Non-viral delivery systems for CRISPR/Cas9-based genome editing: Challenges and opportunities

In the paper

“This approach allows for robust DNA knock-in in both dividing and non-dividing cells in vitro and in vivo [87].”

What the sources say

• Not checked yetcites [87] Suzuki, 2016, In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration, Nature source

In the paper

“More importantly, the editing levels can persist for at least 12 months, despite the transient nature of the delivery system and the editing components.”

What the sources say

• Not checked yetcites [116] Jonathan, 2018, A single administration of CRISPR/Cas9 lipid nanoparticles achieves robust and persistent in vivo genome editing, Cell Rep. source

In the paper

“In another study, Miller et al. reported a non-viral vector based on zwitterionic amino lipids to deliver Cas9 mRNA and sgRNAs. In contrast to transient therapies, they showed permanent editing of LoxP gene in genetically engineered mice containing a homozygous Rosa26 promoter Lox-Stop-Lox tdTomato (tdTO) cassette after intravenous administration [105].”

What the sources say

• Not checked yetcites [105] Miller, 2017, 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. Engl. source

In the paper

“Jiang et al. developed lipid-like nanoparticles for targeted delivery of Cas9 mRNA and sgRNA to the liver. This system achieved disruption of HBV DNA and the proprotein convertase subtilisin/kexin type 9 (pcsk9) gene in vivo [104].”

What the sources say

• Not checked yetcites [104] Jiang, 2017, A non-viral CRISPR/Cas9 delivery system for therapeutically targeting HBV DNA and pcsk9 in vivo, Cell Res. source

In the paper

“Zuris et al. injected Cas9:sgRNA complexes targeting GFP gene into the cochlea of transgenic Atoh1-GFP mice. This system resulted in ~20% Cas9-mediated genome modification in hair cells [69].”

What the sources say

• Not checked yetcites [69] Lehrman, 1999, Virus treatment questioned after gene therapy death, Nature source

In the paper

“Self-assembled DNA nanoclews were constructed for CRISPR/Cas9-based genome editing system delivery. After being loaded with CRISPR RNPs, these nanoclews enabled efficient target disruption of EGFP gene in vitro and in U2OS.EGFP xenograft tumor models in vivo after intratumoral injection [103].”

What the sources say

• Not checked yetcites [103] Sun, 2015, Self-assembled DNA nanoclews for the efficient delivery of CRISPR-Cas9 for genome editing, Angew Chem. Int. Ed. Engl. source

In the paper

“Additionally, unlike viral delivery systems, the synthetic nature of LNP-INT01 allowed for multi-dosing regimens to obtain cumulative editing efficacy without significant changes in cytokine levels or body weight.”

What the sources say

• Not checked yetcites [116] Jonathan, 2018, A single administration of CRISPR/Cas9 lipid nanoparticles achieves robust and persistent in vivo genome editing, Cell Rep. source

In the paper

“With this approach, they edited AAVS1 gene and PTEN gene and achieved effective gene editing efficiency up to 30% in HeLa, HEK-293T and Raw 264.7 cell lines (Fig. 2A) [97].”

What the sources say

• Not checked yetcites [97] Mout, 2017, Direct cytosolic delivery of CRISPR/Cas9-ribonucleoprotein for efficient gene editing, ACS Nano source

In the paper

“After loading with CRISPR/Cas9 targeting polo-like kinase 1 (plk1) gene, LHNPs effectively inhibited U87MG tumor growth [119].”

What the sources say

• Not checked yetcites [119] Chen, 2017, Targeted delivery of CRISPR/Cas9-mediated cancer gene therapy via liposome-templated hydrogel nanoparticlesaptation repertoires, Adv. Funct. Mater. source

In the paper

“In a recent study, graphene oxide-poly(ethylene glycol)-polyethylenimine (GO-PEG-PEI) nanocarrier mediated efficient CRISPR RNPs delivery via physisorption and π-stacking interaction. This strategy induced efficient genome editing in AGS cells with an efficiency of ~39% (Fig. 2D) [102].”

What the sources say

• Not checked yetcites [102] Yue, 2018, Graphene oxide-mediated Cas9/sgRNA delivery for efficient genome editing, Nanoscale source

In the paper

“However, due to the large size of Cas9 protein (~ 160 kD), the positive charge of Cas9 protein and the strong negative charge of sgRNA, the effective delivery of Cas9/sgRNA ribonucleoprotein complexes (RNPs) can be difficult [88–91].”

