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

In the paper

“Although viral vectors have been widely used in the delivery of the CRISPR/Cas9 system in vitro and in vivo, their fundamental shortcomings, such as the risk of carcinogenesis, limited insertion size, immune responses and difficulty in large-scale production, severely limit their further applications.”

• Also stated in: § 1. Introduction · plain

  “Similarly, although highly efficient, the intrinsic drawbacks associated with viral vectors, including the risk of carcinogenesis, limitation of insertion size, immune responses and difficulty in large-scale production, severely limited their applications [69–72].”

What the sources say

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

  The article’s discussion of the Fischer trial and UK gene-therapy trials supports a risk of insertional mutagenesis leading to cancer from retroviral vectors. It does not address limited insertion size, immune responses, large-scale production, or CRISPR/Cas9 delivery, so it does not support the full list of shortcomings or their stated effect on further applications. The indexed copy provides no section or page labels. (LLM, conf=0.88)

  cites [70] Gore, 2003, Adverse effects of gene therapy: gene therapy can cause leukaemia: no shock, mild horror but a probe, Gene Ther. sourceExperimental — accuracy not yet measured
• Partly supported

  The Summary (p. 1125) reports T-cell lymphomas in monkeys exposed to a retroviral preparation contaminated with replication-competent helper virus, supporting a specific carcinogenesis risk. The paper does not establish limited insertion size, adverse immune responses, or difficulty with large-scale production. Its concluding discussion says the findings should not deter future use of retroviral vectors, so it does not support the claim that these shortcomings severely limit further applications. (LLM, conf=0.90)

  cites [71] Donahue, 1992, Helper virus induced T cell lymphoma in nonhuman primates after retroviral mediated gene transfer, J. Exp. Med. sourceExperimental — accuracy not yet measured
• Not checked yetcites [72] Sun, 2003, Immune responses to adeno-associated virus and its recombinant vectors, Gene Ther. source

In the paper

“However, the desired recombination efficacy often occurs infrequently, which presents enormous challenges for robust applications [61, 82–84].”

What the sources say

• Not checked yetcites [61] Wang, 2013, One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering, Cell source
• Not checked yetcites [82] Chu, 2015, Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells, Nat. Biotechnol. source
• Not checked yetcites [83] Yu, 2015, Small molecules enhance CRISPR genome editing in pluripotent stem cells, Cell Stem Cell source
• Not checked yetcites [84] Yang, 2013, One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering, Cell source

In the paper

“Although inhibiting the NHEJ pathway with gene silencing or chemical inhibitors can partially increase the efficiency of HDR pathway, the gene repair efficacy still needs to be further improved for disease therapy [82, 85].”

What the sources say

• Not checked yetcites [82] Chu, 2015, Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells, Nat. Biotechnol. source
• Not checked yetcites [85] Maruyama, 2015, Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining, Nat. Biotechnol. source

In the paper

“This system induced higher targeted gene disruption efficacy than that of Lipofectamine 3000 in vitro. After intraperitoneal administration, they also showed effective disruption of targeted MTH1 gene and significantly inhibited the progression of ovarian tumor in vivo (Fig. 4A) [117].”

What the sources say

• Not checked yetcites [117] Li, 2017, Artificial vrus delivers CRISPR-Cas9 system for genome editing of cells in mice, ACS Nano source

In the paper

“This delivery system enabled effective knock-outs of target gene (Plk-1) and inhibited the growth of melanoma both in vitro and in vivo (Fig. 4B) [118].”

What the sources say

• Not checked yetcites [118] Wang, 2018, Thermo-triggered release of CRISPR-Cas9 system by lipid-encapsulated gold nanoparticles for tumor therapy, Angew Chem. Int. Ed. Engl. source

In the paper

“All these results demonstrated that this system substantially reduced progressive hearing loss in Beethoven mouse model (Fig. 3B) [113].”

What the sources say

• Not checked yetcites [113] Gao, 2018, Treatment of autosomal dominant hearing loss by in vivo delivery of genome editing agents, Nature source

In the paper

“After being injected into the hindlimb muscle of mdx mice simultaneously with cardiotoxin (CTX), this system efficiently corrected the DNA mutation that causes DMD in mice with reduced off-target effect and reduced muscle fibrosis in mdx mice (Fig. 3A) [110].”

