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Cited by 415RNA Interference and Gene DeliveryCRISPR and Genetic EngineeringNanoparticle-Based Drug Delivery

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

Open access
Li, Ling, Hu, Shuo, Chen, Xiaoyuan·2018·Biomaterials
DOI 10.1016/j.biomaterials.2018.04.031PMC PMC5944364
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Abstract

In recent years, CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) genome editing systems have become one of the most robust platforms in basic biomedical research and therapeutic applications. To date, efficient in vivo delivery of the CRISPR/Cas9 system to the targeted cells remains a challenge. 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. Alternative non-viral delivery systems for CRISPR/Cas9 are urgently needed. With the rapid development of non-viral vectors, lipid- or polymer-based nanocarriers have shown great potential for CRISPR/Cas9 delivery. In this review, we analyze the pros and cons of delivering CRISPR/Cas9 systems in the form of plasmid, mRNA, or protein and then discuss the limitations and challenges of CRISPR/Cas9-based genome editing. Furthermore, current non-viral vectors that have been applied for CRISPR/Cas9 delivery in vitro and in vivo are outlined in details. Finally, critical obstacles for non-viral delivery of CRISPR/Cas9 system are highlighted and promising strategies to overcome these barriers are proposed.

AI snapshot

Written from the paper’s full text.

TL;DR

This review argues that efficient in vivo delivery remains a major hurdle for CRISPR/Cas9 genome editing and that non-viral vectors are promising alternatives to viral vectors despite lower delivery efficiency. It surveys plasmid, mRNA, and protein delivery formats, then outlines non-viral vector examples and extracellular/intracellular barriers to clinical translation.

Key findings

  • Non-viral vectors may offer low immunogenicity, absence of endogenous virus recombination, less limitation on large genetic payloads, and ease of large-scale production, but relatively low gene delivery efficiency and transgene expression remain major barriers.
  • The review compares three CRISPR/Cas9 delivery formats: plasmid DNA is simple and stable but large (>7 kb total plasmid) and requires nuclear entry; mRNA gives quick, transient expression but has poor stability and possible low efficiency; Cas9/sgRNA ribonucleoprotein gives the swiftest, most transient editing but is difficult to deliver due to Cas9 size (~160 kD) and charge.
  • Reported non-viral delivery examples include up to 30% editing in HeLa, HEK-293T, and Raw 264.7 cells with arginine-modified gold nanoparticles; 37% reduction in EGFP expression in CHO cells over 4 days with ZIFs; ~39% editing in AGS cells with GO-PEG-PEI; ~20% Cas9-mediated genome modification in cochlear hair cells after Cas9:sgRNA injection; >6% hepatocyte correction in a hereditary tyrosinemia mouse model; and >97% (mice) or >90% (rats) reduction in serum TTR protein levels with LNP-INT01, with editing persisting at least 12 months.
  • The paper highlights barriers including encapsulation difficulty, physiological instability, host immune responses, insufficient targeted accumulation, phagocytic/RES clearance, cellular uptake, endosomal/lysosomal degradation, cytoplasmic mobility, and nuclear import.

Methods

  • Design: narrative review of non-viral delivery systems for CRISPR/Cas9-based genome editing, focusing on challenges and opportunities rather than presenting new primary data.
  • Data/sample: published literature and selected in vitro and in vivo studies on CRISPR/Cas9 delivery using plasmid, mRNA, or protein formats and non-viral vectors.
  • Main analysis: thematic synthesis of delivery formats, non-viral vector examples, disease applications, and extracellular/intracellular delivery barriers, with proposed strategies to overcome them.

