and BHEM-Chol [106]. Liu et al. characterized the diameter, zeta potentials and encapsulation efficiency of these CLANs to screen an optimized formulation for pCas9 delivery. The results indicated that increasing the quantity of the cationic lipid could improve the encapsulation efficiency. Then, they demonstrated that CLANs could deliver pCas9 into CML cells. CML is caused by the fusion between the breakpoint cluster region and Abelson murine leukemia viral oncogene homolog (BCR-ABL) [107]. Thus, Liu et al. designed a gRNA targeting the overhanging fusion region of the BCR-ABL gene (gBCR-ABL), which enabled us to specifically disrupt the BCR-ABL gene while sparing the BCR or ABL gene. After intravenously injecting CLANs encapsulating pX330, which co-expressed Cas9 and gBCR-ABL (CLANpCas9/gBCR-ABL) into the CML mouse model, they efficiently disrupted the BCR-ABL gene of CML cells in both blood and bone marrow and

Journal Pre-proof improved the survival of CML mice. Subsequently, Wang and coauthors further optimized the properties of CLANs for delivering pCas9 into immune cells. They prepared a library of CLANs with different surface PEG densities, surface charges and cationic lipids [108, 109]. After intravenous injection, they screened the optimized CLANs that could be most efficiently internalized by neutrophils and B cells, respectively. Then, Liu et al. constructed a pX330 plasmid co-expressing Cas9 and a gRNA targeting the neutrophil elastase (NE) gene (pCas9/gNE). Using the screened CLAN45 to deliver pCas9/gNE into neutrophils in a type 2 diabetes (T2D) mouse model, they successfully knocked out the NE gene in

of

neutrophils, which mitigated the diabetes symptoms of T2D mice [108]. Li et al. also

ro

encapsulated a pX330 plasmid targeting B-cell activating factor receptor (BAFFR) into CLANs (CLANpCas9/gBAFFR) to edit the genome of B cells in vivo. After injection of

-p

CLANpCas9/gBAFFR into a rheumatoid arthritis (RA) mouse model, CLANpCas9/gBAFFR

re

effectively treated RA via disrupting the BAFFR gene [109]. Unspecific uptake by nontarget cells is an unavoidable problem in nanoparticle delivery

lP

systems. To realize specific genome editing, a promoter of the CD68 molecule, which drives specific CD68 protein expression in macrophages and in monocyte precursors

na

[110], was chosen to replace the chicken β-actin promoter of pX330 and pX458 [70]. The resulting pM330 and pM458 plasmids could specifically drive Cas9 nuclease

Jo ur

expression in macrophages. After intravenous injection of CLANs encapsulating a pM330 plasmid targeting the Ntn1 gene (CLANpM330/gNtn1), CLANpM330/gNtn1 specifically disrupted the Ntn1 gene in macrophages via specific expression of Cas9. With CLANpM330/gNtn1 treatment, Luo et al. mitigated the T2D symptoms [70]. Jiang and coworkers constructed another cationic LNP to deliver a pCas9 targeting polo-like kinase 1 (Plk-1) (pCas9/gPlk-1) for tumor therapy (Fig. 7B) [111]. They complexed the plasmid with chondroitin sulfate and then protamine to condense the plasmid into a tightly bound core. Then, the core was coated by the cationic lipid mixture of DOTAP, DOPE and cholesterol to form an LNP with a core-shell structure. Furthermore, they modified the surface of LNP with DSPE-PEG, which could enhance the stability of the nanoparticle and prevent protein adsorption, to yield the polymerized LNP (PLNP). The core-shell structure of PLNP effectively protected pCas9/gPlk-1 from nuclease degradation and immune recognition in the circulation. Treating A375

Journal Pre-proof tumor-bearing mice with PLNP carrying pCas9/gPlk-1 suppressed tumor growth by disrupting the Plk-1 gene in A375 cells. Alternatively, Teixeira and colleagues constructed cationic lipid-based nanoemulsions with medium-chain triglycerides (MCTs), DOPE, and DOTAP and decorated the nanoemulsion with DSPE-PEG (Fig. 7C) [112]. They delivered pCas9 and donor DNA by either incorporating the DNA/DOTAP complex into the oil core during the preparation of the nanoemulsion or by directly adsorbing DNA onto its surface. Both strategies successfully transfected pCas9 and the donor DNA into the cultured fibroblasts. Cationic lipid-based liposomes have been routinely used for the delivery of nucleic

of

acids. Wei and coauthors constructed a folate receptor-targeted liposome (F-LP) with

ro

DOTAP, cholesterol, methoxy-PEG-succinyl-cholesterol, and folate-PEG-succinylcholesterol (Fig. 7D) [113]. The folate of F-LP could bind to the folate receptor on

-p

tumor cells for tumor targeting. After simple mixing, the CRISPR-Cas plasmid could

re

form lipoplexes with the F-LP via electrostatic interactions. Using the lipoplexes to deliver a pX330 plasmid targeting DNA methyltransferase 1, they inhibited the tumor

lP

growth of ovarian cancers. Li et al. used a multifunctional peptide R8-dGR, which could bind to integrin αvβ3 and neuropilin-1 of various tumor cells, to modify the

na

cationic liposome for tumor-targeted delivery (Fig. 7E) [114]. They first prepared a cationic liposome with DOTAP and cholesterol, and then the pCas targeting hypoxia-

Jo ur

inducible factor-1α (pHIF-1α) was complexed with the liposome with the help of protamine sulfate. Finally, R8-dGR-DSPE-PEG was conjugated to the cationic liposome to provide a PEG sheath on the liposome. Intravenous injection of R8-dGRmodified liposomes carrying pHIF-1α and paclitaxel prolonged the survival in a pancreatic tumor mouse model by disrupting HIF1α. Apart from using synthesized cationic lipids to prepare liposomes, Marepally and coworkers developed liposomes with an asymmetric hydrophobic core using the fatty acids of natural food-grade palmstearin and cholesterol (Fig. 7F) [115]. In vitro transfections demonstrated that liposomes with an asymmetric core could transfect pCas more efficiently than liposomes with a symmetric core.

Jo ur

na

lP

re

-p

ro

of

Journal Pre-proof

Fig. 7. Cationic lipid-based delivery systems of CRISPR-Cas plasmids. (A) Screening CLANs for delivering pCas9 into chronic myeloid leukemia (CML) cell, neutrophil, B cell and macrophage for different disease treatment. T2D: type 2 diabetes (B) Polymerized lipid nanoparticle (PLNP) delivered pCas9/gPlk1 in vivo for A375 tumor inhibition. Adapted with permission from [111]. Copyright (2017) Springer Nature. (C) Delivering pCas9/donor DNA by encapsulating in or adsorbing on nanoemulsion. Adapted with permission from [112]. Copyright (2018) Elsevier. (D) Complexing pCas9 with folate receptor-targeted liposome for in vivo SKOV3 tumor targeted delivery. (E) Decorating the lipoplex of pCas9/gHIF-1α and cationic liposome with R8dGR-DSPE-PEG for in vivo BxPC-3 tumor targeted delivery. (F) Natural cationic lipids for liposome preparation. Adapted with permission from [115]. Copyright (2017) American Chemical Society.

Journal Pre-proof 3.1.2

PEI-based delivery systems

PEI is a gold standard for gene delivery, including short nucleic acids and large plasmids. Zhang et al. demonstrated that the conjugate of PEI (25 kDa) and βcyclodextrin (β-CD) was able to condense pX330 into the nanocomplex at an N/P ratio (the ratio of positively chargeable polymer amine (N) groups to negatively charged nucleic acid phosphate (P) groups) of 60 (Fig. 8A) [116]. Ryu et al. utilized branched PEI (25 kDa) to condense pCas into nanocomplex, and the N/P ratio was reduced to 6 (Fig. 8B) [117]. In addition, PEI with a low molecular weight could also condense the pCas. Yan et al. modified PEI (2 kDa) with dioleoylphosphatidylethanolamine (PE)

of

which can condense pCas at an N/P ratio of 16 (Fig. 8B) [118].

ro

Liang et al. synthesized a PEI-based lipopolymer for pCas9 delivery (Fig. 8C) [119].

-p

They combined branched PEI (1.8 kDa), cholesteryl chloroformate and PEG into the PEG-PEI-Cholesterol (PPC) polymer. Then, the PPC and pCas9 were incubated at an

re

N/P ratio of 11 to form complexes. To develop an osteosarcoma (OS)-targeted delivery system, they screened an OS cell-specific aptamer (LC09) and functionalized the

lP

complexes this aptamer onto the surface. After intravenously injecting the OSconjugated complexes loaded with a pCas9 targeting vascular endothelial growth factor

na

A (VEGFA) into the OS tumor-bearing mice, they demonstrated that LC09 could facilitate pCas9 selectively distribute in the OS tumor.

Jo ur

Liu et al. synthesized a phenylboronic acid (PBA)-modified PEI (2.1 kDa) that could condense the CRISPR-dCas9 plasmid at an N/P ratio of 20 (Fig. 8D) [120]. They synthesized the anionic polymer 2,3-dimethylmaleic anhydride (DMMA)-modified PEG-b-polylysine (mPEG113-b-PLys100/DMMA) to coat the positive polyplex of PBAPEI and CRISPR-dCas9. In acidic endosomes, degradation of DMMA led to the disassembly of the positive mPEG113-b-PLys100 shell from the positive polyplex core due to electrostatic repulsion. They demonstrated that delivering CRISPR-dCas9 to activate miR-524 expression with their delivery system achieved MDA-MB-231 tumor growth inhibition. It has been reported that modifying cationic polymers with fluorinated compounds could efficiently encapsulate nucleic acids [121]. Therefore, Wei and coauthors synthesized a fluorinated polymer (PF33) by modifying heptafluorobutyric anhydride on PEI (1.8 kDa) (Fig. 8E) [122]. After condensing pX330 into nanoparticles with PF33

Journal Pre-proof at a mass ratio of 1:1, they coated the nanoparticles with RGD-R8-PEG-HA (RRPH) polymers to form a “core-shell” structure artificial virus (RRPHC). The negative HA polymer shielded the positive charge of PF33, and the PEG chains improved the stability of RRPHC and the R8-RGD peptide enhanced targeting ability of RRPHC by specifically binding to the integrin αvβ3 receptors on tumor cells. Delivering pX330 targeting MTH1 with RRPHC significantly inhibited ovarian tumor growth in mice. Qi et al. synthesized another fluorinated acid-labile branched hydroxylrich polycation (ARP-F) for pCas9 delivery (Fig. 8F) [123]. The ARP-F could condense pCas9 within the mass ratio of 2:1. Ortho ester linkages in ARP-F could be degraded in acidic

of

endosome for rapid release of pCas9. Intravenous injection of the complex of ARP-F

Jo ur

na

lP

re

-p

ro

and pCas9 targeting surviving (Surv) gene inhibited A549 tumor growth in mice.

Fig. 8. PEI-based delivery systems of CRISPR-Cas plasmids. (A) Nanocomplex formed by the binding of PEI-β-cyclodextrin (PEI-β-CD) with pCas9. Adapted with permission from [116]. Copyright (2018) John Wiley and Sons. (B) Nanocomplex

Journal Pre-proof formed by the binding of PEI (25 kDa) or PEI (2 kDa) with pCas9. (C) LC09 aptamerfunctionalized nanoparticle for OS cell-targeted delivery of pCas9. (D) pCas9-loaded phenylboronic acid-modified PEI (PEI-PBA)-based multistage delivery nanoparticle to inhibit MDA-MB-231 tumor growth in vivo. Adapted with permission from [120]. Copyright (2018) John Wiley and Sons. (E) Fluorinated PEI-based artificial virus for pCas9 delivery to inhibit SKOV3 tumor growth in vivo. Adapted with permission from [122]. Copyright (2017) American Chemical Society. (F) Nanocomplex formed by the binding of fluorinated acid-labile branched hydroxylrich polycation (ARP-F) with pCas9 for inhibiting A549 tumor growth in vivo. Adapted with permission from [123].

ro

3.1.3

of

Copyright (2018) John Wiley and Sons.

Cationic polypeptide-based delivery systems

-p

Both CPPs and NLS peptides are cationic polypeptides, which have been demonstrated

re

to effectively assist in nucleic acid delivery [124]. To utilize cationic polypeptides for pCas9 delivery, Jiang and coauthors modified AuNPs with nucleus-targeting TAT

lP

peptides, allowing pCas9 to condense on the surface via electrostatic interactions (Fig. 9A) [125]. Cationic lipids (DOTAP, DOPE, and cholesterol) were then coated onto

na

AuNP-condensed pCas9 (ACP), followed by decorating with PEG-DSPE to form a PEG sheath on the lipid-encapsulated ACP (LACP). LACP was able to accumulate in

Jo ur

melanoma after intravenous injection into mice. After endocytosis, pCas9 was released from LACP by the laser-triggered thermal effects of AuNPs, and cationic TAT peptides could guide Plk1-specific pCas9 to enter the nucleus to disrupt the Plk1 gene for tumor inhibition.

Sukhorukov and coworkers utilized cationic poly-L-arginine (PARG) and dextran sulfate (DEXS) to prepare (PARG/DEXS)3 capsules using layer-by-layer assembly on CaCO3 cores (Fig. 9B) [126]. The capsules were then coated with hollow SiO2. By simply incubating pCas9 with SiO2-coated capsules, pCas9 was immobilized on the surface via electrostatic interactions. Although they only tested the transfection efficiency of this system in vitro, they demonstrated that cationic PARG could also be used for condensing pCas. Cheng previously developed a cationic α-helical polypeptide poly(γ-4-((2-(piperidin-1yl)ethyl)aminomethyl)benzyl-L-glutamate) (PPABLG) [127]. They demonstrated that

Journal Pre-proof PPABLG could bind and condense plasmids and siRNAs while retaining the helical structure, which further provided membrane-penetrating ability to enhance endocytosis and endosomal escape. Leong and Cheng collaboratively adapted the PPABLG polypeptide to complex pCas9 into nanocomplexes (HNPs), followed by incorporating PEG-Polythymine40 (PEG-T40) to form a PEG sheath for stabilizing the nanocomplexes (P-HNPs) (Fig. 9C) [75]. A local injection of P-HNPs carrying pCas9/gPlk1 in HeLa

na

lP

re

-p

ro

of

tumor-bearing mice significantly inhibited tumor growth.

Fig. 9. Cationic polypeptide-based delivery systems of CRISPR-Cas plasmids. (A)

Jo ur

Condensing pCas9 with TAT-modified AuNP for delivering pCas9 to inhibit melanoma growth in vivo. Adapted with permission from [125]. Copyright (2018) John Wiley and Sons. (B) Complexing pCas9 with SiO2-coated (PARG/DEXS)3 capsule. Adapted with permission from [126]. Copyright (2018) Elsevier. (C) Polypeptide poly(γ-4-((2(piperidin-1-yl)ethyl)aminomethyl)benzyl-L-glutamate) (PPABLG) peptide-based PHNP for delivering pCas9 to inhibit HeLa tumor growth in vivo. Adapted with permission from [75]. Copyright (2018) National Academy of Sciences.

3.1.4

Other cationic material-based delivery systems

Apart from cationic materials (including cationic lipids and PEI) with high charge density, Leong Group also developed a self-assembled micelle with quaternary ammonium-terminated poly(propylene oxide) (PPO-NMe3) and amphiphilic Pluronic F127 to condense pCas9 (Fig. 10A) [128]. Although PPO-NMe3 is a linear polymer

Journal Pre-proof with a low charge density, it has a strong nucleic acid-binding affinity. Mixing PPONMe3, pCas9 and F127 could cause micelle assembly (F127/PPO-NMe3/pCas9) through electrostatic and hydrophobic interactions. Intratumoral injection of F127/PPO-NMe3/pCas9 micelles into HeLa tumor-bearing mice inhibited tumor growth by disrupting the HPV18-E7 oncogene. Zhang et al. utilized a previously synthesized cholesterol (CHO)-terminated ethanolamine-aminated poly(glycidyl methacrylate) (CHO-PGEA) with rich hydroxyl groups to complex pCas9 into nanoparticles via electrostatic interactions (Fig. 10B) [129]. After an intravenous injection of CHO-PGEA encapsulating pCas9/gFbn1 into mice, they achieved efficient

of

knockout of the Fbn1 gene in aortic cells.

ro

Liu et al. demonstrated that protamine sulfate was also able to complex with pCas9 (Fig. 10C) [130]. They then decorated the complex with an endosomolytic peptide

-p

(KALA), and the aptamer AS1411 incorporated carboxymethyl chitosan to the outer

re

layer. The resulting multifunctional nanoparticles could efficiently transfect pCas9 into tumor cells. Wang and colleagues utilized poly(β-amino ester) (PBAE) to form

lP

nanoparticles with pCas9 via electrostatic interactions (Fig. 10D) [131]. They constructed pCas9 targeting HPV16-E7 and encapsulated them into PBAE

na

nanoparticles. The results indicated that pCas9 delivered by PBAE nanoparticles

Jo ur

inhibited tumor growth after intratumoral injection.

