TABLE 2.
Different delivery vehicles for the CRISPR/Cas9 system and important observations
| Delivery vehicle | Cargo | Target | Route | Observations | Reference |
|---|---|---|---|---|---|
| Lipid‐based delivery | |||||
| LHNPs | ‐ | Plk1 | ‐ | LHNPs as a versatile CRISPR/Cas9‐delivery tool to study cancer biology and gene therapy. | [99] |
| LNP (lipoid, cholesterol, DOPE, DSPE‐PEG2k) | ‐ | GFP | ‐ | NP formulation used in diseased models and study of therapeutics. | [100] |
| LNP‐7C3 | mRNA | ICAM‐2 | IV/IM | A system to quantify how more than 100 nm LNPs deliver mRNA, translated into a functional protein. | [101] |
| LNP‐MK571 | mRNA | TSC2 | IV | Enhances intracellular mRNA delivery both in vivo and in vitro, acts as leukotriene‐antagonists, and is approved for asthma treatment and some other lung diseases. | [102] |
| Cationic lipid‐assisted NPs | mRNA | NLRP3 | IV | A promising strategy for treating NIRP3‐dependent inflammatory diseases and an affordable carrier for delivery of CRISPR/Cas9 into a macrophage. Effective genome editing efficiency (≈53% in the Raw264.7 cell line). | [103] |
| Exosome‐liposome hybrid NPs | Plasmid | mRunx2, hCTNNB1 | Can be used to deliver the CRISPR/Cas9 system in MSCs and study of in vivo gene manipulation. | [104] | |
| LNP‐INT01 | mRNA | Ttr | IV | Enabled ≈ 97% knockout of the mouse Ttr gene in the liver. | [105] |
| PEG‐b‐PLGA‐based cationic lipid‐assisted NPs | Plasmid | NE | IV | CLANpCas9 gene disrupted the NE gene and eased the insulin resistance of T2D mice by decreasing the epididymal white adipose tissue inflammation in the rat liver. | [106] |
| Chalcogen‐containing lipidoids | ‐ | GFP | IV | Combinatorial library of chalcogen (O, S, Se) comprising lipidoid NPs for intracellular delivery of anionic Cas9:single‐guide RNA for genome editing. | [107] |
| ZALNPs | mRNA | Luciferase | IV | ZALNPs guide the design of long RNA carriers and a promising safety and utility of genome editing. The in vitro knockout was reported as 95% | [108] |
| Cationic lipid‐assisted PEG‐PLGA NPs | Plasmid | Ntnl | IV | To express Cas9 in macrophages and precursor monocytes, leading to 20% gene knockout in vivo and 30% in vitro. | [109] |
| Polymeric‐based delivery | |||||
| PBAE NPs | Plasmid | E7 | ‐ | These NPs (with PBAE and CRISPR/shRNA) could be potentially developed as PV‐targeting drugs and used in studies on HPV‐related cervical malignancies. | [110] |
| PEG‐PLGA‐based CLANs | Plasmid | BCR‐ABL | IV | A strategy for targeted treatment of CML with an in vitro indel frequency of almost 46%. | [106] |
| PEGylated chitosan | Plasmid | CFTR | ‐ | Delivery of gene‐editing system by PEGylated chitosan nano complexes. | [111] |
| Cationic polymer PC | Plasmid | β‐subunit of Hb, rhomboid 5 homolog 1 (RHBDFI) | ‐ | A strategy for the large plasmid delivery encoding Cas9/sgRNA for efficient genome editing. | [112] |
| Rigid nanoparticle‐based delivery | |||||
| Black phosphorus nanosheets | ‐ | ‐ | IT | 2D delivery biodegradable platform for CRISPR/Cas9 RNP delivery and some bioactive compounds for biomedical applications. Induction of indel frequency in MCF‐7 cells ≈ 32%. | [113] |
| Arg functionalized gold NPs | ‐ | ‐ | ‐ | For the study of fabrication of Cas9En‐RNP/ArgNPs nano‐assembly. | [114], [115] |
| Polymer/inorganic hybrid NPs (protamine sulfate, CaCO3 and CaPO4) | Plasmid | CDKII | ‐ | Effective genome editing and in situ detection of protein expression. | [116] |
| Gold nanocluster, lipid core‐shell nanocarrier | Plasmid | Plkl | Delivery of protein‐nucleic acid hybrids for gene therapy. | [117] | |
| Gold NPs | ‐ | mG1uR5 | IT | Brain‐targeted therapeutics and development of focal brain‐knockout animal models. The protein and mRNA of mGluR5 reduced ≈ 50%. | [118] |