What the sources say

• Not checked yetcites [110] Lee, 2017, Nanoparticle delivery of Cas9 ribonucleoprotein and donor DNA in vivo induces homology-directed DNA repair, Nat. Biomed. Eng. source

In the paper

“Ramakrishna et al. developed cell-penetrating peptide (CPP)-conjugated recombinant Cas9 protein and CPP-complexed guide RNAs for gene disruptions of CCR5 locus in human cell lines, including HEK293T, HeLa, NCCIT (a human pluripotent embryonal carcinoma cell line), human dermal fibroblasts and human embryonic stem cells.”

• Also stated in: § 4.1 Non-viral vectors in vitro and ex vivo · plain

  “This system led to efficient disruptions of CCR5 gene with minimal off-target mutations [96].”

What the sources say

• Not checked yetcites [96] Ramakrishna, 2014, Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA, Genome Res. source

In the paper

“With a single intravenous administration, this system enabled significant editing of the TTR gene in the liver of mice or rats, and resulted in a dramatic reduction (>97% for mice, >90% for rats) in serum TTR protein levels.”

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

“Yin et al. combined lipid nanoparticle-mediated delivery of Cas9 mRNA with AAV encoding sgRNAs and donor templates to repair FAH gene in a mouse model of human hereditary tyrosinemia. The efficiency of correction was >6% of hepatocytes after a single application and the treatment rescued disease symptoms such as weight loss and liver damage [112].”

What the sources say

• Not checked yetcites [112] Yin, 2016, Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo, Nat. Biotechnol. source

In the paper

“However, with the rapid development of novel biomaterials in recent years, efficient delivery of gene payloads to pass the multiple barriers under physiological conditions and promote transgene expression can be achieved.”

In the paper

“Nevertheless, with the rapid development of various synthetic vectors, non-viral vectors are very likely to provide unique advantages over viral vectors in these fields.”

In the paper

“It also offers the most transient functionality of genome editing cassette with reduced off-target effects and toxicity.”

In the paper

“As an alternative, non-viral vectors may offer tantalizing possibility in CRISPR/Cas9 delivery with respect to their low immunogenicity, absence of endogenous virus recombination, less limitation in delivering larger genetic payloads and ease of large-scale production [73–75].”

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 [75] Li, 2015, Challenges in CRISPR/CAS9 delivery: potential roles of nonviral vectors, Hum. Gene Ther. source

In the paper

“Furthermore, CRISPR/Cas9 system holds tremendous promise for gene therapy [46–51]. It can correct causal mutations in the original genome and rescue the disease phenotypes of monogenic disorders permanently, which currently represents the most translatable field in CRISPR/Cas9-mediated disease therapy.”

What the sources say

• Not checked yetcites [46] Yin, 2014, Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype, Nat. Biotechnol. source
• Not checked yetcites [47] Wu, 2013, Correction of a genetic disease in mouse via use of CRISPR-Cas9, Cell Stem Cell source
• Not checked yetcites [48] Lin, 2014, The CRISPR/Cas9 system facilitates clearance of the intrahepatic HBV templates in vivo, Mol. Ther. Nucleic Acids source
• Not checked yetcites [49] Dever, 2016, CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells, Nature source
• Not checked yetcites [50] Gaj, 2017, In vivo genome editing improves motor function and extends survival in a mouse model of ALS, Sci. Adv. source
• Not checked yetcites [51] Bengtsson, 2017, Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy, Nat. Commun. source

In the paper

“However, due to the multiple functions of epigenetics, safety is a big concern that needs special attention when applying these strategies.”

In the paper

“The second obstacle for plasmid-based expression is the requirement of nuclear entry for DNA transcription, which partially decreases the genome editing efficiency and also induces the delay in therapeutic efficacy.”

In the paper

“Furthermore, Cas9 mRNA delivery provides transient expression of Cas9 protein, which may be helpful for decreasing the off-target editing events.”

In the paper

“Like RNPs, Cas9 mRNA administration results in quick onset of genome editing, as Cas9 mRNA bypasses the requirement of nuclear entry for transcription.”

In the paper

“Monogenic diseases can tolerate a low initial gene editing efficiency due to the selective advantages of the corrected cells, while cancer treatment requires relatively high editing efficiency to ensure effective therapy, because the unedited cancer cells possess selective advantages and proliferate quickly.”

In the paper

“In addition, the plasmid-based expression often leads to a longer duration of Cas9 protein, which may result in higher off-target effects and strong immune responses [95].”