What the sources say

• Not checked yetcites [88] Zuris, 2015, Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo, Nat. Biotechnol. source
• Not checked yetcites [89] Subburaj, 2016, Site-directed mutagenesis in Petunia x hybrida protoplast system using direct delivery of purified recombinant Cas9 ribonucleoproteins, Plant Cell Rep. source
• Not checked yetcites [90] Woo, 2015, DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins, Nat. Biotechnol. source
• Not checked yetcites [91] Schumann, 2015, Generation of knock-in primary human T cells using Cas9 ribonucleoproteins, Proc. Natl. Acad. Sci. U. S. A. source

In the paper

“In principle, this format enables the swiftest genome editing as there is no need for transcription and/or translation.”

In the paper

“With the rapid development of non-viral vectors, lipid- or polymer-based nanocarriers have shown great potential for CRISPR/Cas9 delivery.”

In the paper

“Especially, AAV can be flexibly constructed to target specific organs or tissues based on the diverse tissue tropism of AAV serotypes [66, 67].”

What the sources say

• Not checked yetcites [66] Zincarelli, 2008, Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection, Mol. Ther. source
• Not checked yetcites [67] Gao, 2005, New recombinant serotypes of AAV vectors, Curr. Gene Ther. source

In the paper

“So far, relatively low gene delivery efficiency and transgene expression have been the major barriers in non-viral vectors-based gene therapy [76, 77].”

What the sources say

• Not checked yetcites [76] Mintzer, 2009, Nonviral vectors for gene delivery, Chem. Rev. source
• Not checked yetcites [77] Davis, 2002, Non-viral gene delivery systems, Curr. Opin. Biotechnol. source

In the paper

“To date, efficient in vivo delivery of the CRISPR/Cas9 system to the targeted cells remains a challenge.”

• Also stated in: § 1. Introduction · hedged

  “Despite the aforementioned merits, efficient delivery may likely become the main hurdle in the eventual application and clinical translation of CRISPR/Cas9 system.”

• Also stated in: § 2. Limitations and challenges of CRISPR/Cas9-based genome editing · plain

  “Meanwhile, achieving efficient delivery of CRISPR system into particular cell types, tissues or organs for therapeutic benefit is another main ongoing challenge.”

In the paper

“In recent years, viral vectors have been extensively applied to deliver CRISPR cassettes in vitro and in vivo largely owing to their high efficiency in gene delivery and long-term stable transgene expression [34, 58, 65].”

What the sources say

• Not checked yetcites [34] Heckl, 2014, Generation of mouse models of myeloid malignancy with combinatorial genetic lesions using CRISPR-Cas9 genome editing, Nat. Biotechnol. source
• Not checked yetcites [58] Yu, 2017, Nrl knockdown by AAV-delivered CRISPR/Cas9 prevents retinal degeneration in mice, Nat. Commun. source
• Not checked yetcites [65] Shalem, 2014, Genome-scale CRISPR-Cas9 knockout screening in human cells, Science source

In the paper

“In addition, CRISPR/Cas9 system has shown potentials in polygenetic diseases, such as cancer, by inhibiting oncogene expression or deactivating oncogenic virus [52–55].”

What the sources say

• Not checked yetcites [52] Xiao-Jie, 2015, CRISPR-Cas9: a new and promising player in gene therapy, J. Med. Genet. source
• Not checked yetcites [53] Sanchez-Rivera, 2015, Applications of the CRISPR-Cas9 system in cancer biology, Nat. Rev. Cancer source
• Not checked yetcites [54] Castillo, 2016, Gene editing using CRISPR-Cas9 for the treatment of lung cancer, Colomb. Med. (Cali.) source
• Not checked yetcites [55] Maddalo, 2014, In vivo engineering of oncogenic chromosomal rearrangements with the CRISPR/Cas9 system, Nature source

In the paper

“Unfortunately, although physical approaches are often successful in the laboratory, these methods are not very amenable for clinical translation.”

In the paper

“Overexpression of drug resistance-associated genes, such as P-gp and Bcl-2, is one of the most consistent alterations in multidrug resistant cells [131–134].”