Limitations

  • The paper states that non-viral vectors face two main in vivo limitations: serum stability of polyplexes after systemic administration and relatively low transfection efficiency in targeted tissues. It also states that non-viral vectors applied in CRISPR/Cas9 delivery remain very few and that cationic lipid-based vectors, though among the most effective to date, are mainly limited to local administration.
  • The paper states multiple delivery obstacles: effective encapsulation due to large size and charge properties of Cas9 protein, mRNA, and donor DNA; instability under physiological conditions; host immune responses; insufficient accumulation in target tissues; phagocytic clearance and RES blockage; cellular uptake; endosomal/lysosomal degradation; and cytoplasmic mobility/nuclear import.
  • Evident from the text: this is a narrative review with selected examples rather than a systematic review or meta-analysis, so it does not provide quantitative pooled estimates or comprehensive comparative efficacy across all non-viral systems.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1016/j.biomaterials.2018.04.031

Cites

205 of 205 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 167 cited sources checked · 0 supported · 0 flagged

  • Findingcitation

    HITI strategy allows for robust DNA knock-in in both dividing and non-dividing cells in vitro and in vivo.

    “This approach allows for robust DNA knock-in in both dividing and non-dividing cells in vitro and in vivo [87].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [87] Suzuki, 2016, In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration, Nature source

      Not checked in the free run.

  • Findingcitation

    The editing levels from LNP-INT01 can persist for at least 12 months, despite the transient nature of the delivery system and the editing components.

    “More importantly, the editing levels can persist for at least 12 months, despite the transient nature of the delivery system and the editing components.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [116] Jonathan, 2018, A single administration of CRISPR/Cas9 lipid nanoparticles achieves robust and persistent in vivo genome editing, Cell Rep. source

      Not checked in the free run.

  • Findingcitation

    Miller et al.'s zwitterionic amino lipid vector showed permanent editing of LoxP gene in genetically engineered mice after intravenous administration.

    “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].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [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

      Not checked in the free run.

  • Findingcitation

    Jiang et al.'s lipid-like nanoparticles achieved disruption of HBV DNA and pcsk9 gene in vivo.

    “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].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [104] Jiang, 2017, A non-viral CRISPR/Cas9 delivery system for therapeutically targeting HBV DNA and pcsk9 in vivo, Cell Res. source

      Not checked in the free run.

  • Findingcitation

    Zuris et al.'s injection of Cas9:sgRNA complexes targeting GFP gene into the cochlea of transgenic Atoh1-GFP mice resulted in ~20% Cas9-mediated genome modification in hair cells.

    “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].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [69] Lehrman, 1999, Virus treatment questioned after gene therapy death, Nature source

      Not checked in the free run.

  • May combine two claims. 39
  • The quote only partly states this claim. 1
  • Classified as probably not a substantial claim. 19

  • May combine two claims.

    Findingcitation

    The desired recombination efficacy of HDR often occurs infrequently, which presents enormous challenges for robust applications.

    “However, the desired recombination efficacy often occurs infrequently, which presents enormous challenges for robust applications [61, 82–84].”
    Quote in the paperSubstantial claim0 of 4 sources checked
    • cites [61] Wang, 2013, One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering, Cell source

      Not checked in the free run.

    • cites [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 in the free run.

    • cites [83] Yu, 2015, Small molecules enhance CRISPR genome editing in pluripotent stem cells, Cell Stem Cell source

      Not checked in the free run.

    • cites [84] Yang, 2013, One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering, Cell source

      Not checked in the free run.

  • May combine two claims.

    Findingcitation

    Inhibiting the NHEJ pathway with gene silencing or chemical inhibitors can partially increase the efficiency of HDR pathway, but the gene repair efficacy still needs to be further improved for disease therapy.

    “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].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [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 in the free run.

    • cites [85] Maruyama, 2015, Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining, Nat. Biotechnol. source

      Not checked in the free run.

  • May combine two claims.

    Findingcitation

    Li et al.'s core-shell artificial virus induced higher targeted gene disruption efficacy than Lipofectamine 3000 in vitro and inhibited ovarian tumor progression in vivo.

    “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].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [117] Li, 2017, Artificial vrus delivers CRISPR-Cas9 system for genome editing of cells in mice, ACS Nano source

      Not checked in the free run.

  • May combine two claims.

    Findingcitation

    Wang et al.'s LACP delivery system enabled effective knock-outs of Plk-1 and inhibited the growth of melanoma both in vitro and in vivo.