Fig. 10. Other cationic material-based delivery systems of CRISPR-Cas plasmids. (A) Quaternary ammonium-terminated poly(propylene oxide) (PPO-NMe3), Pluronic F127

Journal Pre-proof and pCas9 self-assembled micelle inhibited HeLa tumor growth by disrupting HPV18E7 after intratumoral injection. Adapted with permission from [128]. Copyright (2018) John Wiley and Sons. (B) Complexing pCas9/gFbn1 with cholesterol (CHO)terminated ethanolamine-aminated poly(glycidyl methacrylate) (CHO-PGEA) into nanoparticle for genome editing in aortic cells in vivo. Adapted with permission from [129]. Copyright (2019) John Wiley and Sons. (C) Protamine sulfate-based multifunctional nanoparticles for pCas9 delivery. (D) Complexing pCas9 with poly(βamino ester) (PBAE) to form nanoparticles for disrupting HPV16-E7 in vivo.

Other types of delivery systems

of

3.1.5

ro

Exosomes are natural vesicles of high biocompatibility and low immunogenicity. They have a demonstrated cell-oriented ability and can carry various biomolecules, including

-p

plasmids, siRNAs, and miRNAs. Kim et al. purified exosomes derived from SKOV3

re

tumor cells (SKOV3-Exo) and loaded the exosomes with pCas9 by electroporation (Fig. 11A) [132]. After intravenous injection into SKOV3 tumor mouse model, SKOV3-Exo

lP

selectively accumulated in SKOV3 tumors and inhibited tumor growth by pCas9 targeting poly(ADP-ribose) polymerase-1 (PARP-1)-mediated genome editing. This

na

work conferred cancer cell tropism-dependent targeting delivery with tumor-derived exosomes. To further enhance the encapsulation efficiency of pCas9, Lin et al. first

Jo ur

mixed pCas9 and cationic liposomes into prepared complexes (Fig. 11B) [133]. The complexes were then incubated with exosomes to form hybrid exosomes, which enhanced the loading content of pCas9 with the help of cationic lipids from the liposomes.

In addition, Brinkmann and coauthors prepared artificial chromatin by assembling pCas9 with histones by salt gradient dialysis. The chromatin was then connected to bispecific antibodies via nucleic acid-binding peptide bridges (Fig. 11C) [134]. They demonstrated that the antibody-targeted chromatin could efficiently transfect pCas9 into cultured cells and achieved a 90% transfection efficiency. Moses Group recently synthesized a noncationic tumor-targeted nanolipogel system (tNLG) to deliver pCas9 into triple-negative breast cancer (TNBC) (Fig. 11D) [135]. They first utilized a mixture of phospholipids (DOPC:DSPE-PEG-COOH) to prepare phospholipid film and resuspended the film in DMSO:EtOH solution, and then the

Journal Pre-proof solution was added dropwise into sodium alginate solution containing pCas9 followed by freeze-thaw cycles and extrusion to prepare the nanolipogel. Finally, the nanolipogel was decorated with ICAM1 antibody for tumor targeting delivery. They demonstrated that systemically administered tNLGs efficiently knocked out Lcn2 in TNBC tumor. Furthermore, to realize precise genome editing, Chen and coworkers reported a second near-infrared window (NIR-II) imaging-guided and NIR-light-triggered remote control of CRISPR-Cas9 genome editing (Fig. 11E) [136]. They used a bush-structured semiconducting polymer (SPPF), which was prepared by conjugating semiconducting polymer (SP) with alkyl side chains, PEG, and fluorinated PEI, to complex pCas9 via

of

electrostatic interactions and supramolecular interactions. Dexamethasone (Dex) was

ro

also encapsulated into the hydrophobic core of the SPPF nanoparticle to facilitate pCas9 enter the nucleus. The resulted SPPF-Dex/pCas9 system could achieve NIR light-

Jo ur

na

lP

re

-p

controllable disruption of MTH1 gene in the tumor site.

Fig. 11. Other delivery systems of CRISPR-Cas plasmids. (A) Purifying tumor cellderived exosome for in vivo tumor tropism-dependent delivery of pCas9. (B) Hybrid exosome of cationic liposome and exosome for pCas9 delivery. Adapted with permission from [133]. Copyright (2018) John Wiley and Sons. (C) Antibodyconjugated artificial chromatin for pCas9 delivery. (D) ICAM antibody-conjugated

Journal Pre-proof tumor-targeted nanolipogel system (tNLG) for delivering pCas9 to disrupt Lcn2 for triple-negative breast cancer (TNBC) therapy. Adapted with permission from [135]. Copyright (2019) National Academy of Sciences. (E) SPPF-Dex/pCas9 system for NIR-triggered site-specific genome editing of MTH1 gene in HCT 116 tumor. Adapted with permission from [136]. Copyright (2019) John Wiley and Sons.

3.2 Delivery systems of RNA-based CRISPR-Cas The RNA form of CRISPR-Cas is an excellent candidate for developing in vivo genome

of

editing therapeutics due to its transient, nonintegrating Cas9 expression construct [137]. However, the single-stranded structures of Cas9 mRNA and gRNA are quite unstable,

ro

which raises the requirements of delivery systems. In addition, RNA-based CRISPRCas is composed of a long Cas mRNA and a short gRNA. The delivery systems should

-p

be able to load both of them. Currently reported delivery systems of RNA-based

CLANs for RNA-based CRISPR-Cas delivery

lP

3.2.1

re

CRISPR-Cas can be generally categorized into CLANs, LNP and cell-derived vesicles.

The CLAN platform could also be used for delivering Cas9 mRNA and gRNA for in

na

vivo genome editing (Fig. 12) [138, 139]. Wang and coworkers created a library of CLANs with different properties and then screened the CLANs that could efficiently

Jo ur

encapsulate and deliver Cas9 mRNA/gRNA into macrophages and dendritic cells. Similar to pCas9 encapsulation, Cas9 mRNA and gRNA were mixed together and formed tight complexes with cationic lipids via electrostatic interactions in the first emulsification step. In the second emulsification step, the complexes were efficiently encapsulated into the aqueous core of CLANs. After intravenous injection of CLAN encapsulating Cas9 mRNA and gRNA targeting NLRP3 (gNLRP3), the NLRP3 gene in macrophages was significantly disrupted, which mitigated LPS-induced septic shock, monosodium urate crystal (MSU)-induced peritonitis and high-fat-diet (HFD)-induced T2D (Fig. 12A) [138]. For the genome editing of dendritic cells, we encapsulated Cas9 mRNA and gRNA targeting a costimulatory molecule CD40 (gCD40) into CLANs (CLANmCas9/gCD40). After intravenous injection into the skin-transplanted mice, CLANmCas9/gCD40 disrupted CD40 in dendritic cells, which inhibited T cell activation and prolonged the survival of

Journal Pre-proof transplanted skin (Fig. 12B) [139]. In conclusion, our CLAN platform was able to deliver both plasmid- and RNA-based CRISPR-Cas and can be expanded for delivering

-p

ro

of

other forms of nucleic acid-based CRISPR-Cas.

re

Fig. 12. Cationic lipid-assisted polymeric nanoparticles (CLANs) for RNA-based

lP

CRISPR-Cas9 delivery. (A) Screening CLAN for delivering Cas9 mRNA/gNLRP3 into macrophage, which disrupted NLRP3 gene and mitigated inflammatory diseases.

na

Adapted with permission from [138]. Copyright (2018) Springer Nature. (B) Utilizing CLAN to deliver Cas9 mRNA/gCD40 into dendritic cell for inducing transplant

Elsevier.

3.2.2

Jo ur

tolerance by disrupting CD40. Adapted with permission from [139]. Copyright (2019)

LNP for RNA-based CRISPR-Cas delivery

Using microfluidics to complex Cas9 mRNA/gRNA with lipids into LNP is the most commonly used and scalable strategy [140, 141]. Anderson Group previously screened an ionizable lipid cKK-E12 for selectively delivering siRNA into liver parenchymal cells in vivo [142]. Then, they prepared LNP by mixing an ethanol solution containing cKK-E12, cholesterol, C14-PEG and DOPE with an aqueous solution containing Cas9 mRNA/gRNA by microfluidics (Fig. 13A) [143]. The PEG shell protected the Cas9 mRNA/gRNA LNP from RNases. They demonstrated that LNP could efficiently load and deliver Cas9 mRNA and gRNA targeting Pcsk9 (gPcsk9) into hepatocytes after intravenous

injection,

which

decreased

serum

cholesterol

levels

in

a

hypercholesterolemia model. Furthermore, Finn et al. developed a similar LNP (Fig.

Journal Pre-proof 13B) [144]. They utilized an ionizable lipid (LP01), cholesterol, DSPC and DMG-PEG to complex Cas9 mRNA/gRNA into LNP via microfluidics. They demonstrated that a single administration of LNP encapsulating Cas9 mRNA and gRNA targeting TTR gene was able to achieve high levels of gene disruption in hepatocytes. Wang and Xu collaboratively identified a bioreducible lipid (BAMEA-O16B) containing three disulfide bonds (Fig. 13C) [145]. BAMEA-O16B was mixed with cholesterol, DOPE, and DSPE-PEG was added dropwise to an aqueous solution of DSPE-PEG after filming-rehydration to prepare LNP for Cas9 mRNA/gRNA encapsulation. They demonstrated that Cas9 mRNA/gRNA could be responsively

of

released from BAMEA-O16B LNP after internalization into a reductive intracellular

ro

environment. Intravenous injection of BAMEA-O16B LNP selectively accumulated in

-p

hepatocytes for genome editing.

Several lipid-like materials have also been synthesized. Li et al. prepared a library of

re

lipid-like nanoparticles (LLNs) using N1,N3,N5-tris(2-aminoethyl)benzene-1,3,5tricarboxamide (TT) derivatives, and screened TT3-formulated LLN (TT3 LLN) as the

lP

most efficient formula for mRNA delivery [146]. They further optimized TT3 LLN for Cas9 mRNA/gRNA delivery by changing the formula of TT3 lipid, DOPE, cholesterol

na

and DMG-PEG (Fig. 13D) [147]. They delivered Cas9 mRNA and gRNA targeting HBV DNA with the optimized TT3 LLN to eliminate HBV in hepatocytes of mice. In

Jo ur

addition, they demonstrated that TT3 LLN encapsulating Cas9 mRNA and gPcsk9 was able to disrupt the Pcsk9 gene in hepatocytes. Miller et al. developed a class of zwitterionic amino lipids (ZALs) that could efficiently bind to RNA due to its long carbon alkyl tails and alcohol/ester groups (Fig. 13E) [148]. They used a two-channel microfluidic mixer to prepare ZAL-based nanoparticles (ZNPs) by mixing ethanol solution containing ZA3-Ep10 lipid, cholesterol, and PEG-lipid with aqueous solution containing Cas9 mRNA/gRNA. Intravenous injection of ZNP encapsulating Cas9 mRNA/gRNA could edit the genome in the liver, kidneys and lungs in mice. In addition, Dong and colleagues screened two lead materials, MPA-A with linear ester chains and MPA-Ab with branched ester chains, for encapsulating Cas9 mRNA/gRNA into LLN via microfluidic mixer [149]. They demonstrated that both MPA-A and MPA-Ab-based LLN could deliver Cas9 mRNA/gRNA for in vitro and in vivo genome editing.

lP

re

-p

ro

of

Journal Pre-proof

Fig. 13. LNP for RNA-based CRISPR-Cas9 delivery. (A) cKK-E12-based lipid

na

nanoparticle (LNP) delivering Cas9 mRNA/gRNA to disrupt Pcsk9 in hepatocytes for hypercholesterolemia therapy. (B) LP01-based LNP delivering Cas9 mRNA/gRNA to

Jo ur

disrupt TTR gene in liver for TTR amyloidosis treatment. Adapted with permission from [144]. Copyright (2018) Elsevier. (C) Responsively releasing Cas9 mRNA/gRNA into hepatocytes with BAMEA-O16B-based LNP for genome editing. Adapted with permission from [145]. Copyright (2019) John Wiley and Sons. (D) TT3-based lipidlike nanoparticle (LLN) delivering Cas9 mRNA/gRNA for genome editing in hepatocytes. (E) ZA3-Ep10-based nanoparticles (ZNP) for delivering Cas9 mRNA/gRNA for genome editing in multiple tissues.

3.2.3

Cell-derived vesicle-based delivery systems

Besides plasmids delivery, exosomes can be adapted for delivering RNA-based CRISPR-Cas. Yuan and coauthors directly loaded dCas9 mRNA and gRNA into exosomes when cells were packaging exosomes (Fig. 14A) [150]. They constructed three expression vectors: a vector of fusion protein of an exosomal surface marker CD9 and human antigen R (HuR), which could bind to AU-rich elements (AREs) in RNA; a

Journal Pre-proof vector of dCas9 with AREs cloned downstream of its stop codon; and a vector of gRNA targeting the C/ebpα gene (gC/ebpα). Co-transfecting three vectors with a lentivirus into packaging cells could efficiently load dCas9 mRNA and gRNA into CD9-HuR functionalized exosomes via binding AREs in RNA with HuR. With CD9-HuR functionalized exosomes, they delivered dCas9 mRNA and gC/ebpα into the liver for gene silencing via intravenous administration. Furthermore, the membranes of red blood cells (RBCs) have also been utilized for RNA delivery. Usman et al. established a strategy that could produce large-scale amounts of extracellular vesicles (EVs) with the RBCs. Cas9 mRNA and gRNA could be loaded

of

into the EVs via electroporation (Fig. 14B) [151]. Their work demonstrated that EVs

na

lP

re

-p

ro

from RBCs could be used for both plasmid- and RNA-based CRISPR-Cas transfection.

Fig. 14. Cell-derived vesicle for RNA-based CRISPR-Cas9 delivery. (A) Targeted

Jo ur

delivery of dCas9 mRNA/gRNA with CD9-human antigen R (HuR) functionalized exosome into liver for gene silencing. Adapted with permission from [150]. Copyright (2019) American Chemical Society. (B) Red blood cell (RBC)-derived extracellular vesicle (EV) delivering Cas9 mRNA/gRNA into cancer cell for gene knockout. Adapted with permission from [151]. Copyright (2018) Springer Nature.

3.3 Delivery systems of RNP-based CRISPR-Cas RNPs are composed of a large Cas protein and a short gRNA. gRNA can bind to DNA via Watson–Crick base pairing or the Cas protein can be conjugated to polypeptides, proteins, and PEI. These features can also be used for loading RNP. In addition, RNP can be loaded via electrostatic interactions with positively charged materials due to its negative net charge. These positively charged materials can be cationic lipids, PEI, polypeptides, and metal-organic frameworks (MOFs). Vesicles from cells can also be

Journal Pre-proof used to deliver RNP.

3.3.1

DNA nanoclews for RNP-based CRISPR-Cas delivery

Previously, Gu and coworkers developed yarn-like DNA nanoclews via rolling circle amplification (RCA) for doxorubicin delivery [152]. Inspired by the ability of ssDNA to base-pair with the guide portion of the Cas9-bound gRNA [153], the authors anticipated that the DNA of the nanoclews could also be designed to deliver RNP. Thus, they used DNA nanoclews to be partially complementary to gRNA, and then the Cas9

of

RNP was loaded via base-pairing (Fig. 15) [154]. Subsequently, they coated the complex of Cas9 RNP and DNA nanoclews with PEI for enhanced endosomal escape.

ro

They demonstrated that the PEI-coated DNA nanoclews could efficiently transfect the Cas9 RNP targeting EGFP into U2OS cells for EGFP knockout in vitro. Furthermore,

Jo ur

na

lP

re

tumors in vivo after intratumoral injection.