What the sources say

• Not checked yetcites [95] Hughes, 2009, Immunogenicity of intrathecal plasmid gene delivery: cytokine release and effects on transgene expression, J. Gene Med. source

In the paper

“Generally, CRISPR/Cas9 systems delivered by viral or no-viral vectors are more likely to work well in vitro and ex vivo owing to the direct interaction with cells.”

In the paper

“dCas9-KRAB system, recruiting a diverse array of histone modifiers that reversibly suppresses gene expression through the formation of heterochromatin, significantly reduced the expression of a series of endogenous genes in the genome [125, 128].”

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 [128] Thakore, 2016, Editing the epigenome: technologies for programmable transcription and epigenetic modulation, Nat. Methods source

In the paper

“In recent years, CRISPR/Cas9 systems have been widely applied in manipulating mouse zygotes and cultured cells ex vivo and in vitro.”

In the paper

“Generally, the particles should be nano-sized and the junction of endothelial cells has a great influence on the extravasation of these particles.”

In the paper

“The molecular weight of the commonly used Cas9 protein from streptococcus pyogenes is about 160 KD, larger than that of the most proteins [89].”

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

In the paper

“Following systemic administration, the elements of CRISPR/Cas9 system should remain stable before reaching its target site. Then the elements need to be effectively taken up by the targeted cell types and escape from endo-lysosome system to avoid degradation.”

In the paper

“This issue is very important for targeted gene editing efficacy as well as the off-target probability in other tissues.”

In the paper

“There are two main limitations of non-viral vectors-mediated CRISPR/Cas9 delivery in vivo. The first one is the serum stability of polyplexes after systemic administration [73]. The second one is the relatively low transfection efficiency in the targeted tissues after administration in vivo [76, 77].”

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 [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

“Meanwhile, native Cas9 protein is positively charged, while the conventional proteins are negatively charged [88, 97, 103, 139].”

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 [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

“However, the effects of either siRNA or chemical inhibitor are transient and are limited to the mother cell that takes the drug. If the cell divides, these drugs will lose activity in the daughter cells.”

In the paper

“These issues are not just for non-viral vectors-mediated CRISPR/Cas9 delivery, but for non-viral vectors-mediated gene delivery in general.”

In the paper

“On the one hand, protein, plasmid and RNA are vulnerable to proteases and nucleases in the blood. They can degrade quickly in naked form in systemic circulation.”

In the paper

“Although the newly discovered Cas9 form from Staphylococcus aureus strain (SaCas9) is about 1 kb shorter than SpCas9, the overall genetic size is still too large for application [94, 140].”

What the sources say

• Not checked yetcites [94] Ran, 2015, In vivo genome editing using Staphylococcus aureus Cas9, Nature source
• Not checked yetcites [140] Kleinstiver, 2015, Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition, Nat. Biotechnol. source

In the paper

“PEGylation, the most commonly used strategy to reduce the opsonization and minimize the clearance by RES, may also be applicable for non-viral vectors-mediated CRISPR/Cas9 delivery [144, 145].”

What the sources say

• Not checked yetcites [144] Ogris, 1999, PEGylated DNA/transferrin-PEI complexes: reduced interaction with blood components, extended circulation in blood and potential for systemic gene delivery, Gene Ther. source
• Not checked yetcites [145] Malam, 2011, Current trends in the application of nanoparticles in drug delivery, Curr. Med. Chem. source

In the paper

“In the past decades, there have been extensive efforts on facilitating particles accumulation in the targeted tissues for non-viral delivery vectors-mediated gene therapy [155, 156].”

What the sources say

• Not checked yetcites [155] Liu, 2017, Tumor associated macrophage-targeted microRNA delivery with dual-responsive polypeptide nanovectors for anti-cancer therapy, Biomaterials source
• Not checked yetcites [156] Li, 2017, Targeted Co-delivery of PTX and TR3 siRNA by PTP peptide modified dendrimer for the treatment of pancreatic cancer, Small

In the paper

“Additionally, surface decorating active targeting ligands on the vectors should be another effective method to promote targeted accumulation via receptor-mediated recognition.”