    “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].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [118] Wang, 2018, Thermo-triggered release of CRISPR-Cas9 system by lipid-encapsulated gold nanoparticles for tumor therapy, Angew Chem. Int. Ed. Engl. source

      Not checked in the free run.

  • The quote only partly states this claim.

    Findingcitation

    Local injection of Cas9-Tmc1-mut3 sgRNA-lipid complexes into the cochlea of neonatal Tmc1Bth/+ mice substantially reduced progressive hearing loss in the Beethoven mouse model.

    “All these results demonstrated that this system substantially reduced progressive hearing loss in Beethoven mouse model (Fig. 3B) [113].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [113] Gao, 2018, Treatment of autosomal dominant hearing loss by in vivo delivery of genome editing agents, Nature source

      Not checked in the free run.

  • May combine two claims.

    Findingcitation

    Lee et al.'s CRISPR-Gold system efficiently corrected the DNA mutation that causes DMD in mdx mice with reduced off-target effect and reduced muscle fibrosis.

    “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].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [110] Lee, 2017, Nanoparticle delivery of Cas9 ribonucleoprotein and donor DNA in vivo induces homology-directed DNA repair, Nat. Biomed. Eng. source

      Not checked in the free run.

  • May combine two claims.

    Findingcitation

    Ramakrishna et al. developed CPP-conjugated recombinant Cas9 protein and CPP-complexed guide RNAs for gene disruptions of CCR5 locus in human cell lines, leading to efficient disruptions of CCR5 gene with minimal off-target mutations.

    “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].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [96] Ramakrishna, 2014, Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA, Genome Res. source

      Not checked in the free run.

  • May combine two claims.

    Findingcitation

    LNP-INT01 enabled significant editing of the TTR gene in the liver of mice or rats, resulting in a dramatic reduction (>97% for mice, >90% for rats) in serum TTR protein levels after a single intravenous administration.

    “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.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [116] Jonathan, 2018, A single administration of CRISPR/Cas9 lipid nanoparticles achieves robust and persistent in vivo genome editing, Cell Rep. source

      Not checked in the free run.

  • May combine two claims.

    Findingcitation

    Yin et al.'s lipid nanoparticle-mediated delivery of Cas9 mRNA with AAV encoding sgRNAs and donor templates repaired FAH gene in a mouse model of human hereditary tyrosinemia, with correction efficiency >6% of hepatocytes after a single application and rescued disease symptoms.

    “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].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [112] Yin, 2016, Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo, Nat. Biotechnol. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S4 (based).

    Interpretationnone given

    With the rapid development of novel biomaterials, efficient delivery of gene payloads to pass multiple barriers under physiological conditions and promote transgene expression can be achieved.

    “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.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S2 (checkable), S4 (based).

    Interpretationnone given

    With the rapid development of various synthetic vectors, non-viral vectors are very likely to provide unique advantages over viral vectors for in vivo CRISPR/Cas9 delivery.

    “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.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    Directly delivering native Cas9 protein with sgRNA offers the most transient functionality of genome editing cassette with reduced off-target effects and toxicity.

    “It also offers the most transient functionality of genome editing cassette with reduced off-target effects and toxicity.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationcitation

    Non-viral vectors may offer advantages over viral vectors for CRISPR/Cas9 delivery, including low immunogenicity, absence of endogenous virus recombination, less limitation in delivering larger genetic payloads, and ease of large-scale production.

    “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].”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [73] Pack, 2005, Design and development of polymers for gene delivery, Nat. Rev. Drug Discov. source

      Not checked in the free run.

    • cites [74] Li, 2007, Non-viral is superior to viral gene delivery, J. Control Release source

      Not checked in the free run.

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

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Viral vectors have fundamental shortcomings, including risk of carcinogenesis, limited insertion size, immune responses, and difficulty in large-scale production, which severely limit their further applications.

    “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].”
    Quote in the paperSubstantial claim0 of 4 sources checked
    • cites [69] Lehrman, 1999, Virus treatment questioned after gene therapy death, Nature source

      Not checked in the free run.