-p

the PEI-coated DNA nanoclews could also disrupt EGFP in U2OS.EGFP xenograft

Fig. 15. Delivery of Cas9 RNP with DNA nanoclew prepared by rolling circle amplification (RCA) for genome editing. I) Bind to the cell membrane; II) endocytosis; III) endosome escape; IV) transport into the nucleus; V) introduce genome editing. Adapted with permission from [154]. Copyright (2015) John Wiley and Sons.

3.3.2

In situ polymerized nanocapsule for RNP-based CRISPR-Cas

Journal Pre-proof delivery Recently, Gong Group developed a nanocapsule for Cas9 RNP delivery (Fig. 16) [155]. Due to the heterogeneous surface charges of RNP, they first coated the RNP with both cationic and anionic monomers via electrostatic interactions. An imidazole-containing monomer, glutathione (GSH)-degradable crosslinker, PEG can be absorbed to the surface of the RNP via hydrogen bonding and van der Waals interactions. Then, GSHcleavable nanocapsules were formed around the RNP via in situ free-radical polymerization. In addition, targeting ligands, for example CPPs, can be added into the nanocapsule by conjugation to PEG. They demonstrated that the GSH cleavable

of

nanocapsule could protect Cas9 RNP in the endosome after cellular uptake and could

ro

be quickly cleaved by GSH after escape into the cytoplasm for subsequent genome editing. After local injection of Cas9 RNP nanocapsules, robust gene editing was

na

lP

re

-p

observed in retinal pigment epithelium (RPE) and muscle.

Jo ur

Fig. 16. Delivery of Cas9 RNP with nanocapsule prepared by in situ free-radical polymerization. Adapted with permission from [155]. Copyright (2019) Springer Nature.

3.3.3

AuNP-based delivery systems

AuNPs can be easily modified for tailoring charge, hydrophilicity and functional ligands. Murthy and colleagues developed a vehicle named CRISPR-Gold, which was composed of ssDNA-conjugated AuNPs, donor ssDNA, Cas9 RNP and endosomal disruptive

polymer

poly(N-(N-(2-aminoethyl)-2-aminoethyl)

aspartamide)

(PAsp(DET)) (Fig. 17A) [90]. They first conjugated AuNPs with thiol-terminated ssDNA. Then, the resulting AuNP-ssDNA conjugate was hybridized with the donor ssDNA via partial base pairing. Cas9 RNP was further adsorbed onto the AuNPs through the specific base-pairing of gRNA and the other part of the donor ssDNA.

Journal Pre-proof Finally, the nanoparticles were deposited with a layer of negatively charged silica, and cationic PAsp(DET) was coated onto the nanoparticles to form CRISPR-Gold. By simultaneously delivering Cas9 RNP and donor ssDNA with CRISPR-Gold, they reduced muscle fibrosis in mdx mice by repairing the mutated dystrophin gene. Afterwards, Murthy and Lee cooperatively tested the CRISPR-Gold for brain genome editing (Fig. 17A) [156]. They demonstrated that CRISPR-Gold was able to encapsulate both Cas9 and Cpf1 RNP. Intracranial injection of CRISPR-Gold could edit most cell types in the brain. Furthermore, Adair and coauthors utilized a similar strategy to prepare AuNPs and

of

achieved targeted HDR in hematopoietic stem and progenitor cells (HSPCs) (Fig. 17B)

ro

[157]. In brief, an AuNP core was conjugated with thiol-terminated crRNA with an 18oligo ethylene glycol (OEG) units-spacer (crRNA–18 spacer–SH), and Cas9 or Cpf1

-p

was attached to crRNA via natural affinity. Then, the RNP-loaded AuNPs were coated

re

with PEI, and the ssDNA donor was adsorbed on the surface via electrostatic interactions. They demonstrated that AuNPs could efficiently transfect Cas9 or Cpf1

lP

RNP/ssDNA into HSPCs for genome editing in vitro. Rotello Group modified AuNPs with arginine-functionalized thiol ligand to prepared

na

arginine-functionalized AuNPs (ArgNPs), which could directly transfer into the cytoplasm without the endocytosis process (Fig. 17C) [158]. Moreover, they attached

Jo ur

a glutamate peptide tag (E-tag) to the N-terminus of Cas9 protein (Cas9En), which was incubated with gRNA to form Cas9En RNP. Then, Cas9En RNP was mixed with cationic ArgNPs to form self-assembled nanoassemblies via electrostatic interactions. They demonstrated that these nanoassemblies could directly transfer across the cell membrane of cultured HeLa cells and deliver Cas9En RNP into the nuclei. Subsequently, they mixed Cas9En RNP targeting the SIRP-α gene with ArgNPs to form nanoassemblies for macrophage engineering (Fig. 17C) [159]. They transfected RAW264.7 macrophages with these nanoassemblies in vitro and produced SIRP-α knockout macrophages. These SIRP-α knockout macrophages could efficiently recognize and engulf U2OS-GFP cells.

ro

of

Journal Pre-proof

-p

Fig. 17. AuNP-based delivery systems for RNP-based CRISPR-Cas delivery. (A) Delivery of Cas9 RNP and donor ssDNA for genome editing of muscle or brain.

re

Adapted with permission from [90]. Copyright (2017) Springer Nature. (B) Delivery of Cas9/Cpf1 and donor ssDNA by crRNA-conjugated gold nanoparticle (AuNP) for

lP

genome editing in hematopoietic stem and progenitor cells (HSPCs). Adapted with permission from [157]. Copyright (2019) Springer Nature. (C) Self-assembly of

na

Cas9En and arginine-functionalized AuNP (ArgNP) into nanoassembly for cytosolic delivery of Cas9 RNP. Adapted with permission from [158]. Copyright (2017)

3.3.4

Jo ur

American Chemical Society.

Cationic lipid-based delivery systems

Because the net charge of RNP is negative, cationic liposomes or LNPs can be directly used for RNP transfection. Zuris et al. demonstrated that the Cas9 protein (+22 net charges) can be rendered highly anionic by fusion to a negatively charged GFP (-30 net charges) or complexation with a gRNA. Using Lipofectamine 2000, RNP could be delivered into the hair cells of the mouse inner ear via local injection (Fig. 18A) [88]. Subsequently, Xu and Liu collaboratively explored cationic LNP for Cas9 RNP delivery (Fig. 18B) [160]. They synthesized 12 bioreducible lipids and formulated them into LNPs by mixing them with cholesterol and DOPE and adding dropwise to an aqueous solution of C16-PEG-ceramide after filming-rehydration. The 8-O14B lipid formulated LNP was screened as the most efficient for RNP transfection in vitro by direct mixing.

Journal Pre-proof Similarly, Xu and Wang synthesized a bioreducible lipid (80-O16B) with a similar structure to the 8-O14B and BAMEA-O16B lipids, as we mentioned earlier [145]. They prepared LNP with the same filming-rehydration method and loaded Cas9 RNP by directly mixing with LNP (Fig. 18C). They demonstrated that 80-O16B LNPs selectively accumulated in the liver after intravenous injection, indicating that cationic lipids with similar structures have special affinity to liver [161]. The drawback of delivering the RNP with liposomes or LNPs is that most of the RNP is absorbed on their surface, and they could not protect the RNP from nuclease degradation and immune

re

-p

ro

of

recognition after systemic injection.

lP

Fig. 18. Lipofectamine or LNP for RNP-based CRISPR-Cas delivery. (A) Utilizing Lipofectamine 2000 or RNAiMAX for supernegatively charged Cas9-GFP RNP

na

delivery. Adapted with permission from [88]. Copyright (2014) Springer Nature. (B) Lipid 8-O14B-based lipid nanoparticle (LNP) for supernegatively charged Cas9-GFP

Jo ur

RNP delivery. Adapted with permission from [160]. Copyright (2016) National Academy of Sciences. (C) Disulfide bond-containing cationic lipidoid-based LNP for delivering Cas9 RNP into liver.

3.3.5

PEI-based delivery systems

Positively charged PEI has also been developed for RNP delivery. Yue et al. functionalized graphene oxide (GO) with PEG and PEI (Fig. 19A) [162]. Then, Cas9 RNP was loaded onto GO-PEG-PEI via physisorption and π-stacking interactions. Alternatively, Chung and coworkers covalently modified the Cas9 protein with branched PEI (bPEI) (Fig. 19B) [163]. Then, they mixed Cas9-bPEI with gRNA to form a nanocomplex via electrostatic interactions. Additionally, Yoon and colleagues mixed PEI and Cas9 RNP to form positively charged complexes, and then the complexes were loaded into lecithin-based liposomes mainly via electrostatic

Journal Pre-proof interactions (Fig. 19C) [164]. A DOGS-NTA-Ni lipid was incorporated into the liposomes to maintain the liposomal structure via the interactions between NTA-Ni and the His tag of the Cas9 protein. They demonstrated that lecithin-based liposomes carrying Cas9 RNP could efficiently disrupt the DPP4 gene after intravenous injection,

ro

of

which ameliorated T2D in db/db mice.

-p

Fig. 19. PEI-based delivery systems of Cas9 RNP. (A) PEI-PEG-functionalized graphene oxide (GO) for Cas9 RNP transfection. Adapted with permission from [162].

re

Copyright (2018) Royal Society of Chemistry. (B) bPEI-based nanocomplex for Cas9 RNP transfection. Adapted with permission from [163]. Copyright (2017) American

lP

Chemical Society. (C) Delivering PEI/Cas9 RNP complex by lecithin-based liposome

Polypeptides/proteins-based delivery systems

Jo ur

3.3.6

na

for disrupting DPP4 in vivo to treat type 2 diabetes (T2D).

Conjugation of the Cas9 protein with polypeptides or proteins has been developed to facilitate RNP delivery. For instance, Jang and coworkers fused a Cas9 protein with low molecular weight protamine (Cas9-LMWP). Cas9-LMWP, crRNA and tracrRNA could assemble into a ternary complex via electrostatic interactions (Fig. 20A) [165]. Intratumoral injection of the ternary complex was able to inhibit A549 tumor growth in mice via disrupting KRAS gene. Alternatively, Kim Group used a combined strategy by conjugating the Cas9 protein with CPPs (GGGRRRRRRRRRLLLL, m9R) via a thioether bond and complexing gRNA with CPPs (C3G9R4LC peptide, 9R) to form cationic nanoparticles via electrostatic interactions (Fig. 20B) [166]. Because both Cas9-m9R and gRNA:9R complex were positively charged, they could be easily internalized. Furthermore, Qazi et al. fused the Cas9 protein with the truncated P22 scaffold protein from virus-like particles (VLPs) of the bacteriophage P22 (Fig. 20C) [167]. Using the P22 scaffold protein as a template, the Cas9 protein and gRNA were

Journal Pre-proof encapsulated into the P22 VLPs. They demonstrated that this strategy could protect the Cas9 protein and gRNA from proteases and nucleases. Rouet et al. fused asialoglycoprotein receptor ligands (ASGPrL) with the Cas9 protein for targeting delivery to liver cells and proved that Cas9-ASGPrL RNP could preferentially internalize in HepG2 cell lines in vitro via ASGPr-mediated endocytosis (Fig. 20D) [168]. Subsequently, they utilized an endosomolytic peptide (ppTG21) to enhance the genome editing efficiency of the Cas9-ASGPrL RNP [169]. Apart from conjugation, complexing or encapsulating the RNP with polypeptides was also feasible. Kim Group prepared a Cas9 nanocomplex for genome editing in post-

of

mitotic neurons (Fig. 20E) [170]. The Cas9 nanocomplex was prepared by adding an

ro

amphiphilic R7L10 peptide (NH2-RRRRRRRRLLLLLLLLLL-COOH) to Cas9 RNP to rapidly form stable complexes via electrostatic interactions. They demonstrated that

-p

disrupting Bace1 in neurons via localized injection of Cas9 nanocomplex into hippocampus brain successfully suppressed amyloid beta (Aβ)-associated pathologies

re

in two mouse models of Alzheimer’s disease. Additionally, Shen et al. designed an

lP

amphipathic α-helical peptide called the Endo-Porter (EP) peptide (Fig. 20F) [171]. Then, Cas9 RNP was loaded by complexing with the EP peptide to form nanosize

na

complexes (CriPs). They demonstrated that the CriPs could induce genome editing in peritoneal exudate cells of mice. Montenegro and coauthors utilized an amphiphilic

Jo ur

penetrating peptide for encapsulating Cas9 RNP (Fig. 20G) [172]. The amphiphilic penetrating peptide was composed of a cationic peptide scaffold and a hydrophobic aldehyde tail. The cationic peptide scaffold could bind to Cas9 RNP via electrostatic interactions, and the hydrophobic aldehyde tail formed the outer layer so that Cas9 RNP was encapsulated in the nanostructure. Qiao et al. selected negatively charged RFP and a cationic biopolymer chitosan (CS) for Cas9 RNP delivery (Fig. 20H) [173]. They first coated RFP with preprotonated chitosan to prepare positively charged nanoparticles (RFP@CS). RFP@CS, negatively charged Cas9 RNP and ssDNA donors were then self-assembled into large nanoassemblies (Cas9 RNP-ssDNA-RFP@CS) via electrostatic interactions. They demonstrated that Cas9 RNP-ssDNA-RFP@CS could be efficiently endocytosed by multiple kinds of cells for genome editing. Jiang and colleagues constructed a DNAzyme-controlled CRISPR-Cas9 nanosystem (Fig. 20I) [174]. They first hybridized two ends of a Y-shaped DNA (Y-DNA) to DNAzyme and gRNA via

Journal Pre-proof Watson–Crick base pairing; then, the other end of Y-DNA (with a biotin end) was conjugated to streptavidin, and the Cas9 protein was loaded by binding to gRNA. Using the DNAzyme-controlled nanosystem, the Cas9 RNP could be responsively released by

Jo ur

na

lP

re

-p

ro

of

DNAzyme-mediated cleavage of DNA linkers in the presence of Mn2+.

Fig. 20. Polypeptides/proteins-based delivery systems of Cas9 RNP. (A) A selfassembled ternary complex of fused Cas9 protein with low molecular weight protamine (Cas9-LMWP) and crRNA:tracrRNA hybrid for disrupting KRAS in A549 tumor after intratumoral injection. Adapted with permission from [165]. Copyright (2018) American Chemical Society. (B) Delivering Cas9 RNP by fusing Cas9 with m9R peptide and mixing gRNA with 9R peptide. (C) Packaging Cas9 RNP into P22 viruslike particles (VLP) by E. Coli transfected with pMJ908 and pBAD plasmids. Adapted

Journal Pre-proof with permission from [167]. Copyright (2016) American Chemical Society. (D) Fusing Cas9 with asialoglycoprotein receptor ligands (ASGPrL) for hepatocyte-targeted delivery. Adapted with permission from [168]. Copyright (2018) American Chemical Society. (E) R7L10 peptide-based Cas9 nanocomplex for disrupting Bace1 in neurons after localized injection into hippocampus brain. Adapted with permission from [170]. Copyright (2019) Springer Nature. (F) EP peptide-based CriP complex for in vivo gene disruption after intraperitoneal injection. Adapted with permission from [171]. Copyright (2018) American Society for Biochemistry & Molecular Biology. (G) An amphiphilic penetrating peptide-based nanostructure for Cas9 RNP encapsulation.

of

Adapted with permission from [172]. Copyright (2017) Royal Society of Chemistry.

ro

(H) Utilizing chitosan-coated red fluorescent protein (RFP) to assemble with Cas9 RNP and ssDNA donors into Cas9 RNP-ssDNA-RFP@CS nanoassembly. Adapted with

-p

permission from [173]. Copyright (2019) Royal Society of Chemistry. (I) Loading Cas9 RNP onto a DNAzyme-controlled CRISPR-Cas9 nanosystem by a Y-shape DNA and

re

streptavidin. Adapted with permission from [174]. Copyright (2019) Royal Society of

Other materials-based delivery systems

na

3.3.7

lP

Chemistry.