What the sources say

• Not checked yetcites [131] Borst, 2000, A family of drug transporters: the multidrug resistance-associated proteins, J. Natl. Cancer Inst. source
• Not checked yetcites [132] Thomas, 2003, Overcoming multidrug resistance in cancer: an update on the clinical strategy of inhibiting p-glycoprotein, Cancer Control source
• Not checked yetcites [133] Czabotar, 2014, Control of apoptosis by the BCL-2 protein family: implications for physiology and therapy, Nat. Rev. Mol. Cell Biol. source
• Not checked yetcites [134] Delbridge, 2016, Thirty years of BCL-2: translating cell death discoveries into novel cancer therapies, Nat. Rev. Cancer source

In the paper

“Additionally, due to the instability of the genome of cancer cells, they have evolved many mechanisms to escape CRISPR/Cas9-mediated genome editing, such as mutation of the PAM sequence and the recognition site etc. [120–122].”

What the sources say

• Not checked yetcites [120] Negrini, 2010, Genomic instability–an evolving hallmark of cancer, Nat. Rev. Mol. Cell Biol. source
• Not checked yetcites [121] Shen, 2011, Genomic instability and cancer: an introduction, J. Mol. Cell Biol. source
• Not checked yetcites [122] Tubbs, 2017, Endogenous DNA damage as a source of genomic instability in cancer, Cell source

In the paper

“Most recently, dCas9 fusion proteins-mediated CRISPRi, such as dCas9-KRAB (Krüppel-associated box) and dCas9-LSD1 (lysine-specific histone demethylase 1) system, has been introduced [125–127].”

What the sources say

• Not checked yetcites [125] Thakore, 2015, Highly specific epigenome editing by CRISPR-Cas9 repressors for silencing of distal regulatory elements, Nat. Methods source
• Not checked yetcites [126] Mandegar, 2016, CRISPR interference efficiently induces specific and reversible gene silencing in human iPSCs, Cell Stem Cell source
• Not checked yetcites [127] Kearns, 2015, Functional annotation of native enhancers with a Cas9-histone demethylase fusion, Nat. Methods source

In the paper

“Even more problematic is that the genome editing efficiency of CRISPR/Cas9 system is not high enough under the current conditions.”

In the paper

“Collectively, using non-viral vectors for in vivo delivery of CRISPR/Cas9 system has shown great potential for monogenic disorders treatment.”

In the paper

“However, the large genetic size of Cas9 (~ 4.5 kb) and the total plasmid size (> 7 kb) significantly increase the difficulty of delivery and expression of CRISPR/Cas9 systems [94].”

What the sources say

• Not checked yetcites [94] Ran, 2015, In vivo genome editing using Staphylococcus aureus Cas9, Nature source

In the paper

“However, the efficient in vivo delivery of CRISPR/Cas9 systems is nontrivial due to the increased number of barriers under physiological conditions.”

In the paper

“Acquired drug resistance is the major cause of chemotherapy-based cancer treatment failure [129, 130].”

What the sources say

• Not checked yetcites [129] Kitano, 2004, Cancer as a robust system: implications for anticancer therapy, Nat. Rev. Cancer source
• Not checked yetcites [130] Dean, 2005, Tumour stem cells and drug resistance, Nat. Rev. Cancer source

In the paper

“In the meantime, many non-viral vectors have also been rapidly developed for this purpose.”

In the paper

“Additionally, the relatively poor stability of Cas9 mRNA is also an important obstacle for this type of delivery strategy.”

In the paper

“Moreover, the integration of plasmid into the host genome can be another risk.”

In the paper

“The third option is the plasmid-based CRISPR/Cas9 system [93]. This is an appealing delivery approach owing to the simplicity and low cost of manipulation.”

What the sources say

• Not checked yetcites [93] Ran, 2013, Genome engineering using the CRISPR-Cas9 system, Nat. Protoc. source

In the paper

“Non-viral vectors-mediated transient expression of CRISPR/Cas9 system may be an effective approach to alleviate this problem.”

In the paper

“Many reports have demonstrated that epigenetic alterations play important roles in driving cancer initiation and progression [123, 124].”

What the sources say

• Not checked yetcites [123] Egger, 2004, Epigenetics in human disease and prospects for epigenetic therapy, Nature source
• Not checked yetcites [124] Dawson, 2012, Cancer epigenetics: from mechanism to therapy, Cell source

In the paper

“Thus, the cationic lipids or polymers widely used to encapsulate negatively charged proteins based on electrostatic interaction cannot be applied to deliver the native form of Cas9 protein.”

In the paper

“On the other hand, cationic non-viral vectors used to condense negatively charged CRISPR RNPs and plasmids are prone to interact with plasma proteins and the physiological salt in physiological conditions, resulting in the recognition and clearance by the reticuloendothelial system (RES) [141–143].”