    • cites [70] Gore, 2003, Adverse effects of gene therapy: gene therapy can cause leukaemia: no shock, mild horror but a probe, Gene Ther. source

      Not checked in the free run.

    • cites [71] Donahue, 1992, Helper virus induced T cell lymphoma in nonhuman primates after retroviral mediated gene transfer, J. Exp. Med. source

      Not checked in the free run.

    • cites [72] Sun, 2003, Immune responses to adeno-associated virus and its recombinant vectors, Gene Ther. source

      Not checked in the free run.

  • May combine two claims.

    Interpretationcitation

    CRISPR/Cas9 system holds tremendous promise for gene therapy and can correct causal mutations in the original genome and rescue disease phenotypes of monogenic disorders permanently.

    “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.”
    Quote in the paperSubstantial claim0 of 6 sources checked
    • cites [46] Yin, 2014, Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype, Nat. Biotechnol. source

      Not checked in the free run.

    • cites [47] Wu, 2013, Correction of a genetic disease in mouse via use of CRISPR-Cas9, Cell Stem Cell source

      Not checked in the free run.

    • cites [48] Lin, 2014, The CRISPR/Cas9 system facilitates clearance of the intrahepatic HBV templates in vivo, Mol. Ther. Nucleic Acids source

      Not checked in the free run.

    • cites [49] Dever, 2016, CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells, Nature source

      Not checked in the free run.

    • cites [50] Gaj, 2017, In vivo genome editing improves motor function and extends survival in a mouse model of ALS, Sci. Adv. source

      Not checked in the free run.

    • cites [51] Bengtsson, 2017, Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy, Nat. Commun. source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Interpretationreasoning

    Due to multiple functions of epigenetics, safety is a big concern when applying these strategies.

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

  • May combine two claims.

    Interpretationnone given

    The requirement of nuclear entry for DNA transcription partially decreases the genome editing efficiency and also induces the delay in therapeutic efficacy.

    “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.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationnone given

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

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

  • May combine two claims.

    Interpretationreasoning

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

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

  • May combine two claims.

    Interpretationreasoning

    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.

    “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.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationcitation

    Plasmid-based expression often leads to a longer duration of Cas9 protein, which may result in higher off-target effects and strong immune responses.

    “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].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [95] Hughes, 2009, Immunogenicity of intrathecal plasmid gene delivery: cytokine release and effects on transgene expression, J. Gene Med. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning

    Generally, CRISPR/Cas9 systems delivered by viral or non-viral vectors are more likely to work well in vitro and ex vivo owing to the direct interaction with cells.

    “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.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    dCas9-KRAB system reversibly suppresses gene expression through heterochromatin formation and significantly reduced expression of a series of endogenous genes.

    “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].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [125] Thakore, 2015, Highly specific epigenome editing by CRISPR-Cas9 repressors for silencing of distal regulatory elements, Nat. Methods source

      Not checked in the free run.

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

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Premisenone given

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

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

  • May combine two claims.

    Premisenone given

    Particles should be nano-sized and junction of endothelial cells has great influence on extravasation of these particles.

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

  • May combine two claims.

    Premisecitation

    Cas9 protein from S. pyogenes has molecular weight about 160 KD, larger than most proteins.

    “The molecular weight of the commonly used Cas9 protein from streptococcus pyogenes is about 160 KD, larger than that of the most proteins [89].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [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 in the free run.

  • Probably not a substantial claim: may not pass S4 (based). May combine two claims.

    Interpretationnone given

    Following systemic administration, CRISPR/Cas9 elements must remain stable before reaching target site, be effectively taken up by targeted cell types, and escape endo-lysosome system to avoid degradation.

    “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.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationnone given

    Insufficient accumulation is important for targeted gene editing efficacy as well as off-target probability in other tissues.

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

  • May combine two claims.

    Premisecitation

    Non-viral vectors face two main limitations in systemic delivery in vivo: serum stability of polyplexes after systemic administration and relatively low transfection efficiency in targeted tissues after administration.

    “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].”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [73] Pack, 2005, Design and development of polymers for gene delivery, Nat. Rev. Drug Discov. source

      Not checked in the free run.

