Table 4 Summary of accomplishments and challenges for viral and non-viral delivery approach of CRISPR Cas system
Therapeutic genome editing approaches | Delivery methods | Targets or disease | Genome editing accomplishments | Carrying capacity | Challenges | Strategies | References |
|---|---|---|---|---|---|---|---|
Viral vector | Adenovirus | T cells | CCR5 knockout is in clinical trials | 37 Kb | In vivo, immunogenicity is a major restraint. | Targeting immune privilege organs such as eyes, brain, uterus. | |
AAV in vitro | T cells and HSCs | High genome editing rate as a donor; can be paired with non-viral nuclease delivery | 4.7 kb | HDR donor size is limited by vector carrying capability. | It is possible to generate donor templates for HDR-mediated methods by infecting AAV vectors with a ssDNA vector genome | ||
AAV in vivo | brain, retina, Liver, heart, muscle | In animal models, knockouts and HDR have been produced; this can be used with non-viral nuclease delivery. | 4.7 kb | 1. There are still issues with delivery efficiency and preexisting immunity to natural serotypes. 2. Exposes continuously for a long time after implying in vivo and increase risk of off-targeting 3. Having a small packaging size, 4.7 kbp, while the genomic size of SpCas9 alone is around 4.3 kbp 4. Hepatoxicity | 1.a.To eliminate pre-existing immunity to AAV, it can be employed alone or in conjunction with other approaches. 1.b. Targeting immune privilege organs such as eyes, brain. - 3.a. splicing the Cas9 protein into two AAV vector (AAV-split-Cas9) can be performed. 3.b. Choosing a smaller size of Cas9 protein such as SpCas9 which is 1 kilo base shorter. - | ||
Lentiviral vector | In retina and in vitro | Lentivirus with integrase defects utilized as a donor | 8Â kb | The de novo expression of a protein lacking in the host may result in immune responses leading to the clearance of the transduced cells and the formation of antibodies that inhibit the activity of secreted factors | Cyclosporine, tacrolimus, and cyclophosphamide can inhibit the synthesis and secretion of cytokines and prevent the activation and proliferation of T cells | ||
Non-viral vector | Electroporation | In vitro: T cells, HSCs; in vivo: muscle and kidney | High genome editing efficiency in cells difficult to transfect | - | 1.Only feasible in ex vivo applications; in vivo electroporation is limited to mice, unclear if possible in humans | 1.a. combining the CRISPR/Cas9 system and in utero electroporation is an effective and rapid approach to achieve brain-specific gene knockout in vivo. 1.b. electroporation does not require microinjection skills and can be used to treat 40–50 embryos simultaneously. | |
Lipid-based delivery vehicles | PCSK9, TTR, TMC1 | High NHEJ efficiency for hepatocytes and hair cells in vivo. Minimize immunogenicity Reduce off-targeting | - | 1.a.Cas9 mRNA may activate TLRs. 1.b. Due to the constant positive charge, these formulations induce toxicity, adverse reactions, and immunogenic responses | 1.Lipid nanoparticles (LNPs) based on ionizable cationic lipids were developed to circumvent these restrictions | ||
Microinjection | In vivo: zebrafish Caenorhabditis elegans | Â | - | 1. Cell damage 2. Only a single cell can be targeted in each injection. | 1.To reduce cell damage, a high level of sophistication and manual skills are required. - | ||
iTOP | Â | iTOP transduction is effective for intracellular delivery of the Cas9 protein and sgRNAs independently, or direct delivery of RNPs. | - | Lower efficiency in primary cells. Since it is only soluble at high salt concentrations, it is not adequate for in vivo. | Â | [209] |