MOFs have been developed for Cas9 RNP delivery. Khashab and coworkers loaded

Jo ur

Cas9 RNP into zeolitic imidazolate framework-8 (ZIF-8) via electrostatic interactions (Fig. 21A) [175]. In brief, the 2-methylimidazole (2-MIM) solution was first mixed with Cas9 RNP, and then a zinc nitrate solution was slowly added under agitation. They demonstrated that the Cas9 RNP was successfully encapsulated into ZIF-8 and can be quickly released from ZIF-8 under acidic conditions (pH 5 or 6). Subsequently, Yang et al. developed an ATP-responsive ZIF-90 as a general platform for cytosolic protein delivery (Fig. 21B) [176]. They used imidazole-2-carboxaldehyde (2-ICA) instead of 2-methylimidazole, and the protein was loaded by directly mixing with 2-ICA and Zn2+. They demonstrated that ZIF-90 could encapsulate diverse proteins, including GFP, RNase A-NBC and Cas9, and these proteins could be quickly released in the presence of ATP. Cheng and Ping collaboratively developed a boronic acid-rich dendrimer for cytosolic delivery of Cas9 RNP (Fig. 21C) [177]. They identified a boronic acid-rich dendrimer P4 that could form spherical nanoparticles with Cas9 RNP via electrostatic interactions.

Journal Pre-proof Although they tested only the genome editing efficiency in vitro, P4 could be modified with targeting ligands or other functional moieties for the in vivo delivery of Cas9 RNP. Harvey and colleagues utilized gesicles for Cas9 RNP delivery (Fig. 21D) [178]. They prepared Cas9 RNP-loaded gesicles by transfecting 293T cells with four plasmids expressing CherryPicker Red, vesicular stomatitis virus G (VSV-G), Cas9, and gRNA, in which VSV-G promoted gesicle formation and CherryPicker Red facilitates Cas9 RNP packaging into the gesicles via physical interactions of CherryPicker Red with the Cas9 protein. Lu and colleagues utilized a type of functionalized EVs, namely arrestin domain-containing protein 1 [ARRDC1]-mediated microvesicles (ARMMs),

of

for Cas9 RNP delivery (Fig. 21E) [179]. In brief, the Cas9 protein was first fused with

ro

WW domains of the ITCH protein. Then, the WW-Cas9 fusion protein and gRNA were incorporated into ARMMs via the interaction of ARRDC1 with the WW domains.

-p

They demonstrated that ARMMs could be used as a platform for delivering other

re

cargos, including the p53 protein and RNAs. Similarly, Chen et al. transfected HPVor HBV-specific CRISPR/Cas9 expression plasmids into HeLa cells and HuH7 cells,

lP

respectively (Fig. 21F) [180]. Cas9 RNP targeting HPV or HBV was packaged into naturally produced endogenous exosomes from HeLa cells or HuH7 cells.

na

Pan et al. designed a remote-activated CRISPR-Cas9 system using near-infrared (NIR) light-activatable upconversion nanoparticles (UCNPs) (Fig. 21G) [181]. They first

Jo ur

conjugated Cas9 RNP onto UCNPs by photocleavable 4-(hydroxymethyl)-3nitrobenzoic acid (ONA) molecules. PEI was then coated onto the surface to facilitate endosomal escape. The resulting UCNPs-Cas9@PEI could responsively release Cas9 RNP via NIR-mediated ONA cleavage. Intratumoral injection of UCNPs-Cas9@PEI targeting Plk1 could induce controllable knockout of the Plk1 gene for inhibiting A549 tumors in mice. In addition, two-dimensional black phosphorus nanosheets (BPs) were also demonstrated to be able to deliver Cas9 RNP. Since both BPs and Cas9 RNP were negatively charged, Zhou et al. engineered a Cas9 protein with three NLSs (Cas9N3) to render the Cas9N3 RNP positively charged (Fig. 21H) [182]. Then, Cas9N3 RNP was loaded onto the BPs via electrostatic interactions with a loading capacity of 98.7%. They demonstrated that intratumoral injection of Cas9N3-BPs was able to disrupt EGFP expression in A549/EGFP xenografted tumors.

na

lP

re

-p

ro

of

Journal Pre-proof

Fig. 21. Other materials-based delivery systems of Cas9 RNP. (A) Acid responsive ZIF-

Jo ur

8 for Cas9 RNP delivery. 2-MIM: 2-methylimidazole. (B) ATP-responsive ZIF-90 for Cas9 RNP encapsulating. 2-ICA: imidazole-2-carboxaldehyde. Adapted with permission from [176]. Copyright (2019) American Chemical Society. (C) Boronic acid-rich dendrimer P4-based spherical nanoparticle for Cas9 RNP delivery. Adapted with permission from [177]. Copyright (2019) American Association for the Advancement of Science. (D) CherryPicker Red-expressed gesicle for Cas9 RNP packaging. VSV-G: vesicular stomatitis virus G. (E) Loading WW-Cas9 into ARMM via the interaction between ARRDC1 and the WW domains of the ITCH protein. (F) Delivery of Cas9 RNP by naturally produced endogenous exosomes. (G) Responsively release of Cas9 RNP from upconversion nanoparticle (UCNP)-Cas9@PEI via nearinfrared (NIR)-mediated nitrobenzoic acid (ONA) cleavage. Adapted with permission from [181]. Copyright (2019) American Association for the Advancement of Science. (H) Delivery of Cas9N3 RNP by two-dimensional black phosphorus nanosheet (BP). Adapted with permission from [182]. Copyright (2018) John Wiley and Sons.

Journal Pre-proof 3.4 Delivery systems of other forms of CRISPR-Cas Other than three forms of CRIPSR-Cas mentioned above, CRISPR-Cas tools can be delivered in other modified or combined forms. For example, Cas proteins have been mutated or fused with other functional proteins to impart more kinds of gene manipulations. Liu and Leong have utilized their carriers for delivering these reengineered CRISPR-Cas tools in plasmid forms, which could be delivered by same carriers of pCas9 [75, 120]. In addition, Buchholz and coworkers generated a variety of Cas9 fusion proteins, including Cas9 fused to GFP and dCas9 fused to transcription domains (VP64-Rta) [183]. They demonstrated that these Cas9 fusion proteins could

of

also form RNPs with gRNAs, which can be transfected using Lipofectamine 2000.

ro

In addition, two groups delivered the combined CRISPR-Cas9 system of Cas9 protein

-p

and DNA. Wang et al. designed a nanocarrier that could deliver the Cas9 protein and a gRNA plasmid (Fig. 22A) [184]. They first modified gold nanoclusters (GNs) with TAT

re

peptides (TAT-GNs), and the Cas9 protein and the gRNA plasmid were attached on TAT-GNs to form complexes. The complexes were then encapsulated by the lipid

lP

mixture of DOTAP, DOPE, cholesterol and DSPE-PEG. The resulting nanocarriers were denoted as LGCPs. Their results proved that TAT peptides could facilitate Cas9

na

protein and gRNA plasmid entry into the nucleus, and intratumoral injection of LGCPs could inhibit A375 melanoma growth in mice by disrupting the Plk1 gene. Zhou and

Jo ur

colleagues constructed liposome-templated hydrogel nanoparticles (LHNPs) with a core-shell structure to encapsulate Cas9 protein and minicircle DNA (Fig. 22B) [185]. Briefly, the Cas9 protein was mixed with cyclodextrin (CD)-engrafted PEI (PEI-CD) and adamantine (AD)-engrafted PEI (PEI-AD) in aqueous solution, then DOTAP, cholesterol and DSPE-PEG-maleimide were resuspended in the aqueous solution to prepare LHNPs via the filming-rehydration method. Minicircle DNA was loaded by incubating with the LHNPs, and targeting ligands (mHph3 and iRGD) were conjugated on the surface of LHNPs. Utilizing this system, they effectively co-delivered the Cas9 protein and minicircle DNA to U87 tumor. Ahn and colleagues creatively constructed poly-gRNA/siRNA/Cas9 RNP nanoparticles by rolling circle transcription (RCT) (Fig. 22C) [186]. A circular DNA template composed of the complementary sequence of gRNA and siRNA was firstly constructed. Circular DNA was then used to produce continuous RNA composed of gRNA and

Journal Pre-proof siRNA sequences via RCT. In the meantime, Cas9 protein was added to bind the gRNA during RCT. The poly-gRNA/siRNA/Cas9 RNPs were self-assembled into nanoparticles due to the highly concentrated RNA strands, and could be directly used for transfection. When internalized by target cells, the siRNA sequence could be digested by Dicer, which turned poly-gRNA/siRNA/Cas9 RNP into monomic Cas9/gRNA RNP for genome editing. Anderson Group combined viral and nonviral carriers to deliver the components of CRISPR-Cas9 into liver (Fig. 22D) [187]. They prepared LNP encapsulating Cas9 mRNA (nano.Cas9 mRNA) with mixed lipids composed of C12-200, cholesterol, C14-

of

PEG, DOPE and arachidonic acid via the microfluidic method. AAV2/8 was used to

ro

carry the gRNA expression cassette and HDR template of the Fah gene (AAV-HDR). Intravenous injection of nano.Cas9 mRNA and AAV-HDR in tyrosinemia mice

-p

corrected a Fah mutation in more than 6% of hepatocytes, which rescued liver damage.

re

Zhu et al. designed a combined system of magnetic nanoparticles (MNPs) and recombinant baculoviral vectors (BVs) to deliver CRISPR-Cas9 (Fig. 22E) [188]. The

lP

MNPs were modified with cationic TAT peptides (MNP-TAT), and the MNP-TAT were bound to the BVs via electrostatic interactions. The resulting MNP-BVs could

na

efficiently enter target cells to express Cas9 and gRNA for genome editing in a magnetic

Jo ur

field after the local injection.

lP

re

-p

ro

of

Journal Pre-proof

Fig. 22. Delivery systems of other forms of CRISPR-Cas. (A) Simultaneous delivery of Cas9 protein and a gRNA plasmid with gold nanocluster (GN)-based LGCP

na

nanoparticle. Adapted with permission from [184]. Copyright (2017) John Wiley and Sons. (B) Delivery of Cas9 protein and a minicycle DNA with PEI-based liposome-

Jo ur

templated hydrogel nanoparticle (LHNP). Adapted with permission from [185]. Copyright (2017) John Wiley and Sons. (C) Preparation of poly-gRNA/siRNA/Cas9 RNP nanoparticles by rolling circle transcription (RCT). Adapted with permission from [186]. Copyright (2017) Elsevier. (D) Combining lipid nanoparticle (LNP)encapsulated Cas9 mRNA and Adeno-associated viruse (AAV)-expressed homologydirected repair (HDR) template/gRNA for correcting gene mutation in hepatocytes in vivo. (E) Utilizing magnetic nanoparticle (MNP)-baculoviral vectors (BV) for magnetic field-controlled genome editing.

4.

Rational design of CRISPR-Cas delivery systems for clinical use

Although CRISPR-Cas-mediated genome editing can arbitrarily manipulate all genes in human cells, the top priority of genome editing in the clinic is to treat genetic diseases, including SCID, β-thalassemia, sickle cell anemia, hemophilia, key enzyme

Journal Pre-proof deficiencies and cancers, which usually lack safe and effective therapeutics. According to the database of Online Mendelian Inheritance in Man (OMIM), mutations in over 3,000 genes have been reported to cause diseases [189]. However, only a small number of genetic diseases can be treated by traditional drugs. The versatility of CRISPR-Casinduced NHEJ, HDR and base editing enables us to potentially treat all genetic diseases by disrupting or correcting deleterious mutations, inserting therapeutic transgenes or introducing protective mutations. For example, a gain-of-function mutation may cause overexpression of the pathogenic product, which can be treated by inactivating the mutant gene via NHEJ to terminate pathogenic overexpression or to correct the

of

mutation via HDR to restore the physiological level of gene expression. A loss-of-

ro

function point mutation may cause a deficiency of the functional protein, and it can be precisely repaired via HDR or base editing. A large loss-of-function mutation can be

-p

treated by inserting a copy of the therapeutic transgene into the mutated region or “safe harbor” locus. Some people are susceptible to infections or genetic diseases; in such

re

cases, protective mutations can be introduced for prevention, such as an NHEJmediated mutation of the CCR5 gene to prevent an HIV infection or an HDR-mediated

lP

change in the APP gene (A673T) to prevent Alzheimer's disease [190, 191].

na

4.1 The clinical requirements of in vivo genome editing

Jo ur

For clinical applications of CRISPR-Cas-mediated genome editing, both efficacy and safety issues should be carefully taken into account. Appropriate strategies should be designed for each disease. Generally, therapeutic genome editing in the clinic can be divided into ex vivo and in vivo approaches [192]. In ex vivo editing approaches, target cells are isolated from patients and modified with CRISPR-Cas. The edited cells are transplanted back into patients. However, the target cells of ex vivo editing are usually restricted to stem cells and immune cells, such as hematopoietic stem cells, T cells and NK cells, which can be cultured and transplanted into the patient. Thus, ex vivo editing approaches are widely tested for SCID, βthalassemia, sickle cell anemia and CAR-T therapy in the clinic. The majority of genetic diseases are caused by target cells that can only be modified by in vivo editing. In in vivo editing approaches, CRISPR-Cas need to be directly delivered into target cells in the body and modify the genome in situ, which requires no isolation

Journal Pre-proof of target cells and allows for the simultaneous modification of target cells in multiple tissues. Nevertheless, there are more concerns to be addressed in this scenario. First, only target cells should be modified after delivering CRISPR-Cas tools in vivo. Editing of normal tissues and cells may cause damage to their physiological functions. Unavoidable editing of untargeted cells that cause no serious harm and safety risks may be acceptable, however, editing the germ cells causes ethical problems and is forbidden [193]. Second, the off-target effects at the genomic level should be carefully evaluated and minimized to the best [194]. Because genetic modifications are permanent, offtarget editing may cause tumorigenesis or malfunction of target cells. Third, the

of

efficiency of genome editing should be sufficiently high to achieve therapeutic effects.

ro

Although the requirements of genome editing efficiency for different diseases vary, higher efficiency generally improves the therapeutic outcomes. Fourth, an appropriate

-p

CRISPR-Cas tool should be selected to reduce changes in genomes. Typically, one disease can be treated by multiple genome editing strategies, and it is necessary to

re

choose the most conservative genome editing strategy to make gentle modifications to the genome. For instance, knocking out a gene by introducing a stop codon by base

lP

editing is safer than NHEJ-induced random indels [57]. Fifth, the carriers for in vivo delivery of CRISPR-Cas should be biocompatible, protect CRISPR-Cas from enzymes

na

and immune systems in physiological environments, and efficiently deliver CRISPRCas into target cells. Therefore, proper selection of CRISPR-Cas tools and rational

Jo ur

design of delivery systems are both important for balancing the benefits and risks of in vivo genome editing.

4.2 Selection of CRISPR-Cas tools Because this review focuses on the delivery systems of CRISPR-Cas, we only discuss some major aspects in the selection of CRISPR-Cas tools. For diseases that can be treated by activating or repressing gene expression, CRISPRa or CRISPRi, which can regulate gene expression without introducing changes to the genome, is the most suitable tool for clinical use. Next, base editors that introduce no DSBs into the genome are also preferable choices for diseases that can be treated by point mutations. In addition, CRISPR-Cas nickases that cut only single strands of genomic DNA offer higher specificity than CRISPR-Cas9 nucleases in HDR-mediated genome repair and can potentially decrease the frequency of unwanted indel mutations [4]. The native

Journal Pre-proof CRISPR-Cas9 and other nucleases that induce DSBs should be the last option because they are associated with high frequency of unwanted mutations. Furthermore, the form of CRISPR-Cas tools should also be carefully chosen. Because genetic modifications are permanent and the long-term existence of Cas nuclease in the target cells can increase the off-target effects, it is better to directly deliver CRISPRCas in the form of RNP, whose dosage can be precisely controlled. Delivery of Cas mRNA to transiently translate into Cas nuclease is also a preferable choice for in vivo genome editing. Because the CRISPR-Cas plasmid can express Cas nuclease for a long time and can potentially integrate into the genome, the plasmid form of CRISPR-Cas

of

is not a good choice for in vivo clinical genome editing. However, plasmids can be

ro

equipped with tissue or cell-specific promoters to drive site-specific expression of Cas nuclease and gRNA. In addition, drug, light- or other-responsive promoters can also be

re

-p

constructed into plasmids to control the expression of Cas nuclease and gRNA.