What the sources say

• Not checked yetcites [141] Li, 2015, Polymeric nanocarriers for non-viral gene delivery, J. Biomed. Nanotechnol. source
• Not checked yetcites [142] Dash, 1999, Factors affecting blood clearance and in vivo distribution of polyelectrolyte complexes for gene delivery, Gene Ther. source
• Not checked yetcites [143] Xu, 2011, Drug delivery trends in clinical trials and translational medicine: challenges and opportunities in the delivery of nucleic acid-based therapeutics, J. Pharmacol. Sci. source

In the paper

“However, the accompanying decreased cellular uptake after PEGylation cannot be neglected.”

In the paper

“For example, as a unique feature of solid tumors, the enhanced permeability and retention (EPR) effect can be exploited to promote particles accumulation into tumors.”

In the paper

“Encapsulating these elements in the interior of the vectors would significantly prevent this recognition and subsequent clearance.”

In the paper

“However, some reports also demonstrated the generation of PEG-specific antibodies after administration of PEG-coated liposomes [153, 154].”

What the sources say

• Not checked yetcites [153] Ishida, 2007, PEGylated liposomes elicit an anti-PEG IgM response in a T cell-independent manner, J. Control Release source
• Not checked yetcites [154] Wang, 2007, Anti-PEG IgM elicited by injection of liposomes is involved in the enhanced blood clearance of a subsequent dose of PEGylated liposomes, J. Control Release source

In the paper

“Generally, after being sheltered by non-viral vectors, the recognition by the host immune system would be reduced to some extent.”

In the paper

“The fourth obstacle is the insufficient accumulation of non-viral vector-mediated CRISPR/Cas9 systems in the targeted tissues.”

In the paper

“Like other gene therapy strategies, environmental stimuli-triggered detachment may be a feasible solution to this problem [146].”

What the sources say

• Not checked yetcites [146] Kuai, 2011, Targeted delivery of cargoes into a murine solid tumor by a cell-penetrating peptide and cleavable poly(ethylene glycol) comodified liposomal delivery system via systemic administration, Mol. Pharm. source

In the paper

“Due to the permanent gene editing effect of CRISPR/Cas9 systems in chromosome, the daughter cells will inherit the edited gene sequences of the mother cells.”

In the paper

“Similar to the protein form, Cas9 mRNA and DNA (~ 4.5 kb) also exhibit large size, which increases the difficulty of efficient encapsulation.”

In the paper

“The first obstacle is effective encapsulation of the editing tools in delivery vectors due to the large size and different charge properties of Cas9 protein, mRNA and donor DNA.”

What the sources say

• Not checked yetcites [89] Subburaj, 2016, Site-directed mutagenesis in Petunia x hybrida protoplast system using direct delivery of purified recombinant Cas9 ribonucleoproteins, Plant Cell Rep. source
• Not checked yetcites [88] Zuris, 2015, Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo, Nat. Biotechnol. source
• Not checked yetcites [97] Mout, 2017, Direct cytosolic delivery of CRISPR/Cas9-ribonucleoprotein for efficient gene editing, ACS Nano source
• Not checked yetcites [103] Sun, 2015, Self-assembled DNA nanoclews for the efficient delivery of CRISPR-Cas9 for genome editing, Angew Chem. Int. Ed. Engl. source
• Not checked yetcites [139] Jinek, 2014, Structures of Cas9 endonucleases reveal RNA-mediated conformational activation, Science source

In the paper

“Some reports have demonstrated that EPR effect can provide 20–30% increase in delivery compared with critical normal organs [157].”

What the sources say

• Not checked yetcites [157] Kobayashi, 2013, Improving conventional enhanced permeability and retention (EPR) effects; what is the appropriate target?, Theranostics source

In the paper

“Besides, in contrast with viral vectors, cumulative editing efficacy can be obtained after multiple administrations of non-viral vectors-mediated CRISPR/Cas9 delivery owing to limited immune responses [73, 74, 116, 138].”

What the sources say

• Not checked yetcites [73] Pack, 2005, Design and development of polymers for gene delivery, Nat. Rev. Drug Discov. source
• Not checked yetcites [74] Li, 2007, Non-viral is superior to viral gene delivery, J. Control Release source
• Not checked yetcites [116] Jonathan, 2018, A single administration of CRISPR/Cas9 lipid nanoparticles achieves robust and persistent in vivo genome editing, Cell Rep. source
• Not checked yetcites [138] Yin, 2014, Non-viral vectors for gene-based therapy, Nat. Rev. Genet. source

In the paper

“The third obstacle is the host immune responses triggered by the elements of CRISPR/Cas9 system, which is derived from bacteria.”