    • cites [76] Mintzer, 2009, Nonviral vectors for gene delivery, Chem. Rev. source

      Not checked in the free run.

    • cites [77] Davis, 2002, Non-viral gene delivery systems, Curr. Opin. Biotechnol. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Native Cas9 protein is positively charged, while conventional proteins are negatively charged.

    “Meanwhile, native Cas9 protein is positively charged, while the conventional proteins are negatively charged [88, 97, 103, 139].”
    Quote in the paperSubstantial claim0 of 4 sources checked
    • cites [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 in the free run.

    • cites [97] Mout, 2017, Direct cytosolic delivery of CRISPR/Cas9-ribonucleoprotein for efficient gene editing, ACS Nano source

      Not checked in the free run.

    • cites [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 in the free run.

    • cites [139] Jinek, 2014, Structures of Cas9 endonucleases reveal RNA-mediated conformational activation, Science source

      Not checked in the free run.

  • May combine two claims.

    Interpretationnone given

    Effects of either siRNA or chemical inhibitor are transient and limited to mother cell that takes drug; if cell divides, drugs lose activity in daughter cells.

    “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.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Interpretationnone given

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

    “These issues are not just for non-viral vectors-mediated CRISPR/Cas9 delivery, but for non-viral vectors-mediated gene delivery in general.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisenone given

    Protein, plasmid and RNA are vulnerable to proteases and nucleases in blood and can degrade quickly in naked form in systemic circulation.

    “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.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    SaCas9 is about 1 kb shorter than SpCas9, but overall genetic size is still too large for application.

    “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].”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [94] Ran, 2015, In vivo genome editing using Staphylococcus aureus Cas9, Nature source

      Not checked in the free run.

    • cites [140] Kleinstiver, 2015, Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition, Nat. Biotechnol. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    PEGylation is most commonly used strategy to reduce opsonization and minimize clearance by RES, may be applicable for non-viral vectors-mediated CRISPR/Cas9 delivery.

    “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].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [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 in the free run.

    • cites [145] Malam, 2011, Current trends in the application of nanoparticles in drug delivery, Curr. Med. Chem. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Extensive efforts on facilitating particle accumulation in targeted tissues for non-viral delivery vectors-mediated gene therapy exist.

    “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].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [155] Liu, 2017, Tumor associated macrophage-targeted microRNA delivery with dual-responsive polypeptide nanovectors for anti-cancer therapy, Biomaterials source

      Not checked in the free run.

    • cites [156] Li, 2017, Targeted Co-delivery of PTX and TR3 siRNA by PTP peptide modified dendrimer for the treatment of pancreatic cancer, Small

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing), S4 (based).

    Interpretationnone given

    Surface decorating active targeting ligands on vectors should be another effective method to promote targeted accumulation via receptor-mediated recognition.

    “Additionally, surface decorating active targeting ligands on the vectors should be another effective method to promote targeted accumulation via receptor-mediated recognition.”
    Quote in the paperNot a substantial claim
    • The authors state this as an assumption: Strategies for facilitating particle accumulation in targeted tissues for non-viral delivery vectors-mediated gene therapy can also be applied to non-viral vectors-mediated CRISPR/Cas9 genome editing.

  • May combine two claims.

    Premisecitation

    Liu et al. fabricated pH-responsive PLGA nanoparticles encapsulating NH4HCO3 to trigger antigen release from endo-lysosomes; NH4HCO3 interacted with protons, produced CO2 and NH3 to disrupt endo-lysosomes and promote antigen release into cytosol.

    “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].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [183] Liu, 2015, pH-responsive poly(D,L-lactic-co-glycolic acid) nanoparticles with rapid antigen release behavior promote immune response, ACS Nano source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    These peptides undergo structural transformation from random coil to α-helical conformation in lower pH environments, promoting endosomal fusion.

    “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.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    For CRISPR/Cas9-based plasmids, they need to overcome more barriers than CRISPR RNPs and mRNA; they must enter nucleus for transcription and traffic back to cytosol for translation.