4.3 Rational design of in vivo delivery systems

lP

The precondition of applying in vivo editing for clinical therapy is the rational design of delivery systems that can deliver CRISPR-Cas into target cells while causing the

na

least safety risks. Although viral vectors are still the main delivery systems of in vivo genome editing, their tissue-specific tropism, immunogenicity and tumorigenic risks

Jo ur

limit their clinical applications [195, 196]. Hence, the development of nonviral carriers to deliver CRISPR-Cas into diverse target cells is the general trend of clinical in vivo uses.

First, in vivo delivery systems for the clinic are expected to be biocompatible with low toxicity and low immunogenicity. For example, PLA or PLGA has been widely used in many FDA-approved drug products [197]. Using these materials, if appropriate, for in vivo delivery of CRISPR-Cas can facilitate the translation process in clinics. Second, personalized delivery systems are ideal for efficiently packaging and protecting plasmid, RNA, RNP or other combined forms of CRISPR-Cas according to their features. The integrity and biological activity of plasmid, RNA and RNP need to be maintained after packaging. The delivery systems are encouraging to shield CRISPRCas from nucleases, proteases, and immune cells. For example, encapsulation of CRISPR-Cas inside the delivery systems and modifying a PEG layer on the surface of

Journal Pre-proof delivery systems have been proven to be effective by previous studies. Third, delivery systems of each form of CRISPR-Cas should be able to transport and release them into target intracellular sites to exert their proper function. For example, the delivery systems of RNP-based CRISPR-Cas should facilitate endosomal escape and localize into nuclei for genome editing. The delivery systems of RNA-based CRISPR-Cas should release Cas mRNA into the cytoplasm for translation. Plasmidbased CRISPR-Cas delivery systems should be able to escape from endosomes and translocate into the nucleus for transcription. As mentioned above, cationic polypeptides, including CPPs and NLS, can be used to improve endosomal escape and

of

nuclear entrance.

ro

Fourth, site-specific delivery of CRISPR-Cas would greatly enhance the safety of

-p

therapeutic genome editing. Apart from equipping pCas9 with tissue or cell-specific promoters for site-specific expression of Cas nuclease and gRNA, stimuli-responsive

lP

RNA- and RNP-based CRISPR-Cas.

re

delivery systems can also be engineered to realize precise genome editing of plasmid-,

Furthermore, specific delivery routes should be selected based on different diseases, while the systemic delivery with targeting ligands is always a direction. Alternatively,

na

the local administration could be highly appropriate for treating diseases in muscle and

5.

Jo ur

eye tissues.

Conclusions

CRISPR-Cas-based genome editing has shown enormous potential in treating diverse genetic diseases, including cancers, β-thalassaemia and tyrosinemia [198, 199]. In addition, many CRISPR-Cas-based tools have been created for regulating gene expression, engineering posttranscription, and imaging nucleic acids [3]. Therefore, tremendous momentums are placed on CRISPR-Cas for the development of clinical therapeutics today [13]. Although β-haemoglobinopathies have been shown to be treated by transplantation of hematopoietic stem cells whose mutated HBB gene have been corrected ex vivo [198], and PD-1-deficient T cells have been tested for the treatment of metastatic non-small cell lung cancer in clinical trials [200], many other diseases can only be treated by editing the genomes of the target cells in their native tissues. Thus, developing efficient and safe delivery systems to transport CRISPR-Cas

Journal Pre-proof into target cells in vivo remains the major challenge of their clinical use [1, 18]. From the perspective of delivery, CRISPR-Cas can be divided into plasmids, mRNA/ gRNA or RNP, which are biomacromolecules with large sizes. Many kinds of nonviral delivery systems have been demonstrated to be effective in introducing CRISPR-Cas into target cells ex vivo and in vivo. As mentioned above, the materials in these delivery systems are mainly cationic lipids, cationic polymers, cationic polypeptides, DNA nanostructures, AuNPs, cell-derived vesicles and other organic materials. Delivery systems that are not stable in physiological environments, for example

of

Lipofectamine 2000 or PEI, can be used for ex vivo genome editing. Apart from exosomes, which are naturally stable in physiological environments, other in vivo

ro

delivery systems are usually modified with a surface layer of PEG to prevent the

-p

adsorption of serum proteins. For example, CLAN nanoparticles and the LNPs were both PEGylated, and they could deliver plasmids or RNA-based CRISPR-Cas into

re

diverse immune cells or hepatocytes after intravenous injection. Because many nontarget cells may also endocytose these systemically injected nanoparticles [201], the

lP

in vivo fate of these nanoparticles should be carefully studied, and the safety risks of inducing genome editing in normal cells, such as hematopoietic stem cells and germ

na

cells, should be evaluated [202, 203].

There are also some delivery systems that may not be suitable for systemic injection,

Jo ur

which could be locally injected into the tumor, muscle or brain to induce local genome editing. For example, DNA nanoclews could deliver the CRISPR-Cas RNP to edit tumor genes after intratumoral injection [154]. AuNPs could deliver CRISPR-Cas RNPs and donor DNA to repair the mutated dystrophin gene after intramuscular injection [90]. Although the target cells of local genome editing are limited, the safety risks of this strategy are relatively low. Given that both the pathogenesis of each disease and the in vivo delivery barriers of each target cell are quite different, it is not suggested to develop versatile delivery systems that could deliver all three forms of CRISPR-Cas into multiple target cells. One delivery system may not be optimal for all cells and tissues. In conclusion, the delivery strategies should be cautiously selected and the delivery systems should be optimized for the clinic according to the features of each CRISPR-Cas tool.

Journal Pre-proof Acknowledgments This work was supported by the grants from the start-up packages of UCLA, National Key R&D Program of China (2017YFA0205600), the National Natural Science Foundation of China (81801825, 81901875, 81971723 and 51633008), the Program for Guangdong Introducing Innovative and Entrepreneurial Teams (2017ZT07S054), Guangdong Provincial Pearl River Talents Program (2017GC010713), Outstanding Scholar Program of Guangzhou Regenerative Medicine and Health Guangdong Laboratory (2018GZR110102001), and the Fundamental Research Funds for the

ro

of

Central Universities.

-p

Additional information

re

References

Jo ur

na

lP

[1] G.J. Knott, J.A. Doudna, CRISPR-Cas guides the future of genetic engineering, Science 361 (2018) 866-869. [2] L. Cong, F.A. Ran, D. Cox, S.L. Lin, R. Barretto, N. Habib, P.D. Hsu, X.B. Wu, W.Y. Jiang, L.A. Marraffini, F. Zhang, Multiplex genome engineering using CRISPR/Cas systems, Science 339 (2013) 819-823. [3] M. Adli, The CRISPR tool kit for genome editing and beyond, Nat. Commun. 9 (2018) 1911. [4] F.A. Ran, P.D. Hsu, J. Wright, V. Agarwala, D.A. Scott, F. Zhang, Genome engineering using the CRISPR-Cas9 system, Nat. Protoc. 8 (2013) 2281-2308. [5] P.D. Hsu, E.S. Lander, F. Zhang, Development and applications of CRISPR-Cas9 for genome engineering, Cell 157 (2014) 1262-1278. [6] J.H. Hu, S.M. Miller, M.H. Geurts, W. Tang, L. Chen, N. Sun, C.M. Zeina, X. Gao, H.A. Rees, Z. Lin, D.R. Liu, Evolved Cas9 variants with broad PAM compatibility and high DNA specificity, Nature 556 (2018) 57-63. [7] O. Shalem, N.E. Sanjana, F. Zhang, High-throughput functional genomics using CRISPR-Cas9, Nat. Rev. Genet. 16 (2015) 299-311. [8] C.E. Nelson, Y. Wu, M.P. Gemberling, M.L. Oliver, M.A. Waller, J.D. Bohning, J.N. Robinson-Hamm, K. Bulaklak, R.M. Castellanos Rivera, J.H. Collier, A. Asokan, C.A. Gersbach, Long-term evaluation of AAV-CRISPR genome editing for duchenne muscular dystrophy, Nat. Med. 25 (2019) 427-432. [9] Y. Wu, J. Zeng, B.P. Roscoe, P. Liu, Q. Yao, C.R. Lazzarotto, K. Clement, M.A. Cole, K. Luk, C. Baricordi, A.H. Shen, C. Ren, E.B. Esrick, J.P. Manis, D.M. Dorfman, D.A. Williams, A. Biffi, C. Brugnara, L. Biasco, C. Brendel, L. Pinello, S.Q. Tsai, S.A. Wolfe, D.E. Bauer, Highly efficient therapeutic gene editing of human hematopoietic

Journal Pre-proof

Jo ur

na

lP

re

-p

ro

of

stem cells, Nat. Med. 25 (2019) 776-783. [10] M. Pavel-Dinu, V. Wiebking, B.T. Dejene, W. Srifa, S. Mantri, C.E. Nicolas, C. Lee, G. Bao, E.J. Kildebeck, N. Punjya, C. Sindhu, M.A. Inlay, N. Saxena, S.S. DeRavin, H. Malech, M.G. Roncarolo, K.I. Weinberg, M.H. Porteus, Gene correction for SCID-X1 in long-term hematopoietic stem cells, Nat. Commun. 10 (2019) 2021. [11] H. de Buhr, R.J. Lebbink, Harnessing CRISPR to combat human viral infections, Curr. Opin. Immunol. 54 (2018) 123-129. [12] D.T. Ewart, E.J. Peterson, C.J. Steer, Gene editing for inflammatory disorders, Ann. Rheum. Dis. 78 (2019) 6-15. [13] H. Yin, W. Xue, D.G. Anderson, CRISPR-Cas: a tool for cancer research and therapeutics, Nat. Rev. Clin. Oncol. 16 (2019) 281-295. [14] Q. Gao, X. Dong, Q. Xu, L. Zhu, F. Wang, Y. Hou, C.C. Chao, Therapeutic potential of CRISPR/Cas9 gene editing in engineered T-cell therapy, Cancer Med. 8 (2019) 4254-4264. [15] L.S. Qi, M.H. Larson, L.A. Gilbert, J.A. Doudna, J.S. Weissman, A.P. Arkin, W.A. Lim, Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression, Cell 152 (2013) 1173-1183. [16] J. Tan, F. Zhang, D. Karcher, R. Bock, Engineering of high-precision base editors for site-specific single nucleotide replacement, Nat. Commun. 10 (2019) 439. [17] X. Wu, S. Mao, Y. Ying, C.J. Krueger, A.K. Chen, Progress and challenges for live-cell imaging of genomic loci using CRISPR-based platforms, Genomics, Proteomics Bioinf. 17 (2019) 119-128. [18] D. Wilbie, J. Walther, E. Mastrobattista, Delivery aspects of CRISPR/Cas for in vivo genome editing, Acc. Chem. Res. 52 (2019) 1555-1564. [19] T. Wan, D. Niu, C.B. Wu, F.J. Xu, G. Church, Y. Ping, Material solutions for delivery of CRISPR/Cas-based genome editing tools: Current status and future outlook, Mater. Today 26 (2019) 40-66. [20] J. Eoh, L. Gu, Biomaterials as vectors for the delivery of CRISPR-Cas9, Biomater. Sci. 7 (2019) 1240-1261. [21] L. Li, S. Hu, X.Y. Chen, Non-viral delivery systems for CRISPR/Cas9-based genome editing: challenges and opportunities, Biomaterials 171 (2018) 207-218. [22] H.X. Wang, M. Li, C.M. Lee, S. Chakraborty, H.W. Kim, G. Bao, K.W. Leong, CRISPR/Cas9-based genome editing for disease modeling and therapy: challenges and opportunities for nonviral delivery, Chem. Rev. 117 (2017) 9874-9906. [23] B.E. Givens, Y.W. Naguib, S.M. Geary, E.J. Devor, A.K. Salem, Nanoparticlebased delivery of CRISPR/Cas9 genome-editing therapeutics, AAPS J. 20 (2018) 108. [24] A.S. Piotrowski-Daspit, P.M. Glaze, W.M. Saltzman, Debugging the genetic code: non-viral in vivo delivery of therapeutic genome editing technologies, Curr. Opin. Biomed. Eng. 7 (2018) 24-32. [25] R. Mout, M. Ray, Y.W. Lee, F. Scaletti, V.M. Rotello, In vivo delivery of CRISPR/Cas9 for therapeutic gene editing: progress and challenges, Bioconjugate Chem. 28 (2017) 880-884. [26] L. Wang, W.F. Zheng, S.Q. Liu, B. Li, X.Y. Jiang, Delivery of CRISPR/Cas9 by novel strategies for gene therapy, Chembiochem 20 (2019) 634-643.

Journal Pre-proof

Jo ur

na

lP

re

-p

ro

of

[27] H. Yin, K.J. Kauffman, D.G. Anderson, Delivery technologies for genome editing, Nat. Rev. Drug Discovery 16 (2017) 387-399. [28] Q. Ul Ain, J.Y. Chung, Y.H. Kim, Current and future delivery systems for engineered nucleases: ZFN, TALEN and RGEN, J. Controlled Release 205 (2015) 120127. [29] J.S. LaFountaine, K. Fathe, H.D. Smyth, Delivery and therapeutic applications of gene editing technologies ZFNs, TALENs, and CRISPR/Cas9, Int. J. Pharm. 494 (2015) 180-194. [30] C.A. Lino, J.C. Harper, J.P. Carney, J.A. Timlin, Delivering CRISPR: a review of the challenges and approaches, Drug delivery 25 (2018) 1234-1257. [31] Y. Ishino, M. Krupovic, P. Forterre, History of CRISPR-Cas from encounter with a mysterious repeated sequence to genome editing technology, J. Bacteriol. 200 (2018) e00580-17. [32]Y. Ishino, H. Shinagawa, K. Makino, M. Amemura, A. Nakata, Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product, J. Bacteriol. 169 (1987) 54295433. [33] F.J. Mojica, C. Diez-Villasenor, E. Soria, G. Juez, Biological significance of a family of regularly spaced repeats in the genomes of archaea, bacteria and mitochondria, Mol. Microbiol., 36 (2000) 244-246. [34] F.J.M. Mojica, C. Diez-Villasenor, J. Garcia-Martinez, E. Soria, Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements, J. Mol. Evol. 60 (2005) 174-182. [35] C. Pourcel, G. Salvignol, G. Vergnaud, CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies, Microbiology 151 (2005) 653-663. [36] A. Bolotin, B. Ouinquis, A. Sorokin, S.D. Ehrlich, Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin, Microbiology 151 (2005) 2551-2561. [37] K.S. Makarova, L. Aravind, N.V. Grishin, I.B. Rogozin, E.V. Koonin, A DNA repair system specific for thermophilic archaea and bacteria predicted by genomic context analysis, Nucleic Acids Res. 30 (2002) 482-496. [38] R. Jansen, J.D.A. van Embden, W. Gaastra, L.M. Schouls, Identification of genes that are associated with DNA repeats in prokaryotes, Mol. Microbiol. 43 (2002) 15651575. [39] R. Barrangou, C. Fremaux, H. Deveau, M. Richards, P. Boyaval, S. Moineau, D.A. Romero, P. Horvath, CRISPR provides acquired resistance against viruses in prokaryotes, Science 315 (2007) 1709-1712. [40] S.J. Brouns, M.M. Jore, M. Lundgren, E.R. Westra, R.J. Slijkhuis, A.P. Snijders, M.J. Dickman, K.S. Makarova, E.V. Koonin, J. van der Oost, Small CRISPR RNAs guide antiviral defense in prokaryotes, Science 321 (2008) 960-964. [41] L.A. Marraffini, E.J. Sontheimer, CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA, Science 322 (2008) 1843-1845. [42] J.E. Garneau, M.E. Dupuis, M. Villion, D.A. Romero, R. Barrangou, P. Boyaval,