In the paper

“Despite all these, the immunogenicity of non-viral vectors is still considered to be much lower than that of viral vectors [74].”

What the sources say

• Not checked yetcites [74] Li, 2007, Non-viral is superior to viral gene delivery, J. Control Release source

In the paper

“Manipulating drug resistance-associated genes using CRISPR/Cas9 systems together with chemotherapy may be a preferable approach to cancer treatment over conventional combination therapy.”

In the paper

“Attaching PEG or other hydrophilic polymers to the surface of the nanoparticles can partially shield them from immune recognition [151, 152].”

What the sources say

• Not checked yetcites [151] Moghimi, 2002, Chemical camouflage of nanospheres with a poorly reactive surface: towards development of stealth and target-specific nanocarriers, Biochim. Biophys. Acta source
• Not checked yetcites [152] Haque, 2016, Combination strategy of multi-layered surface camouflage using hyperbranched polyethylene glycol and immunosuppressive drugs for the prevention of immune reactions against transplanted porcine islets, Biomaterials source

In the paper

“However, it is also worth noting that partial shielding via simple electrostatic interaction may not be effective enough to eliminate the recognition by host immune cells owing to the exposure of neutral domains of these elements.”

In the paper

“The conventional strategy is to inhibit the expression or activity of these genes through small interfering RNA (siRNA) or chemical inhibitors [135–137].”

What the sources say

• Not checked yetcites [135] He, 2014, Nanoscale metal-organic frameworks for the co-delivery of cisplatin and pooled siRNAs to enhance therapeutic efficacy in drug-resistant ovarian cancer cells, J. Am. Chem. Soc. source
• Not checked yetcites [136] Li, 2014, Co-delivery of siRNAs and anti-cancer drugs using layered double hydroxide nanoparticles, Biomaterials source
• Not checked yetcites [137] Huang, 2017, Nanomedicine-based combination anticancer therapy between nucleic acids and small-molecular drugs, Adv. Drug Deliv. Rev. source

In the paper

“Additionally, the properties such as size, surface charge, hydrophobicity/hydrophilicity, and the steric effects of particle coating have influence on the compatibility of nanoparticles with the immune system [147–150].”

What the sources say

• Not checked yetcites [147] Zolnik, 2010, Nanoparticles and the immune system, Endocrinology source
• Not checked yetcites [148] Dobrovolskaia, 2008, Preclinical studies to understand nanoparticle interaction with the immune system and its potential effects on nanoparticle biodistribution, Mol. Pharm. source
• Not checked yetcites [149] Dobrovolskaia, 2007, Immunological properties of engineered nanomaterials, Nat. Nanotechnol. source
• Not checked yetcites [150] Aggarwal, 2009, Nanoparticle interaction with plasma proteins as it relates to particle biodistribution, biocompatibility and therapeutic efficacy, Adv. Drug Deliv. Rev. source

In the paper

“The second obstacle is the stability of complex under physiological conditions.”

In the paper

“It is worth noting that formation of specific antibodies against nanoparticles may affect the efficacy and the safety of nanoparticle-based therapeutics [149, 150].”

What the sources say

• Not checked yetcites [149] Dobrovolskaia, 2007, Immunological properties of engineered nanomaterials, Nat. Nanotechnol. source
• Not checked yetcites [150] Aggarwal, 2009, Nanoparticle interaction with plasma proteins as it relates to particle biodistribution, biocompatibility and therapeutic efficacy, Adv. Drug Deliv. Rev. source

In the paper

“Inspired by the active transportation process of proteins to nucleus, nuclear localization sequences (NLSs) have been introduced into the vectors to facilitate transportation to nucleus [195–197].”

What the sources say

• Not checked yetcites [195] Haberland, 2005, Nuclear proteins as gene-transfer vectors, Biotechnol. Appl. Biochem. source
• Not checked yetcites [196] Chen, 2017, A gene delivery system containing nuclear localization signal: increased nucleus import and transfection efficiency with the assistance of RanGAP1, Acta Biomater. source
• Not checked yetcites [197] Maity, 2016, Efficient subcellular targeting to the cell nucleus of Quantum Dots densely decorated with a nuclear localization sequence peptide, ACS Appl. Mater. Interfaces source

In the paper

“Briefly, 2° and 3° amines on polymer chains can be protonated inside endo-lysosomes and then counterbalance chloride ions flow into the vesicles. This process creates an osmotic gradient between the exterior and interior of the endo-lysosomes, leading to swelling and bursting of these vesicles and releasing the polyplex into the cytosol [178].”