    “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.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Threshold for rapid renal excretion is about 5.5 nm; hydrodynamic diameters of prepared nanoparticles need to be larger than this size.

    “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].”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [168] Choi, 2007, Renal clearance of quantum dots, Nat. Biotechnol. source

      Not checked in the free run.

    • cites [169] Longmire, 2008, Clearance properties of nano-sized particles and molecules as imaging agents: considerations and caveats, Nanomedicine (Lond) source

      Not checked in the free run.

    • cites [170] Choi, 2010, Design considerations for tumour-targeted nanoparticles, Nat. Nanotechnol. source

      Not checked in the free run.

  • May combine two claims.

    Premisenone given

    Fifth obstacle: phagocytic clearance, blockage of RES in targeted tissues especially liver and spleen, and negatively charged components of extracellular matrix.

    “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.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Nanoparticles smaller than 9 nm can passively diffuse through nuclear pore complexes, while larger ones require assistance of import proteins (e.g., importins) to actively shuttle them into nucleus.

    “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].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [73] Pack, 2005, Design and development of polymers for gene delivery, Nat. Rev. Drug Discov. source

      Not checked in the free run.

  • May combine two claims.

    Premisenone given

    Hyperthermia can increase membrane permeability of compounds and enhance cellular uptake, but temperature and duration need careful control.

    “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.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Interpretationreasoning

    To balance penetration and renal excretion, environmental stimuli-responsive size changeable tactics would be possible compromising solution.

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

  • May combine two claims.

    Premisenone given

    Cationic lipid-based vectors were one of the most effective delivery approaches for CRISPR/Cas9 system to date, but mainly limited to local administration.

    “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.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Different cell types or targeted genes can affect efficiency of CRISPR/Cas9 system.

    “Meanwhile, different cell types or targeted genes can also affect the efficiency of the CRISPR/Cas9 system [93, 199].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [93] Ran, 2013, Genome engineering using the CRISPR-Cas9 system, Nat. Protoc. source

      Not checked in the free run.

    • cites [199] Hou, 2013, Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis, Proc. Natl. Acad. Sci. U. S. A. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    NLSs can be recognized by importin α, nuclear import receptor, and trigger subsequent transportation from cytosol to nucleus.

    “NLSs can be recognized by importin α, the nuclear import receptor, and trigger the subsequent transportation from the cytosol to the nucleus [198].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [198] Fontes, 2000, Structural basis of recognition of monopartite and bipartite nuclear localization sequences by mammalian importin-α, J. Mol. Biol. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S2 (checkable).

    Interpretationreasoning

    Efficiency will need to be improved for in vivo application, especially in correcting majority of genetic disorders.

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

  • Classified as probably not a substantial claim.

    Premisenone given

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

    “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.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Premisenone given

    For off-target effects in non-targeted tissues or cells, tissue-specific promoters in CRISPR/Cas9-based plasmid have been introduced to partially solve this problem.

    “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.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisenone given

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

    “Most NLSs are characterized by short basic and cationic peptide sequences.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S2 (checkable), S4 (based).

    Interpretationnone given

    Specificity of CRISPR/Cas9 technology will need to be further improved to decrease potential off-target probability for increasing safety.

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

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Interpretationreasoning

    Targeted delivery of CRISPR/Cas9 by tissue-specific AAV type or non-viral vectors could be another effective solution to off-target effects in non-targeted tissues or cells.

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

  • Classified as probably not a substantial claim.

    Interpretationreasoning

    In vivo application would particularly benefit from non-viral vectors.

    “The latter would particularly benefit from non-viral vectors.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S4 (based). May combine two claims.

    Interpretationnone given

    Proper selection of targeting site, rational design of sgRNA and using paired Cas9 nickases could be effective approaches to alleviate off-target effects at non-targeted sites in genome of targeted cells.

    “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.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S2 (checkable).

    Interpretationreasoning

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

    “These issues may be resolved by enhancing activity and delivery efficiency of the CRISPR-Cas9 system.”
    Quote in the paperNot a substantial claim
57 of 57

  • The authors state this as an assumption.