Journal Pre-proof

Jo ur

na

lP

re

-p

ro

of

C. Fremaux, P. Horvath, A.H. Magadan, S. Moineau, The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA, Nature 468 (2010) 67-71. [43] M. Jinek, K. Chylinski, I. Fonfara, M. Hauer, J.A. Doudna, E. Charpentier, A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity, Science 337 (2012) 816-821. [44] P. Mali, L.H. Yang, K.M. Esvelt, J. Aach, M. Guell, J.E. DiCarlo, J.E. Norville, G.M. Church, RNA-guided human genome engineering via Cas9, Science 339 (2013) 823-826. [45] J.C. Grieger, R.J. Samulski, Packaging capacity of adeno-associated virus serotypes: impact of larger genomes on infectivity and postentry steps, J. Virol. 79 (2005) 9933-9944. [46] Z. Wu, H. Yang, P. Colosi, Effect of genome size on AAV vector packaging, Mol. Ther. 18 (2010) 80-86. [47] E. Kim, T. Koo, S.W. Park, D. Kim, K. Kim, H.Y. Cho, D.W. Song, K.J. Lee, M.H. Jung, S. Kim, J.H. Kim, J.H. Kim, J.S. Kim, In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni, Nat. Commun. 8 (2017) 14500. [48] 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, F. Zhang, RNA targeting with CRISPR-Cas13, Nature 550 (2017) 280-284. [49] L.S. Qi, M.H. Larson, L.A. Gilbert, J.A. Doudna, J.S. Weissman, A.P. Arkin, W.A. Lim, Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression, Cell 152 (2013) 1173-1183. [50] L.A. Gilbert, M.H. Larson, L. Morsut, Z.R. Liu, G.A. Brar, S.E. Torres, N. SternGinossar, O. Brandman, E.H. Whitehead, J.A. Doudna, W.A. Lim, J.S. Weissman, L.S. Qi, CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes, Cell 154 (2013) 442-451. [51] M.L. Maeder, S.J. Linder, V.M. Cascio, Y.F. Fu, Q.H. Ho, J.K. Joung, CRISPR RNA-guided activation of endogenous human genes, Nat. Methods 10 (2013) 977-979. [52] P. Perez-Pinera, D.D. Kocak, C.M. Vockley, A.F. Adler, A.M. Kabadi, L.R. Polstein, P.I. Thakore, K.A. Glass, D.G. Ousterout, K.W. Leong, F. Guilak, G.E. Crawford, T.E. Reddy, C.A. Gersbach, RNA-guided gene activation by CRISPR-Cas9based transcription factors, Nat. Methods 10 (2013) 973-976. [53] A. Chavez, J. Scheiman, S. Vora, B.W. Pruitt, M. Tuttle, E.P.R. Iyer, S.L. Lin, S. Kiani, C.D. Guzman, D.J. Wiegand, D. Ter-Ovanesyan, J.L. Braff, N. Davidsohn, B.E. Housden, N. Perrimon, R. Weiss, J. Aach, J.J. Collins, G.M. Church, Highly efficient Cas9-mediated transcriptional programming, Nat. Methods 12 (2015) 326-U365. [54] M.E. Tanenbaum, L.A. Gilbert, L.S. Qi, J.S. Weissman, R.D. Vale, A proteintagging system for signal amplification in gene expression and fluorescence imaging, Cell 159 (2014) 635-646. [55] J.G. Zalatan, M.E. Lee, R. Almeida, L.A. Gilbert, E.H. Whitehead, M. La Russa, J.C. Tsai, J.S. Weissman, J.E. Dueber, L.S. Qi, W.A. Lim, Engineering complex synthetic transcriptional programs with CRISPR RNA scaffolds, Cell 160 (2015) 339350. [56] S. Konermann, M.D. Brigham, A.E. Trevino, J. Joung, O.O. Abudayyeh, C.

Journal Pre-proof

Jo ur

na

lP

re

-p

ro

of

Barcena, P.D. Hsu, N. Habib, J.S. Gootenberg, H. Nishimasu, O. Nureki, F. Zhang, Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex, Nature 517 (2015) 583-U332. [57] A.C. Komor, Y.B. Kim, M.S. Packer, J.A. Zuris, D.R. Liu, Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage, Nature 533 (2016) 420-424. [58] N.M. Gaudelli, A.C. Komor, H.A. Rees, M.S. Packer, A.H. Badran, D.I. Bryson, D.R. Liu, Programmable base editing of A.T to G.C in genomic DNA without DNA cleavage, Nature 551 (2017) 464-471. [59] C. Kuscu, M. Parlak, T. Tufan, J.K. Yang, K. Szlachta, X.L. Wei, R. Mammadov, M. Adli, CRISPR-STOP: gene silencing through base-editing-induced nonsense mutations, Nat. Methods 14 (2017) 710-712. [60] P. Billon, E.E. Bryant, S.A. Joseph, T.S. Nambiar, S.B. Hayward, R. Rothstein, A. Ciccia, CRISPR-mediated base editing enables efficient disruption of eukaryotic genes through induction of STOP codons, Mol. Cell 67 (2017) 1068-1079. [61] C. Kuscu, M. Adli, CRISPR-Cas9-AID base editor is a powerful gain-of-function screening tool, Nat. Methods 13 (2016) 983-984. [62] Y. Lei, X.T. Zhang, J.Z. Su, M. Jeong, M.C. Gundry, Y.H. Huang, Y.B. Zhou, W. Li, M.A. Goodell, Targeted DNA methylation in vivo using an engineered dCas9-MQ1 fusion protein, Nat. Commun. 8 (2017) 16026. [63] S. Morita, H. Noguchi, T. Horii, K. Nakabayashi, M. Kimura, K. Okamura, A. Sakai, H. Nakashitna, K. Hata, K. Nakashima, I. Hatada, Targeted DNA demethylation in vivo using dCas9-peptide repeat and scFv-TET1 catalytic domain fusions, Nat. Biotechnol. 34 (2016) 1060-1065. [64] N.A. Kearns, H. Pham, B. Tabak, R.M. Genga, N.J. Silverstein, M. Garber, R. Maehr, Functional annotation of native enhancers with a Cas9-histone demethylase fusion, Nat. Methods 12 (2015) 401-403. [65] D.Y. Kwon, Y.T. Zhao, J.M. Lamonica, Z. Zhou, Locus-specific histone deacetylation using a synthetic CRISPR-Cas9-based HDAC, Nat. Commun. 8 (2017) 15315. [66] F.S. Liang, W.Q. Ho, G.R. Crabtree, Engineering the ABA plant stress pathway for regulation of induced proximity, Sci. Signaling 4 (2011) rs2. [67] P.W. Qin, M. Parlak, C. Kuscu, J. Bandaria, M. Mir, K. Szlachta, R. Singh, X. Darzacq, A. Yildiz, M. Adli, Live cell imaging of low- and non-repetitive chromosome loci using CRISPR-Cas9, Nat. Commun. 8 (2017) 14725. [68] M. Tsoi, T.T. Do, V. Tang, J.A. Aguilera, C.C. Perry, J.R. Milligan, Characterization of condensed plasmid DNA models for studying the direct effect of ionizing radiation, Biophys. Chem. 147 (2010) 104-110. [69] J. Fritz, E.B. Cooper, S. Gaudet, P.K. Sorger, S.R. Manalis, Electronic detection of DNA by its intrinsic molecular charge, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 1414214146. [70] Y.L. Luo, C.F. Xu, H.J. Li, Z.T. Cao, J. Liu, J.L. Wang, X.J. Du, X.Z. Yang, Z. Gu, J. Wang, Macrophage-specific in vivo gene editing using cationic lipid-assisted polymeric nanoparticles, ACS Nano 12 (2018) 994-1005.

Journal Pre-proof

Jo ur

na

lP

re

-p

ro

of

[71] H. Hemmi, O. Takeuchi, T. Kawai, T. Kaisho, S. Sato, H. Sanjo, M. Matsumoto, K. Hoshino, H. Wagner, K. Takeda, S. Akira, A toll-like receptor recognizes bacterial DNA, Nature 408 (2000) 740-745. [72] C.J. Smoyer, S.L. Jaspersen, Breaking down the wall: the nuclear envelope during mitosis, Curr. Opin. Cell Biol., 26 (2014) 1-9. [73] L.J. Branden, A.J. Mohamed, C.I.E. Smith, A peptide nucleic acid-nuclear localization signal fusion that mediates nuclear transport of DNA, Nat. Biotechnol. 17 (1999) 784-787. [74] D.M. Copolovici, K. Langel, E. Eriste, U. Langel, Cell-penetrating peptides: design, synthesis, and applications, ACS Nano 8 (2014) 1972-1994. [75] H.X. Wang, Z. Song, Y.H. Lao, X. Xu, J. Gong, D. Cheng, S. Chakraborty, J.S. Park, M. Li, D. Huang, L. Yin, J. Cheng, K.W. Leong, Nonviral gene editing via CRISPR/Cas9 delivery by membrane-disruptive and endosomolytic helical polypeptide, Proc. Natl. Acad. Sci. U. S. A. 115 (2018) 4903-4908. [76] U. Sahin, K. Kariko, O. Tureci, mRNA-based therapeutics--developing a new class of drugs, Nat. Rev. Drug Discovery 13 (2014) 759-780. [77] B. Wu, C. Eliscovich, Y.J. Yoon, R.H. Singer, Translation dynamics of single mRNAs in live cells and neurons, Science 352 (2016) 1430-1435. [78] S. Vaidyanathan, K.T. Azizian, A.K.M.A. Haque, J.M. Henderson, A. Hendel, S. Shore, J.S. Antony, R.I. Hogrefe, M.S.D. Kormann, M.H. Porteus, A.P. McCaffrey, Uridine depletion and chemical modification increase Cas9 mRNA activity and reduce immunogenicity without HPLC purification, Mol. Ther. 12 (2018) 530-542. [79] B.R. Anderson, H. Muramatsu, B.K. Jha, R.H. Silverman, D. Weissman, K. Kariko, Nucleoside modifications in RNA limit activation of 2'-5'-oligoadenylate synthetase and increase resistance to cleavage by RNase L, Nucleic Acids Res. 39 (2011) 93299338. [80] R.O. Bak, A. Hendel, J.T. Clark, A.B. Kennedy, D.E. Ryan, S. Roy, I. Steinfeld, B.D. Lunstad, R.J. Kaiser, A.B. Wilkens, R. Bacchetta, A. Tsalenko, D. Dellinger, L. Bruhn, M.H. Porteus, Chemically modified guide RNAs enhance CRISPR/Cas genome editing in human primary cells, Hum. Gene Ther. 26 (2015) A11-A12. [81] M.A. DeWitt, J.E. Corn, D. Carroll, Genome editing via delivery of Cas9 ribonucleoprotein, Methods 121-122 (2017) 9-15. [82] J.A. Gagnon, E. Valen, S.B. Thyme, P. Huang, L. Akhmetova, A. Pauli, T.G. Montague, S. Zimmerman, C. Richter, A.F. Schier, Efficient mutagenesis by Cas9 protein-mediated oligonucleotide insertion and large-scale assessment of single-guide RNAs, PLoS One 9 (2014) e98186. [83] S. Kim, D. Kim, S.W. Cho, J. Kim, J.S. Kim, Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins, Genome Res. 24 (2014) 1012-1019. [84] H. Nishimasu, F.A. Ran, P.D. Hsu, S. Konermann, S.I. Shehata, N. Dohmae, R. Ishitani, F. Zhang, O. Nureki, Crystal structure of Cas9 in complex with guide RNA and target DNA, Cell 156 (2014) 935-949. [85] G. Palermo, J.S. Chen, C.G. Ricci, I. Rivalta, M. Jinek, V.S. Batista, J.A. Doudna, J.A. McCammon, Key role of the REC lobe during CRISPR-Cas9 activation by

Journal Pre-proof

Jo ur

na

lP

re

-p

ro

of

'sensing', 'regulating', and 'locking' the catalytic HNH domain, Q. Rev. Biophys. 51 (2018) e91. [86] A.V. Wright, S.H. Sternberg, D.W. Taylor, B.T. Staahl, J.A. Bardales, J.E. Kornfeld, J.A. Doudna, Rational design of a split-Cas9 enzyme complex, Proc. Natl. Acad. Sci. U. S. A. 112 (2015) 2984-2989. [87] F. Jiang, J.A. Doudna, CRISPR-Cas9 structures and mechanisms, Annu. Rev. Biophys. 46 (2017) 505-529. [88] J.A. Zuris, D.B. Thompson, Y. Shu, J.P. Guilinger, J.L. Bessen, J.H. Hu, M.L. Maeder, J.K. Joung, Z.Y. Chen, D.R. Liu, Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo, Nat. Biotechnol. 33 (2015) 73-80. [89] M. Hansen-Bruhn, B.E. de Avila, M. Beltran-Gastelum, J. Zhao, D.E. RamirezHerrera, P. Angsantikul, K. Vesterager Gothelf, L. Zhang, J. Wang, Active intracellular delivery of a Cas9/sgRNA complex using ultrasound-propelled nanomotors, Angew. Chem. Int. Ed. 57 (2018) 2657-2661. [90] K. Lee, M. Conboy, H.M. Park, F. Jiang, H.J. Kim, M.A. Dewitt, V.A. Mackley, K. Chang, A. Rao, C. Skinner, T. Shobha, M. Mehdipour, H. Liu, W.C. Huang, F. Lan, N.L. Bray, S. Li, J.E. Corn, K. Kataoka, J.A. Doudna, I. Conboy, N. Murthy, Nanoparticle delivery of Cas9 ribonucleoprotein and donor DNA in vivo induces homology-directed DNA repair, Nat. Biomed. Eng. 1 (2017) 889-901. [91] C.T. Charlesworth, P.S. Deshpande, D.P. Dever, J. Camarena, V.T. Lemgart, M.K. Cromer, C.A. Vakulskas, M.A. Collingwood, L. Zhang, N.M. Bode, M.A. Behlke, B. Dejene, B. Cieniewicz, R. Romano, B.J. Lesch, N. Gomez-Ospina, S. Mantri, M. PavelDinu, K.I. Weinberg, M.H. Porteus, Identification of preexisting adaptive immunity to Cas9 proteins in humans, Nat. Med. 25 (2019) 249-254. [92] J.M. Crudele, J.S. Chamberlain, Cas9 immunity creates challenges for CRISPR gene editing therapies, Nat. Commun. 9 (2018) 3497. [93] T. Maruyama, S.K. Dougan, M.C. Truttmann, A.M. Bilate, J.R. Ingram, H.L. Ploegh, Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining, Nat. Biotechnol. 33 (2015) 538-542. [94] J. Song, D. Yang, J. Xu, T. Zhu, Y.E. Chen, J. Zhang, RS-1 enhances CRISPR/Cas9- and TALEN-mediated knock-in efficiency, Nat. Commun. 7 (2016) 10548. [95] C. Yu, Y. Liu, T. Ma, K. Liu, S. Xu, Y. Zhang, H. Liu, M. La Russa, M. Xie, S. Ding, L.S. Qi, Small molecules enhance CRISPR genome editing in pluripotent stem cells, Cell Stem Cell 16 (2015) 142-147. [96] N. Savic, G. Schwank, Advances in therapeutic CRISPR/Cas9 genome editing, Transl. Res. 168 (2016) 15-21. [97] Y. Cao, E. Ma, S. Cestellos-Blanco, B. Zhang, R. Qiu, Y. Su, J.A. Doudna, P. Yang, Nontoxic nanopore electroporation for effective intracellular delivery of biological macromolecules, Proc. Natl. Acad. Sci. U. S. A. 116 (2019) 7899-7904. [98] W. Wu, Z. Lu, F. Li, W. Wang, N. Qian, J. Duan, Y. Zhang, F. Wang, T. Chen, Efficient in vivo gene editing using ribonucleoproteins in skin stem cells of recessive dystrophic epidermolysis bullosa mouse model, Proc. Natl. Acad. Sci. U. S. A. 114