What the sources say

• Not checked yetcites [178] Behr, 1997, The proton sponge: a trick to enter cells the viruses did not exploit, Chim. Int. J. Chem. source

In the paper

“Smaller-sized nanoparticles may be preferred to enhance permeability and penetration over large-sized ones [166, 167].”

What the sources say

• Not checked yetcites [166] Dong, 2017, Microfluidic preparation of drug-loaded PEGylated liposomes, and the impact of liposome size on tumour retention and penetration, J. Liposome Res.
• Not checked yetcites [167] Sun, 2017, Enhancing tumor penetration of nanomedicines, Biomacromolecules source

In the paper

“CRISPR/Cas9 system must be delivered to the nucleus to initiate genome editing effect.”

In the paper

“Cytoplasmic mobility and nuclear import is another big obstacle in the intracellular environment.”

In the paper

“In addition to cellular uptake, endosomal and lysosomal degradation is also a critical challenge for efficient CRISPR/Cas9 delivery due to the acidic environment (pH 5.0–6.2) and hydrolases.”

In the paper

“In principle, naked CRISPR plasmid and sgRNA cannot directly transport through the lipid bilayer of the cell membrane due to the hydrophilic and anionic properties.”

In the paper

“However, smaller-sized nanoparticles can also be rapidly cleared from the systemic circulation through the kidneys.”

In the paper

“A variety of non-viral vectors, including but not limited to polyethylenimine (PEI), poly[2-(dimethylamino) ethyl methacrylate] (PDMAEMA), have “proton-sponge effect”.”

In the paper

“In fact, direct transmembrane delivery of the particles into the cytosol bypassing the endo-lysosomes may be an alternative way [187, 188].”

What the sources say

• Not checked yetcites [187] Deng, 2016, One-step gene delivery into the cytoplasm in a fusion-dependent manner based on a new membrane fusogenic lipid, Chem. Commun. (Camb) source
• Not checked yetcites [188] Tang, 2013, Direct delivery of functional proteins and enzymes to the cytosol using nanoparticle-stabilized nanocapsules, ACS Nano source

In the paper

“Non-viral vectors that compact plasmid into small particles may aid the movement to the nucleus.”

In the paper

“Virus such as herpes simplex virus and some serotypes of adenovirus could travel through the cytoplasm via microtubule-mediated transport [76, 193, 194].”

What the sources say

• Not checked yetcites [76] Mintzer, 2009, Nonviral vectors for gene delivery, Chem. Rev. source
• Not checked yetcites [193] Sodeik, 1997, Microtubule-mediated transport of incoming herpes simplex virus 1 capsids to the nucleus, J. Cell Biol. source
• Not checked yetcites [194] Leopold, 2000, Dynein- and microtubule-mediated translocation of adenovirus serotype 5 occurs after endosomal lysis, Hum. Gene Ther. source

In the paper

“Recently, gas-generating nanoparticles have also been designed to boost endo-lysosomal escape in some studies.”

In the paper

“Meanwhile, some compounds, such as chloroquine, imidazole, and zinc chloride, have also been combined with cationic vectors to further enhance endosomal escape [179–182].”

What the sources say

• Not checked yetcites [179] Hughes, 1996, Evaluation of adjuvants that enhance the effectiveness of antisense oligodeoxynucleotides, Pharm. Res. source
• Not checked yetcites [180] Varkouhi, 2011, Endosomal escape pathways for delivery of biologicals, J. Control Release source
• Not checked yetcites [181] Pichon, 2002, Zinc improves gene transfer mediated by DNA/cationic polymer complexes, J. Gene Med. source
• Not checked yetcites [182] Cho, 2003, Polycation gene delivery systems: escape from endosomes to cytosol, J. Pharm. Pharmacol. source

In the paper

“In addition, inspired by the mechanism of viral infection, some membrane fusion peptides, such as GALA peptides and acid-activatable melittin, have been introduced into non-viral vectors to facilitate endosomal escape [184–186].”

What the sources say

• Not checked yetcites [184] Li, 2004, GALA: a designed synthetic pH-responsive amphipathic peptide with applications in drug and gene delivery, Adv. Drug Deliv. Rev. source
• Not checked yetcites [185] Hou, 2013, Mechanisms of nanoparticle-mediated siRNA transfection by melittin-derived peptides, ACS Nano source
• Not checked yetcites [186] Moore, 2008, The effect of endosomal escape peptides on in vitro gene delivery of polyethylene glycol-based vehicles, J. Gene Med. source

In the paper

“Cellular uptake is the next obstacle owing to the selective transmission of the hydrophobic and negatively charged plasma membrane.”