    Strategies for facilitating particle accumulation in targeted tissues for non-viral delivery vectors-mediated gene therapy can also be applied to non-viral vectors-mediated CRISPR/Cas9 genome editing.

    “We assume that these strategies can also be applied to non-viral vectors-mediated CRISPR/Cas9 genome editing.”
1 of 1

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Genome editing components must reach the cell nucleus to achieve therapeutic effects, requiring overcoming extracellular and intracellular barriers including enzymatic degradation in blood, RES clearance, cell membrane penetration, endosome escape, and nuclear membrane infiltration.

    “Firstly, genome editing components must reach the cell nucleus to achieve therapeutic effects. Thus, extracellular and intracellular barriers need to be overcome, including avoiding enzymatic degradation in the blood, circumventing clearance by the reticuloendothelial system (RES), penetrating cell membranes, escaping endosome entrapment, and infiltrating nuclear membranes.”

    From RNA delivery biomaterials for the treatment of genetic and rare diseases (2019) · cites this paper as [208]

  • The complexity and large molecular weight of genome editing components are significant challenges for delivery materials.

    “Secondly, the complexity and large molecular weight of these components are significant challenges for delivery materials.”

    From RNA delivery biomaterials for the treatment of genetic and rare diseases (2019) · cites this paper as [208]

  • Genome editing components should be delivered to target tissues and edit specific sequences in a controllable manner to prevent off-target events and immune responses.

    “Thirdly, genome editing components should be delivered to target tissues and edit specific sequences in a controllable manner to prevent off-target events and immune responses.”

    From RNA delivery biomaterials for the treatment of genetic and rare diseases (2019) · cites this paper as [208]

  • Non-viral materials for delivery of genome editing tools are similar to those used for mRNA delivery.

    “These materials are similar to those used for mRNA delivery.[202, 205, 207–211]”

    From RNA delivery biomaterials for the treatment of genetic and rare diseases (2019) · cites this paper as [208]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Zuris study is cited as an unrelated reference

    Minor

    The text attributes the cochlear genome-editing result to Zuris et al. and cites reference 69, but reference 69 lists Lehrman's article about a gene-therapy death. The bibliography lists the Zuris et al. delivery study as reference 88.

1 of 1

Cites

1 retracted

Flagged first, then by citations

1 of the 206 references couldn’t be matched to a known paper.

Cited by

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Related

Authors
Li, L., Hu, S., & Chen, X.
Affiliations
  • National Institutes of Health (US)
  • Central South University (CN)
Corresponding author
  • Hu, Shuo
  • Chen, Xiaoyuan
ORCID
Venue
Biomaterials
Publisher
Elsevier BV
Published
2018-07
Volume
171
Pages
207-218
ISSN
0142-9612 (ISSN-L)
DOI
10.1016/j.biomaterials.2018.04.031
PMC
PMC5944364
OpenAlex
W2802060782
Cited by
415
References
206
Citations by year
2026: 31 · 2025: 48 · 2024: 59 · 2023: 64 · 2022: 57 · 2021: 71 · 2020: 43 · 2019: 34 · 2018: 8
FWCI
3.83
Type
journal article
Language
en
Open access
Licence
Locations
  • Biomaterials (acceptedVersion)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (acceptedVersion)
  • PubMed Central (acceptedVersion)
Funders
  • National Natural Science Foundation of China (81771873)
  • National Institute of Biomedical Imaging and Bioengineering
  • National Institutes of Health
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Physical Sciences › Materials Science › Biomaterials › Nanoparticle-Based Drug Delivery
Keywords
CRISPR/Cas9, genome editing, non-viral delivery, gene delivery vectors, lipid nanoparticles, polymeric nanoparticles, plasmid DNA delivery, mRNA delivery, protein delivery, in vivo delivery, CRISPR therapeutics
MeSH terms
Gene Editing, CRISPR-Associated Protein 9, Disease, Viruses, Gene Transfer Techniques, CRISPR-Cas Systems

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-10.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-10.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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