| Nanoparticle coupled to specific ligand structures | |||||
| pVLPs | Plasmid | ‐ | ‐ | Penetration through the cellular membrane to deliver genetic cargos within the nucleus through the viral entry route. | [119] |
| Arg NPs | ‐ | AAVSI, PTEN | ‐ | Cytoplasmic delivery of Cas9/sgRNA RNP through the co‐engineering of Cas9 protein and Arg NPs. | [114], [115] |
| Cas9 protein and sgRNA‐coated endoporter | Plasmid | CDKII | ‐ | Effective Cas9 RNP delivery initiating targeted gene products in cultured cells and in vivo. | [120] |
| Amphiphilic penetrating peptide NPs | ‐ | EGFP | ‐ | The amphiphilic vectors can deliver Cas9 with low toxicity and good efficiency. | [121] |
| Arg NPs | ‐ | SIRP‐a | ‐ | Can be used as weaponized macrophages for cancer immunotherapy. | [122] |
| TAT peptide‐modified Au NPs | Plasmid | Plkl | IV | CRISPR/Cas9 delivery and targeted genome editing for different diseases. In A375 cells, the irradiation of LACP led to ≈ 65% down‐regulation of the Plk‐l. An intra‐tumoral injection in xenograft models of human melanoma showed tumor volume of the LACP group (no irradiation) ≈ 42% of the volume of the control group. | [53] |
| CPP‐nanoscale ZIFs | mRNA | EGFP | ‐ | CPP‐ZIFs work as easily scaled‐up with excellent loading capacity for co‐delivery of intact Cas9 protein and sgRNA. Possessing ≈ 30% gene knockout | [12] |
| Amino‐ester (MPA‐A, MPA‐Ab) NPs | mRNA | EGFP | ‐ | Biodegradable lipid‐like NPs used as genome‐editing delivery tools for biological and therapeutic applications. | [123] |
| Aptamer AS 1411/ACMC/KALA NPs | Plasmid | CDKII | ‐ | Multi‐functional delivery system NPs for the delivery of plasmid into cancer cell nuclei. | [124] |
| Self‐assembled DNA nanoclews | RNP | EGFP | IT | Delivery of functional nucleic acids and DNA‐binding proteins | [11] |
“‐” refers to: not reported.
Abbreviations: LHNPs, Liposome‐templated hydrogel nanoparticles; PlKI, polo‐like kinase 1; LNP, lipid nanoparticle; DOPE, dioleoyl‐phosphatidyl ethanol‐amine; DSPE‐PEG2k, 1,2‐distearoyl‐sn‐glycero‐3‐phosphoethanolamine‐N‐[amino(polyethylene glycol)‐2000] (ammonium salt); GFP, green fluorescent protein; LNP‐7C3, lipid nanoparticle with specifc lipid composition; ICAM‐2, intracellular adhesion molecule‐2; IV/IM. Intravenous/intramuscular; nm, nanomolar; LNP‐MK571, lipid nanoparticle containing leukotriene antagonsit; TSC2, tuberous sclerosis complex 2; NLRP3, pyrin‐like protein containing a pyrin domain; mRunx2, mouse Runt‐related transcription factor 2; hCTNNB1, human catenin β1; MSCs, mesenchymal stem cells; LNP‐INT01, a biodegradable and ionizable lipid; Ttr, trans‐thyretin; PEG‐b‐PLGA, polyethylene glycol‐β‐poly(lactic‐co‐glycolic acid); CLANpCas9, cationic lipid‐assisted polymeric NPs containing Cas9; T2D, type 2 diabetes; ZALNPs, zwitterionic amino lipid nanoparticles; PBAE NPs, poly(β‐amino ester) nanoparticles; CLANs, cationic lipid‐assisted polymeric nanoparticles; BCR‐ABL, breakpoint cluster region‐Abelson murine leukemia; CML, chronic myeloid leukemia; PEGylated, poly ethylene glycated; CFTR, cystic fibrosis transmembrane conductance regulator; PC, polyethyleneimine‐β‐cyclodextrin; RHBDFI, Rhomboid 5 Homolog 1; MCF‐7, Michigan Cancer Foundation‐7; CaCO3, calcium carbonate; CaPO4, calcium phosphate; CDKII, cyclin dependent kinase II; Plkl, polo‐like kinase 1; pVLPs, peptidyl virus‐like particles; AAVSI, adeno‐associated virus integration site 1; PTEN, phosphate and tensin homolog; EGFP, enhanced green fluorescent protein; SIRP‐a, signal regulatory protein‐alpha; TAT, trans‐activator of transcription; CPP, cell penetrating peptide; ZIFs, zeolitic imidazole Frameworks; MPA‐A, N‐methyl‐1,3‐propanediamine‐A; AS 1411, guanosine rich oligonucleotide aptamer; NE, neutrophil elastase.