In the paper

“Liu et al. fabricated pH-responsive poly(D,L-lactic-co-glycolic acid) (PLGA) nanoparticles encapsulating ammonium bicarbonate (NH4HCO3) to trigger antigen release from endo-lysosomes. NH4HCO3 interacted with protons in endo-lysosomes, then produced CO2 and NH3 to disrupt the endo-lysosomes and promote antigen release into the cytosol [183].”

What the sources say

• Not checked yetcites [183] Liu, 2015, pH-responsive poly(D,L-lactic-co-glycolic acid) nanoparticles with rapid antigen release behavior promote immune response, ACS Nano source

In the paper

“One underlying mechanism is these peptides undergo a structural transformation from a random coil to an α-helical conformation in lower pH environments, thus promoting endosomal fusion.”

In the paper

“For CRISPR/Cas9-based plasmids, they need to overcome more barriers than that of CRISPR RNPs and mRNA. They have to enter the nucleus for transcription and traffic back to the cytosol for translation.”

In the paper

“Due to the threshold for rapid renal excretion being about 5.5 nm, the hydrodynamic diameters of the prepared nanoparticles need to be larger than this size [168–170].”

What the sources say

• Not checked yetcites [168] Choi, 2007, Renal clearance of quantum dots, Nat. Biotechnol. source
• Not checked yetcites [169] Longmire, 2008, Clearance properties of nano-sized particles and molecules as imaging agents: considerations and caveats, Nanomedicine (Lond) source
• Not checked yetcites [170] Choi, 2010, Design considerations for tumour-targeted nanoparticles, Nat. Nanotechnol. source

In the paper

“The fifth encountered obstacle is the phagocytic clearance, blockage of RES in targeted tissues especially in liver and spleen, and the negatively charged components of extracellular matrix.”

In the paper

“Nanoparticles smaller than 9 nm can passively diffuse through nuclear pore complexes (NPCs), while larger ones requiring the assistance of import proteins (e.g., importins) to actively shuttle them into the nucleus [73].”

What the sources say

• Not checked yetcites [73] Pack, 2005, Design and development of polymers for gene delivery, Nat. Rev. Drug Discov. source

In the paper

“Some studies have shown that hyperthermia can increase the membrane permeability of compounds and enhance cellular uptake. But the temperature and duration of this approach need to be carefully controlled.”

In the paper

“To balance the effect of penetration and renal excretion, environmental stimuli-responsive size changeable tactics would be a possible compromising solution.”

In the paper

“It seems like that the cationic lipid-based vectors were one of the most effective delivery approaches for CRISPR/Cas9 system to date. But it is mainly limited to local administration.”

In the paper

“Meanwhile, different cell types or targeted genes can also affect the efficiency of the CRISPR/Cas9 system [93, 199].”

What the sources say

• Not checked yetcites [93] Ran, 2013, Genome engineering using the CRISPR-Cas9 system, Nat. Protoc. source
• Not checked yetcites [199] Hou, 2013, Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis, Proc. Natl. Acad. Sci. U. S. A. source

In the paper

“NLSs can be recognized by importin α, the nuclear import receptor, and trigger the subsequent transportation from the cytosol to the nucleus [198].”

What the sources say

• Not checked yetcites [198] Fontes, 2000, Structural basis of recognition of monopartite and bipartite nuclear localization sequences by mammalian importin-α, J. Mol. Biol. source

In the paper

“Next, the efficiency will also need to be improved for in vivo application, especially in correcting majority of genetic disorders.”

In the paper

“Moreover, unlike commonly delivery approaches for siRNA or protein, whose action site is in the cytosol, CRISPR/Cas9 systems must be delivered to the nucleus to initiate genome editing in the chromosome.”

In the paper

“For the former off-target effects, tissue-specific promoters in CRISPR/Cas9-based plasmid have been introduced into the system to partially solve this problem.”

In the paper

“Most NLSs are characterized by short basic and cationic peptide sequences.”

In the paper

“However, the specificity of this technology will need to be further improved to decrease the potential off-target probability for increasing safety.”

In the paper

“Meanwhile, targeted delivery of CRISPR/Cas9 system by tissue-specific AAV type or non-viral vectors could be another effective solution to this issue.”

In the paper

“The latter would particularly benefit from non-viral vectors.”

In the paper

“In addition, proper selection of the targeting site, rational design of sgRNA and using paired Cas9 nickases could be effective approaches to alleviate the latter off-target effects.”

In the paper

“These issues may be resolved by enhancing activity and delivery efficiency of the CRISPR-Cas9 system.”