Journal Pre-proof

Jo ur

na

lP

re

-p

ro

of

(2017) 1660-1665. [99] R. Maresch, S. Mueller, C. Veltkamp, R. Ollinger, M. Friedrich, I. Heid, K. Steiger, J. Weber, T. Engleitner, M. Barenboim, S. Klein, S. Louzada, R. Banerjee, A. Strong, T. Stauber, N. Gross, U. Geumann, S. Lange, M. Ringelhan, I. Varela, K. Unger, F. Yang, R.M. Schmid, G.S. Vassiliou, R. Braren, G. Schneider, M. Heikenwalder, A. Bradley, D. Saur, R. Rad, Multiplexed pancreatic genome engineering and cancer induction by transfection-based CRISPR/Cas9 delivery in mice, Nat. Commun. 7 (2016) 10770. [100] X. Han, Z. Liu, M.C. Jo, K. Zhang, Y. Li, Z. Zeng, N. Li, Y. Zu, L. Qin, CRISPRCas9 delivery to hard-to-transfect cells via membrane deformation, Sci. Adv. 1 (2015) e1500454. [101] T. Wirth, N. Parker, S. Yla-Herttuala, History of gene therapy, Gene 525 (2013) 162-169. [102] R.J. Samulski, N. Muzyczka, AAV-Mediated Gene Therapy for Research and Therapeutic Purposes, Annu. Rev. Virol. 1 (2014) 427-451. [103] C.F. Xu, S. Iqbal, S. Shen, Y.L. Luo, X. Yang, J. Wang, Development of "CLAN" nanomedicine for nucleic acid therapeutics, Small 15 (2019) e1900055. [104] X.Z. Yang, S. Dou, T.M. Sun, C.Q. Mao, H.X. Wang, J. Wang, Systemic delivery of siRNA with cationic lipid assisted PEG-PLA nanoparticles for cancer therapy, J. Controlled Release 156 (2011) 203-211. [105] C.F. Xu, H.B. Zhang, C.Y. Sun, Y. Liu, S. Shen, X.Z. Yang, Y.H. Zhu, J. Wang, Tumor acidity-sensitive linkage-bridged block copolymer for therapeutic siRNA delivery, Biomaterials 88 (2016) 48-59. [106] Y. Liu, G. Zhao, C.F. Xu, Y.L. Luo, Z.D. Lu, J. Wang, Systemic delivery of CRISPR/Cas9 with PEG-PLGA nanoparticles for chronic myeloid leukemia targeted therapy, Biomater. Sci. 6 (2018) 1592-1603. [107] B.A. Burke, M. Carroll, BCR-ABL: a multi-faceted promoter of DNA mutation in chronic myelogeneous leukemia, Leukemia 24 (2010) 1105-1112. [108] Y. Liu, Z.T. Cao, C.F. Xu, Z.D. Lu, Y.L. Luo, J. Wang, Optimization of lipidassisted nanoparticle for disturbing neutrophils-related inflammation, Biomaterials 172 (2018) 92-104. [109] M. Li, Y.N. Fan, Z.Y. Chen, Y.L. Luo, Y.C. Wang, Z.X. Lian, C.F. Xu, J. Wang, Optimized nanoparticle-mediated delivery of CRISPR-Cas9 system for B cell intervention, Nano Res. 11 (2018) 6270-6282. [110] C.L. Holness, D.L. Simmons, Molecular cloning of CD68, a human macrophage marker related to lysosomal glycoproteins, Blood 81 (1993) 1607-1613. [111] L. Zhang, P. Wang, Q. Feng, N. Wang, Z. Chen, Y. Huang, W. Zheng, X. Jiang, Lipid nanoparticle-mediated efficient delivery of CRISPR/Cas9 for tumor therapy, NPG Asia Mater. 9 (2017) e441-e441. [112] R.S. Schuh, T.G. de Carvalho, R. Giugliani, U. Matte, G. Baldo, H.F. Teixeira, Gene editing of MPS I human fibroblasts by co-delivery of a CRISPR/Cas9 plasmid and a donor oligonucleotide using nanoemulsions as nonviral carriers, Eur. J. Pharm. Biopharm. 122 (2018) 158-166. [113] Z.Y. He, Y.G. Zhang, Y.H. Yang, C.C. Ma, P. Wang, W. Du, L. Li, R. Xiang, X.R. Song, X. Zhao, S.H. Yao, Y.Q. Wei, In vivo ovarian cancer gene therapy using CRISPR-

Journal Pre-proof

Jo ur

na

lP

re

-p

ro

of

Cas9, Hum. Gene Ther. 29 (2018) 223-233. [114] M. Li, H. Xie, Y. Liu, C. Xia, X. Cun, Y. Long, X. Chen, M. Deng, R. Guo, Z. Zhang, Q. He, Knockdown of hypoxia-inducible factor-1 alpha by tumor targeted delivery of CRISPR/Cas9 system suppressed the metastasis of pancreatic cancer, J. Controlled Release 304 (2019) 204-215. [115] P. Dharmalingam, H.K.R. Rachamalla, B. Lohchania, B. Bandlamudi, S. Thangavel, M.K. Murugesan, R. Banerjee, A. Chaudhuri, C. Voshavar, S. Marepally, Green transfection: cationic lipid nanocarrier system derivatized from vegetable fat, palmstearin enhances nucleic acid transfections, ACS Omega 2 (2017) 7892-7903. [116] Z. Zhang, T. Wan, Y. Chen, Y. Chen, H. Sun, T. Cao, Z. Songyang, G. Tang, C. Wu, Y. Ping, F.J. Xu, J. Huang, Cationic polymer-mediated CRISPR/Cas9 plasmid delivery for genome editing, Macromol. Rapid Commun. 40 (2019) e1800068. [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, U.K. Kim, Effective PEI-mediated delivery of CRISPR-Cas9 complex for targeted gene therapy, Nanomedicine 14 (2018) 2095-2102. [118] M. Yan, J. Wen, M. Liang, Y. Lu, M. Kamata, I.S. Chen, Modulation of gene expression by polymer nanocapsule delivery of DNA cassettes encoding small RNAs, PLoS One 10 (2015) e0127986. [119] C. Liang, F. Li, L. Wang, Z.K. Zhang, C. Wang, B. He, J. Li, Z. Chen, A.B. Shaikh, J. Liu, X. Wu, S. Peng, L. Dang, B. Guo, X. He, D.W.T. Au, C. Lu, H. Zhu, B.T. Zhang, A. Lu, G. Zhang, Tumor cell-targeted delivery of CRISPR/Cas9 by aptamerfunctionalized lipopolymer for therapeutic genome editing of VEGFA in osteosarcoma, Biomaterials 147 (2017) 68-85. [120] Q. Liu, K. Zhao, C. Wang, Z. Zhang, C. Zheng, Y. Zhao, Y. Zheng, C. Liu, Y. An, L. Shi, C. Kang, Y. Liu, Multistage delivery nanoparticle facilitates efficient CRISPR/dCas9 activation and tumor growth suppression in vivo, Adv. Sci. 6 (2019) 1801423. [121] M. Wang, H. Liu, L. Li, Y. Cheng, A fluorinated dendrimer achieves excellent gene transfection efficacy at extremely low nitrogen to phosphorus ratios, Nat. Commun. 5 (2014) 3053. [122] L. Li, L. Song, X. Liu, X. Yang, X. Li, T. He, N. Wang, S. Yang, C. Yu, T. Yin, Y. Wen, Z. He, X. Wei, W. Su, Q. Wu, S. Yao, C. Gong, Y. Wei, Artificial virus delivers CRISPR-Cas9 system for genome editing of cells in mice, ACS Nano 11 (2017) 95111. [123] Y. Qi, H. Song, H. Xiao, G. Cheng, B. Yu, F.J. Xu, Fluorinated acid-labile branched hydroxyl-rich nanosystems for flexible and robust delivery of plasmids, Small 14 (2018) e1803061. [124] T. Lehto, K. Ezzat, M.J.A. Wood, S. El Andaloussi, Peptides for nucleic acid delivery, Adv. Drug Delivery Rev. 106 (2016) 172-182. [125] P. Wang, L. Zhang, W. Zheng, L. Cong, Z. Guo, Y. Xie, L. Wang, R. Tang, Q. Feng, Y. Hamada, K. Gonda, Z. Hu, X. Wu, X. Jiang, Thermo-triggered release of CRISPR-Cas9 system by lipid-encapsulated gold nanoparticles for tumor therapy, Angew. Chem. Int. Ed. 57 (2018) 1491-1496. [126] A.S. Timin, A.R. Muslimov, K.V. Lepik, O.S. Epifanovskaya, A.I. Shakirova, U.

Journal Pre-proof

Jo ur

na

lP

re

-p

ro

of

Mock, K. Riecken, M.V. Okilova, V.S. Sergeev, B.V. Afanasyev, B. Fehse, G.B. Sukhorukov, Efficient gene editing via non-viral delivery of CRISPR-Cas9 system using polymeric and hybrid microcarriers, Nanomedicine 14 (2018) 97-108. [127] H. Lu, J. Wang, Y. Bai, J.W. Lang, S. Liu, Y. Lin, J. Cheng, Ionic polypeptides with unusual helical stability, Nat. Commun. 2 (2011) 206. [128] Y.H. Lao, M. Li, M.A. Gao, D. Shao, C.W. Chi, D. Huang, S. Chakraborty, T.C. Ho, W. Jiang, H.X. Wang, S. Wang, K.W. Leong, HPV oncogene manipulation using nonvirally delivered CRISPR/Cas9 or natronobacterium gregoryi argonaute, Adv. Sci. 5 (2018) 1700540. [129] X. Zhang, C. Xu, S. Gao, P. Li, Y. Kong, T. Li, Y. Li, F.J. Xu, J. Du, CRISPR/Cas9 delivery mediated with hydroxyl-rich nanosystems for gene editing in aorta, Adv. Sci. 6 (2019) 1900386. [130] B.Y. Liu, X.Y. He, R.X. Zhuo, S.X. Cheng, Tumor targeted genome editing mediated by a multi-functional gene vector for regulating cell behaviors, J. Controlled Release 291 (2018) 90-98. [131] D. Zhu, H. Shen, S. Tan, Z. Hu, L. Wang, L. Yu, X. Tian, W. Ding, C. Ren, C. Gao, J. Cheng, M. Deng, R. Liu, J. Hu, L. Xi, P. Wu, Z. Zhang, D. Ma, H. Wang, Nanoparticles based on poly (beta-amino ester) and HPV16-targeting CRISPR/shRNA as potential drugs for HPV16-related cervical malignancy, Mol. Ther. 26 (2018) 24432455. [132] S.M. Kim, Y. Yang, S.J. Oh, Y. Hong, M. Seo, M. Jang, Cancer-derived exosomes as a delivery platform of CRISPR/Cas9 confer cancer cell tropism-dependent targeting, J. Controlled Release 266 (2017) 8-16. [133] Y. Lin, J.H. Wu, W.H. Gu, Y.L. Huang, Z.C. Tong, L.J. Huang, J.L. Tan, Exosome-liposome hybrid nanoparticles deliver CRISPR/Cas9 system in MSCs, Adv. Sci. 5 (2018) 1700611. [134] T. Killian, A. Buntz, T. Herlet, H. Seul, O. Mundigl, G. Langst, U. Brinkmann, Antibody-targeted chromatin enables effective intracellular delivery and functionality of CRISPR/Cas9 expression plasmids, Nucleic Acids Res. 47 (2019) e55. [135] P. Guo, J. Yang, J. Huang, D.T. Auguste, M.A. Moses, Therapeutic genome editing of triple-negative breast tumors using a noncationic and deformable nanolipogel, Proc. Natl. Acad. Sci. U. S. A. 116 (2019) 18295-18303. [136] L. Li, Z. Yang, S. Zhu, L. He, W. Fan, W. Tang, J. Zou, Z. Shen, M. Zhang, L. Tang, Y. Dai, G. Niu, S. Hu, X. Chen, A rationally designed semiconducting polymer brush for NIR-II imaging-guided light-triggered remote control of CRISPR/Cas9 genome editing, Adv. Mater. 31 (2019) e1901187. [137] H.X. Zhang, Y. Zhang, H. Yin, Genome editing with mRNA encoding ZFN, TALEN, and Cas9, Mol. Ther. 27 (2019) 735-746. [138] C.F. Xu, Z.D. Lu, Y.L. Luo, Y. Liu, Z.T. Cao, S. Shen, H.J. Li, J. Liu, K.G. Chen, Z.Y. Chen, X.Z. Yang, Z. Gu, J. Wang, Targeting of NLRP3 inflammasome with gene editing for the amelioration of inflammatory diseases, Nat. Commun. 9 (2018) 4092. [139] Y. Zhang, S. Shen, G. Zhao, C.F. Xu, H.B. Zhang, Y.L. Luo, Z.T. Cao, J. Shi, Z.B. Zhao, Z.X. Lian, J. Wang, In situ repurposing of dendritic cells with CRISPR/Cas9based nanomedicine to induce transplant tolerance, Biomaterials 217 (2019) 119302.

Journal Pre-proof

Jo ur

na

lP

re

-p

ro

of

[140] Z. Liu, X. Han, L. Qin, Recent progress of microfluidics in translational applications, Adv. Healthcare Mater. 5 (2016) 871-888. [141] M. Maeki, N. Kimura, Y. Sato, H. Harashima, M. Tokeshi, Advances in microfluidics for lipid nanoparticles and extracellular vesicles and applications in drug delivery systems, Adv. Drug Delivery Rev. 128 (2018) 84-100. [142] Y. Dong, K.T. Love, J.R. Dorkin, S. Sirirungruang, Y. Zhang, D. Chen, R.L. Bogorad, H. Yin, Y. Chen, A.J. Vegas, C.A. Alabi, G. Sahay, K.T. Olejnik, W. Wang, A. Schroeder, A.K. Lytton-Jean, D.J. Siegwart, A. Akinc, C. Barnes, S.A. Barros, M. Carioto, K. Fitzgerald, J. Hettinger, V. Kumar, T.I. Novobrantseva, J. Qin, W. Querbes, V. Koteliansky, R. Langer, D.G. Anderson, Lipopeptide nanoparticles for potent and selective siRNA delivery in rodents and nonhuman primates, Proc. Natl. Acad. Sci. U. S. A. 111 (2014) 3955-3960. [143] H. Yin, C.Q. Song, S. Suresh, Q. Wu, S. Walsh, L.H. Rhym, E. Mintzer, M.F. Bolukbasi, L.J. Zhu, K. Kauffman, H. Mou, A. Oberholzer, J. Ding, S.Y. Kwan, R.L. Bogorad, T. Zatsepin, V. Koteliansky, S.A. Wolfe, W. Xue, R. Langer, D.G. Anderson, Structure-guided chemical modification of guide RNA enables potent non-viral in vivo genome editing, Nat. Biotechnol. 35 (2017) 1179-1187. [144] 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, D.V. Morrissey, A single administration of CRISPR/Cas9 lipid nanoparticles achieves robust and persistent in vivo genome editing, Cell Rep. 22 (2018) 2227-2235. [145] J. Liu, J. Chang, Y. Jiang, X. Meng, T. Sun, L. Mao, Q. Xu, M. Wang, Fast and efficient CRISPR/Cas9 genome editing in vivo enabled by bioreducible lipid and messenger RNA nanoparticles, Adv. Mater. 31 (2019) e1902575. [146] B. Li, X. Luo, B. Deng, J. Wang, D.W. McComb, Y. Shi, K.M. Gaensler, X. Tan, A.L. Dunn, B.A. Kerlin, Y. Dong, An orthogonal array optimization of lipid-like nanoparticles for mRNA delivery in vivo, Nano Lett. 15 (2015) 8099-8107. [147] C. Jiang, M. Mei, B. Li, X. Zhu, W. Zu, Y. Tian, Q. Wang, Y. Guo, Y. Dong, X. Tan, A non-viral CRISPR/Cas9 delivery system for therapeutically targeting HBV DNA and pcsk9 in vivo, Cell Res. 27 (2017) 440-443. [148] J.B. Miller, S. Zhang, P. Kos, H. Xiong, K. Zhou, S.S. Perelman, H. Zhu, D.J. Siegwart, Non-viral CRISPR/Cas gene editing in vitro and in vivo enabled by synthetic nanoparticle co-delivery of Cas9 mRNA and sgRNA, Angew. Chem. Int. Ed. 56 (2017) 1059-1063. [149] X. Zhang, B. Li, X. Luo, W. Zhao, J. Jiang, C. Zhang, M. Gao, X. Chen, Y. Dong, Biodegradable amino-ester nanomaterials for Cas9 mRNA delivery in vitro and in vivo, ACS Appl. Mater. Interfaces 9 (2017) 25481-25487. [150] Z. Li, X. Zhou, M. Wei, X. Gao, L. Zhao, R. Shi, W. Sun, Y. Duan, G. Yang, L. Yuan, In vitro and in vivo RNA inhibition by CD9-HuR functionalized exosomes encapsulated with miRNA or CRISPR/dCas9, Nano Lett. 19 (2019) 19-28. [151] W.M. Usman, T.C. Pham, Y.Y. Kwok, L.T. Vu, V. Ma, B.Y. Peng, Y. San Chan, L.K. Wei, S.M. Chin, A. Azad, A.B.L. He, A.Y.H. Leung, M.S. Yang, N. Shyh-Chang,