In the paper

“Disruption or reorganization of the actin cytoskeleton, or depletion of keratin and vimentin in cells, significantly increases the diffusion of gene payloads [190–192].”

What the sources say

• Not checked yetcites [190] Lechardeur, 2006, Nucleocytoplasmic transport of plasmid DNA: a perilous journey from the cytoplasm to the nucleus, Hum. Gene Ther. source
• Not checked yetcites [191] Dauty, 2005, Actin cytoskeleton as the principal determinant of size-dependent DNA mobility in cytoplasm: a new barrier for non-viral gene delivery, J. Biol. Chem. source
• Not checked yetcites [192] Geiger, 2006, Cyclic stretch-induced reorganization of the cytoskeleton and its role in enhanced gene transfer, Gene Ther. source

In the paper

“Cationic polyplexes could move along microtubules by non-specifically interacting with anionic microtubules or motor proteins [76].”

What the sources say

• Not checked yetcites [76] Mintzer, 2009, Nonviral vectors for gene delivery, Chem. Rev. source

In the paper

“Additionally, ultrasound has also been implicated as a potential approach to promote cell entry owing to ultrasound-induced mild hyperthermia and cell membrane deformation [176, 177].”

What the sources say

• Not checked yetcites [176] Cho, 2002, Ultrasound-induced mild hyperthermia as a novel approach to increase drug uptake in brain microvessel endothelial cells, Pharm. Res. source
• Not checked yetcites [177] Wang, 2010, Low-intensity pulsed ultrasound increases cellular uptake of superparamagnetic iron oxide nanomaterial: results from human osteosarcoma cell line U2OS, J. Magn. Reson. Imag. source

In the paper

“The “proton-sponge effect” of polymers with proton buffer capacity can be exploited to promote endo-lysosomal escape.”

In the paper

“Some auxiliary means, such as topical hyperthermia, radiotherapy and high intensity focused ultrasound, etc., can also be used to enhance this effect [158–160].”

What the sources say

• Not checked yetcites [158] Kong, 2000, Hyperthermia enables tumor-specific nanoparticle delivery: effect of particle size, Cancer Res.
• Not checked yetcites [159] Lammers, 2007, Effect of radiotherapy and hyperthermia on the tumor accumulation of HPMA copolymer-based drug delivery systems, J. Control Release source
• Not checked yetcites [160] Ranjan, 2012, Image-guided drug delivery with magnetic resonance guided high intensity focused ultrasound and temperature sensitive liposomes in a rabbit Vx2 tumor model, J. Control Release source

In the paper

“Once released into the cytosol, non-viral vectors loaded with CRISPR/Cas9 system need to overcome additional barriers in the cytosol, including the mesh-like cytoskeleton and nucleolytic enzymes within the cytosolic milieu [190].”

What the sources say

• Not checked yetcites [190] Lechardeur, 2006, Nucleocytoplasmic transport of plasmid DNA: a perilous journey from the cytoplasm to the nucleus, Hum. Gene Ther. source

In the paper

“Besides, for the fibrotic lesion, degradation of the extracellular matrix using collagenase or hyaluronidase can be effective strategies to enhance accumulation [161, 162].”

What the sources say

• Not checked yetcites [161] Eikenes, 2004, Collagenase increases the transcapillary pressure gradient and improves the uptake and distribution of monoclonal antibodies in human osteosarcoma xenografts, Cancer Res. source
• Not checked yetcites [162] Eikenes, 2005, Hyaluronidase induces a transcapillary pressure gradient and improves the distribution and uptake of liposomal doxorubicin (Caelyx) in human osteosarcoma xenografts, Br. J. Cancer source

In the paper

“Besides, cell penetrating peptides (CPP), such as TAT and R8 peptide, have also been extensively used to facilitate cell entry based on the positive charge and amphipathic in nature [174, 175].”

What the sources say

• Not checked yetcites [174] Torchilin, 2008, Tat peptide-mediated intracellular delivery of pharmaceutical nanocarriers, Adv. Drug Deliv. Rev. source
• Not checked yetcites [175] Torchilin, 2008, Cell penetrating peptide-modified pharmaceutical nanocarriers for intracellular drug and gene delivery, Biopolymers source

In the paper

“For cationic complexes, cellular uptake is partly induced through electrostatic interaction between the positive charge of the complexes and the negative charge of the cell membrane.”

