Table 2.

Pros and cons of the CRISPR/Cas9 delivery methods described in this review.

Method	Advantages	Disadvantages
Microinjection	Liberated right into the cell High efficacy	Time-consuming Technique expertise
Electroporation	Normalized open-access protocols High effectiveness with plasmids	In vitro and ex vivo cell restriction Cell cytotoxicity Not all cells are susceptible
Lipofection	Works in many cell types Easy manipulation Inexpensive Reduced off-targets	Exclusive for cell culture Lysosome degradation
Nanodiamonds	Highly efficient delivery High biocompatibility Water solubility Fully accessible surface Inexpensive	Genotoxicity High pressure and temperature for synthesis Tissue distribution problems
AAVs	Low immunogenicity and cytotoxicity Reduced off-targets High efficacy Low immune response detected Infect both dividing and non-dividing cells	Limited cargo capacity (3.5–4 kb) High cost Technique expertise Safety obstacle Not easy to scale-up
Lentivirus	Expression stability Can be applied in a broad range of cell types Higher efficacy if constructs are shortened Low immune response detected Infect both dividing and non-dividing cells	Limited cargo capacity (8–9 kb) Arbitrary integration Technique expertise Safety obstacle Not easy to scale-up
Microinjection delivery is based on the use of a 0.5–5.0 µm diameter needle to deliver components into a cell or intercellular space, in this case the Cas9 protein and sgRNAs in any form. The electroporation method requires high voltage currents with the purpose of opening nanopores in the cellular membrane to inlet the CRISPR components resuspended in a specific buffer. Nucleofection is a specific electroporation-based method that allows the direct entry of the components into the nucleus. Lipofection consists in the introduction of the DNA components via a liposome-based transfection, in which synthetic cationic lipids aggregate around the negatively-charged DNA molecules. Cellular uptake is based on the fusion of these liposome-like structures with the phospholipidic membrane. Nanodiamonds are carbon nanomaterials which are suspended in a colloidal solution that allow the binding with or coating of biological material for cell transfection, mainly penetrating by the clathrin-mediated endocytosis pathway. The viral transduction method leverages the natural potential of viruses to infect cells, where the vectors have been deprived of the essential pathogenic genes in their replication. The most commonly used are AAVs and lentivirus. AAVs, which consist of single-stranded DNA, present several serotype versions (allowing tissue-specific transduction) and are considered a safe option, given that they are not associated to human diseases showing low immunogenicity and entailing low cellular toxicity (as they do not integrate into the host genome). Lentiviruses are retroviruses derived from a provirus of HIV that proffer stable expression in both dividing and post-mitotic cells due to their host genome integration, and that additionally accommodate cargos up to 5–6 kb in size.