Journal Pre-proof

Jo ur

na

lP

re

-p

ro

of

W.C. Cho, J.H. Shi, M.T.N. Le, Efficient RNA drug delivery using red blood cell extracellular vesicles, Nat. Commun. 9 (2018) 2359. [152] W. Sun, T. Jiang, Y. Lu, M. Reiff, R. Mo, Z. Gu, Cocoon-like self-degradable DNA nanoclew for anticancer drug delivery, J. Am. Chem. Soc. 136 (2014) 1472214725. [153] G. Gasiunas, R. Barrangou, P. Horvath, V. Siksnys, Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria, Proc. Natl. Acad. Sci. U. S. A. 109 (2012) E2579-2586. [154] W. Sun, W. Ji, J.M. Hall, Q. Hu, C. Wang, C.L. Beisel, Z. Gu, Self-assembled DNA nanoclews for the efficient delivery of CRISPR-Cas9 for genome editing, Angew. Chem. Int. Ed. 54 (2015) 12029-12033. [155] G. Chen, A.A. Abdeen, Y. Wang, P.K. Shahi, S. Robertson, R. Xie, M. Suzuki, B.R. Pattnaik, K. Saha, S. Gong, A biodegradable nanocapsule delivers a Cas9 ribonucleoprotein complex for in vivo genome editing, Nat. Nanotechnol. 14 (2019) 974-980. [156] B. Lee, K. Lee, S. Panda, R. Gonzales-Rojas, A. Chong, V. Bugay, H.M. Park, R. Brenner, N. Murthy, H.Y. Lee, Nanoparticle delivery of CRISPR into the brain rescues a mouse model of fragile X syndrome from exaggerated repetitive behaviours, Nat. Biomed. Eng. 2 (2018) 497-507. [157] R. Shahbazi, G. Sghia-Hughes, J.L. Reid, S. Kubek, K.G. Haworth, O. Humbert, H.P. Kiem, J.E. Adair, Targeted homology-directed repair in blood stem and progenitor cells with CRISPR nanoformulations, Nat. Mater. 18 (2019) 1124-1132. [158] R. Mout, M. Ray, G. Yesilbag Tonga, Y.W. Lee, T. Tay, K. Sasaki, V.M. Rotello, Direct cytosolic delivery of CRISPR/Cas9-ribonucleoprotein for efficient gene editing, ACS Nano 11 (2017) 2452-2458. [159] M. Ray, Y.W. Lee, J. Hardie, R. Mout, G. Yesilbag Tonga, M.E. Farkas, V.M. Rotello, CRISPRed macrophages for cell-based cancer immunotherapy, Bioconjugate Chem. 29 (2018) 445-450. [160] M. Wang, J.A. Zuris, F.T. Meng, H. Rees, S. Sun, P. Deng, Y. Han, X. Gao, D. Pouli, Q. Wu, I. Georgakoudi, D.R. Liu, Q.B. Xu, Efficient delivery of genome-editing proteins using bioreducible lipid nanoparticles, Proc. Natl. Acad. Sci. U. S. A. 113 (2016) 2868-2873. [161] Y. Li, J. Bolinger, Y. Yu, Z. Glass, N. Shi, L. Yang, M. Wang, Q. Xu, Intracellular delivery and biodistribution study of CRISPR/Cas9 ribonucleoprotein loaded bioreducible lipidoid nanoparticles, Biomater. Sci. 7 (2019) 596-606. [162] H.H. Yue, X.M. Zhou, M. Cheng, D. Xing, Graphene oxide-mediated Cas9/sgRNA delivery for efficient genome editing, Nanoscale 10 (2018) 1063-1071. [163] Y.K. Kang, K. Kwon, J.S. Ryu, H.N. Lee, C. Park, H.J. Chung, Nonviral genome editing based on a polymer-derivatized CRISPR nanocomplex for targeting bacterial pathogens and antibiotic resistance, Bioconjugate Chem. 29 (2018) 3936-3936. [164] E.Y. Cho, J.Y. Ryu, H.A.R. Lee, S.H. Hong, H.S. Park, K.S. Hong, S.G. Park, H.P. Kim, T.J. Yoon, Lecithin nano-liposomal particle as a CRISPR/Cas9 complex delivery system for treating type 2 diabetes, J. Nanobiotechnol. 17 (2019) 19. [165] S.M. Kim, S.C. Shin, E.E. Kim, S.H. Kim, K. Park, S.J. Oh, M. Jang, Simple in

Journal Pre-proof

Jo ur

na

lP

re

-p

ro

of

vivo gene editing via direct self-assembly of Cas9 ribonucleoprotein complexes for cancer treatment, ACS Nano 12 (2018) 7750-7760. [166] S. Ramakrishna, A.K. Dad, J. Beloor, R. Gopalappa, S.K. Lee, H. Kim, Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA, Genome Res. 24 (2014) 1020-1027. [167] S. Qazi, H.M. Miettinen, R.A. Wilkinson, K. McCoy, T. Douglas, B. Wiedenheft, Programmed self-assembly of an active P22-Cas9 nanocarrier system, Mol. Pharmaceutics 13 (2016) 1191-1196. [168] 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 Onate, J. Compte Barron, 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, J.A. Doudna, Receptor-mediated delivery of CRISPR-Cas9 endonuclease for cell-type-specific gene editing, J. Am. Chem. Soc. 140 (2018) 6596-6603. [169] R. Rouet, D. Christ, Efficient intracellular delivery of CRISPR-Cas ribonucleoproteins through receptor mediated endocytosis, ACS Chem. Biol. 14 (2019) 554-561. [170] H. Park, J. Oh, G. Shim, B. Cho, Y. Chang, S. Kim, S. Baek, H. Kim, J. Shin, H. Choi, J. Yoo, J. Kim, W. Jun, M. Lee, C.J. Lengner, Y.K. Oh, J. Kim, In vivo neuronal gene editing via CRISPR-Cas9 amphiphilic nanocomplexes alleviates deficits in mouse models of Alzheimer's disease, Nat. Neurosci. 22 (2019) 524-528. [171] Y. Shen, J.L. Cohen, S.M. Nicoloro, M. Kelly, B. Yenilmez, F. Henriques, E. Tsagkaraki, Y.J.K. Edwards, X. Hu, R.H. Friedline, J.K. Kim, M.P. Czech, CRISPRdelivery particles targeting nuclear receptor-interacting protein 1 (Nrip1) in adipose cells to enhance energy expenditure, J. Biol. Chem. 293 (2018) 17291-17305. [172] I. Lostale-Seijo, I. Louzao, M. Juanes, J. Montenegro, Peptide/Cas9 nanostructures for ribonucleoprotein cell membrane transport and gene edition, Chem. Sci. 8 (2017) 7923-7931. [173] J. Qiao, W. Sun, S. Lin, R. Jin, L. Ma, Y. Liu, Cytosolic delivery of CRISPR/Cas9 ribonucleoproteins for genome editing using chitosan-coated red fluorescent protein, Chem. Commun. 55 (2019) 4707-4710. [174] X. Zhu, M.M. Lv, J.W. Liu, R.Q. Yu, J.H. Jiang, DNAzyme activated proteinscaffolded CRISPR-Cas9 nanoassembly for genome editing, Chem. Commun. 55 (2019) 6511-6514. [175] S.K. Alsaiari, S. Patil, M. Alyami, K.O. Alamoudi, F.A. Aleisa, J.S. Merzaban, M. Li, N.M. Khashab, Endosomal escape and delivery of CRISPR/Cas9 genome editing machinery enabled by nanoscale zeolitic imidazolate framework, J. Am. Chem. Soc. 140 (2018) 143-146. [176] X. Yang, Q. Tang, Y. Jiang, M. Zhang, M. Wang, L. Mao, Nanoscale ATPresponsive zeolitic imidazole framework-90 as a general platform for cytosolic protein delivery and genome Editing, J. Am. Chem. Soc. 141 (2019) 3782-3786. [177] C. Liu, T. Wan, H. Wang, S. Zhang, Y. Ping, Y. Cheng, A boronic acid-rich dendrimer with robust and unprecedented efficiency for cytosolic protein delivery and CRISPR-Cas9 gene editing, Sci. Adv. 5 (2019) eaaw8922.

Journal Pre-proof

Jo ur

na

lP

re

-p

ro

of

[178] L.A. Campbell, L.M. Coke, C.T. Richie, L.V. Fortuno, A.Y. Park, B.K. Harvey, Gesicle-mediated delivery of CRISPR/Cas9 ribonucleoprotein complex for inactivating the HIV provirus, Mol. Ther. 27 (2019) 151-163. [179] Q. Wang, J. Yu, T. Kadungure, J. Beyene, H. Zhang, Q. Lu, ARMMs as a versatile platform for intracellular delivery of macromolecules, Nat. Commun. 9 (2018) 960. [180] R. Chen, H. Huang, H. Liu, J. Xi, J. Ning, W. Zeng, C. Shen, T. Zhang, G. Yu, Q. Xu, X. Chen, J. Wang, F. Lu, Friend or foe? evidence indicates endogenous exosomes can deliver functional gRNA and Cas9 protein, Small 15 (2019) e1902686. [181] Y. Pan, J. Yang, X. Luan, X. Liu, X. Li, J. Yang, T. Huang, L. Sun, Y. Wang, Y. Lin, Y. Song, Near-infrared upconversion-activated CRISPR-Cas9 system: A remotecontrolled gene editing platform, Sci. Adv. 5 (2019) eaav7199. [182] W. Zhou, H. Cui, L. Ying, X.F. Yu, Enhanced cytosolic delivery and release of CRISPR/Cas9 by black phosphorus nanosheets for genome editing, Angew. Chem. Int. Ed. 57 (2018) 10268-10272. [183] J. Mircetic, I. Steinebrunner, L. Ding, J.-F. Fei, A. Bogdanova, D. Drechsel, F. Buchholz, Purified Cas9 fusion proteins for advanced genome manipulation, Small Methods 1 (2017) 1600052. [184] P. Wang, L. Zhang, Y. Xie, N. Wang, R. Tang, W. Zheng, X. Jiang, Genome editing for cancer therapy: delivery of Cas9 protein/sgRNA plasmid via a gold nanocluster/lipid core-shell nanocarrier, Adv. Sci. 4 (2017) 1700175. [185] Z. Chen, F. Liu, Y. Chen, J. Liu, X. Wang, A.T. Chen, G. Deng, H. Zhang, J. Liu, Z. Hong, J. Zhou, Targeted delivery of CRISPR/Cas9-mediated cancer gene therapy via liposome-templated hydrogel nanoparticles, Adv. Funct. Mater. 27 (2017) 1703036. [186] J.S. Ha, J.S. Lee, J. Jeong, H. Kim, J. Byun, S.A. Kim, H.J. Lee, H.S. Chung, J.B. Lee, D.R. Ahn, Poly-sgRNA/siRNA ribonucleoprotein nanoparticles for targeted gene disruption, J. Controlled Release 250 (2017) 27-35. [187] 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, D.G. Anderson, Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo, Nat. Biotechnol. 34 (2016) 328-333. [188] H. Zhu, L. Zhang, S. Tong, C.M. Lee, H. Deshmukh, G. Bao, Spatial control of in vivo CRISPR-Cas9 genome editing via nanomagnets, Nat. Biomed. Eng. 3 (2019) 126-136. [189] D.B. Cox, R.J. Platt, F. Zhang, Therapeutic genome editing: prospects and challenges, Nat. Med. 21 (2015) 121-131. [190] R. Liu, W.A. Paxton, S. Choe, D. Ceradini, S.R. Martin, R. Horuk, M.E. MacDonald, H. Stuhlmann, R.A. Koup, N.R. Landau, Homozygous defect in HIV-1 coreceptor accounts for resistance of some multiply-exposed individuals to HIV-1 infection, Cell 86 (1996) 367-377. [191] T. Jonsson, J.K. Atwal, S. Steinberg, J. Snaedal, P.V. Jonsson, S. Bjornsson, H. Stefansson, P. Sulem, D. Gudbjartsson, J. Maloney, K. Hoyte, A. Gustafson, Y. Liu, Y. Lu, T. Bhangale, R.R. Graham, J. Huttenlocher, G. Bjornsdottir, O.A. Andreassen, E.G. Jonsson, A. Palotie, T.W. Behrens, O.T. Magnusson, A. Kong, U. Thorsteinsdottir, R.J.

Journal Pre-proof

Jo ur

na

lP

re

-p

ro

of

Watts, K. Stefansson, A mutation in APP protects against Alzheimer's disease and agerelated cognitive decline, Nature 488 (2012) 96-99. [192] H. Bachtarzi, Ex vivo and in vivo genome editing: a regulatory scientific framework from early development to clinical implementation, Regener. Med. 12 (2017) 8. [193] K.E. Ormond, Y. Bombard, V.L. Bonham, L. Hoffman-Andrews, H. Howard, R. Isasi, K. Musunuru, K.A. Riggan, M. Michie, M. Allyse, The clinical application of gene editing: ethical and social issues, Pers. Med. 16 (2019) 337-350. [194] S.W. Cho, S. Kim, Y. Kim, J. Kweon, H.S. Kim, S. Bae, J.S. Kim, Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases, Genome Res. 24 (2014) 132-141. [195] M.A. Kotterman, D.V. Schaffer, Engineering adeno-associated viruses for clinical gene therapy, Nat. Rev. Genet. 15 (2014) 445-451. [196] N. Bessis, F.J. GarciaCozar, M.C. Boissier, Immune responses to gene therapy vectors: influence on vector function and effector mechanisms, Gene Ther. 11 (2004) S10-17. [197] F. Qi, J. Wu, H. Li, G. Ma, Recent research and development of PLGA/PLA microspheres/nanoparticles: a review in scientific and industrial aspects, Front. Chem. Sci. Eng. 13 (2018) 14-27. [198] D.P. Dever, R.O. Bak, A. Reinisch, J. Camarena, G. Washington, C.E. Nicolas, M. Pavel-Dinu, N. Saxena, A.B. Wilkens, S. Mantri, N. Uchida, A. Hendel, A. Narla, R. Majeti, K.I. Weinberg, M.H. Porteus, CRISPR/Cas9 beta-globin gene targeting in human haematopoietic stem cells, Nature 539 (2016) 384-389. [199] S. Baliou, M. Adamaki, A.M. Kyriakopoulos, D.A. Spandidos, M. Panayiotidis, I. Christodoulou, V. Zoumpourlis, CRISPR therapeutic tools for complex genetic disorders and cancer, Int. J. Oncol. 53 (2018) 443-468. [200] F. Baylis, M. McLeod, First-in-human Phase 1 CRISPR gene editing cancer trials: are we ready? Curr. Gene Ther. 17 (2017) 309-319. [201] J. Tang, S. Baxter, A. Menon, A. Alaarg, B.L. Sanchez-Gaytan, F. Fay, Y. Zhao, M. Ouimet, M.S. Braza, V.A. Longo, D. Abdel-Atti, R. Duivenvoorden, C. Calcagno, G. Storm, S. Tsimikas, K.J. Moore, F.K. Swirski, M. Nahrendorf, E.A. Fisher, C. PerezMedina, Z.A. Fayad, T. Reiner, W.J. Mulder, Immune cell screening of a nanoparticle library improves atherosclerosis therapy, Proc. Natl. Acad. Sci. U. S. A. 113 (2016) E6731-E6740. [202] P. Jouannet, CRISPR-Cas9, germinal cells and human embryo, Biol. Aujourd'hui 211 (2017) 207-213. [203] D.P. Dever, M.H. Porteus, The changing landscape of gene editing in hematopoietic stem cells: a step towards Cas9 clinical translation, Curr. Opin. Hematol. 24 (2017) 481-488.

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

Figure 18

Figure 19

Figure 20

Figure 21

Figure 22