In the paper

“For example, due to the overexpression of αvβ3 integrin receptors on tumor vasculature, the nanoparticles decorated with arginine-glycine-glutamic acid (RGD) peptide, a ligand for αvβ3 integrin receptors, have more access to the targeted tumor tissues [163–165].”

What the sources say

• Not checked yetcites [163] Zhang, 2018, 68Ga-BBN-RGD PET/CT for GRPR and integrin αvβ3 imaging in patients with breast cancer, Theranostics source
• Not checked yetcites [164] Li, 2016, Multifunctional "core-shell" nanoparticles-based gene delivery for treatment of aggressive melanoma, Biomaterials source
• Not checked yetcites [165] Amin, 2015, Development of a novel cyclic RGD peptide for multiple targeting approaches of liposomes to tumor region, J. Control Release source

In the paper

“Meanwhile, many types of ligands, such as folate, transferrin, antibody and aptamer, etc., can also be incorporated into the non-viral vectors to boost cellular uptake via receptor-mediated endocytosis [171–173].”

What the sources say

• Not checked yetcites [171] Molas, 2003, Receptor-mediated gene transfer vectors: progress towards genetic pharmaceuticals, Curr. Gene Ther. source
• Not checked yetcites [172] Peer, 2007, Nanocarriers as an emerging platform for cancer therapy, Nat. Nanotechnol. source
• Not checked yetcites [173] Steichen, 2013, A review of current nanoparticle and targeting moieties for the delivery of cancer therapeutics, Eur. J. Pharmaceut. Sci. source

In the paper

“Generally, after complexation with non-viral vectors to form nanoparticles, the cellular uptake would be promoted.”

In the paper

“The recently developed HITI strategy exhibits much higher knock-in efficiency than HDR both in vitro and in vivo [86, 87].”

What the sources say

• Not checked yetcites [86] He, 2016, Knock-in of large reporter genes in human cells via CRISPR/Cas9-induced homology-dependent and independent DNA repair, Nucleic Acids Res. source
• Not checked yetcites [87] Suzuki, 2016, In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration, Nature source

In the paper

“Since the corrected cells have no selection advantages in most genetic disorders, the correction efficiency will undoubtedly need to improve.”

In the paper

“Besides, due to the recognition preference of some specific bases of CRISPR/Cas9 system and the diverse epigenetic landscape, different targeted gene loci may exhibit distinctive different gene editing efficiencies [140, 201–203].”

What the sources say

• Not checked yetcites [140] Kleinstiver, 2015, Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition, Nat. Biotechnol. source
• Not checked yetcites [201] Nishimasu, 2017, Structural basis for the altered PAM recognition by engineered CRISPR-Cpf1, Mol. Cell source
• Not checked yetcites [202] Chylinski, 2014, Classification and evolution of type II CRISPR-Cas systems, Nucleic Acids Res. source
• Not checked yetcites [203] Wang, 2008, Combinatorial patterns of histone acetylations and methylations in the human genome, Nat. Genet. source

In the paper

“Novel effective non-viral vector for CRISPR/Cas9 delivery is still one of the bottlenecks for the successful clinical translation of CRISPR/Cas9-based genome editing to date.”

In the paper

“In addition, delivery alternatives are another main challenge for the application of CRISPR/Cas9 gene editing system both in vitro and in vivo.”

In the paper

“Although numerous non-viral vectors have been developed and exploited in the field of gene therapy over the past decades, the non-viral vectors applied in CRISPR/Cas9 delivery are still very few so far.”

In the paper

“One possible reason is that the large-sized Cas9 protein or plasmid is difficult to be efficiently encapsulated and delivered.”

In the paper

“The mitotic state of the targeted cells is another major concern.”

In the paper

“More importantly, HITI keeps its high efficiency in the non-mitotic cells, which are present in most adult tissues [87].”

What the sources say

• Not checked yetcites [87] Suzuki, 2016, In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration, Nature source

In the paper

“Non-mitotic cells are very inefficient for HDR-based repair, which is the main pathway to correct genetic disorders to date [87, 200].”

What the sources say

• Not checked yetcites [87] Suzuki, 2016, In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration, Nature source
• Not checked yetcites [200] Orthwein, 2015, A mechanism for the suppression of homologous recombination in G1 cells, Nature source