Table 2 Summary of common delivery strategies for CRISPR/Cas9 systems
Delivery systems | CRISPR/Cas9 formats | Delivery efficiency | Advantages | Disadvantages | Applications |
|---|---|---|---|---|---|
Viral delivery | |||||
 AAV | pDNA |  +  +  | Low immunogenicity, non-pathogenic source, broad cell tropism | Limited packaging capacity, prolonged Cas9 expression, pre-existing neutralizing antibodies, high cost | In vivo |
 LV | pDNA |  +  +  +  | Large packaging capacity, low immunogenicity, broad cell tropism | Potential for insertional mutagenesis, prolonged Cas9 expression | In vitro and ex vivo |
 AV | pDNA |  +  +  | Large packaging capacity, minimal genomic integration risk | High immunogenicity, difficult for large-scale production, pre-existing neutralizing antibodies | In vivo |
Physical delivery | |||||
 Electroporation | pDNA, mRNA and RNP |  +  +  +  | Suitable for multiple cell types, simple operation | Severe cell damage, nonspecific transfection | In vitro and ex vivo |
 Microinjection | pDNA, mRNA and RNP |  +  +  +  | Highly specific and reproducible | Severe cell damage, low throughput, requirement of sophistication and manual skills | In vitro and ex vivo |
 Hydrodynamic injection | pDNA and RNP |  +  | Feasible for in vivo gene editing in small animals, highly efficient for liver transduction | Not suitable for large animals and clinical applications, non-specific and low efficiency, traumatic to tissues | In vivo |
Non-viral delivery | |||||
 Lipid NPs | pDNA, mRNA and RNP |  +  | Good biocompatibility, minimal immunogenicity, feasible for large-scale production, temporal release of CRISPR-Cas9, all-in-one delivery, low toxicity | Lower delivery efficiency | In vitro and in vivo |
 Polymeric NPs | pDNA, mRNA and RNP |  +  | Minimal immunogenicity, feasible for large-scale production, relatively flexible functionalization, good pharmacokinetic control, large packaging capacity, all-in-one delivery | Lower delivery efficiency, variable biocompatibility and toxicity | In vitro and in vivo |
 Gold nanomaterials | pDNA and RNP |  +  +  | Higher delivery efficiency, good biocompatibility, unique optical properties and photothermal effect, relatively flexible functionalization | Potential toxicity in vivo at high concentrations | In vitro and in vivo |
 EVs | RNP |  +  | Excellent biocompatibility and negligible immunogenicity, high biostability relatively flexible functionalization, low toxicity | Tedious preparation process | In vitro and in vivo |