TABLE 5.
Delivery methods.
| Delivery methods | Cargo | Species/cell target | Location | Advantages | Limitations | References |
| Physical delivery | ||||||
| Microinjection | DNA donor - dsDNA (linear/plasmid) - dsDNA encoding gene-specific nucleases - lsDNA (>200nt) - ssODN (∼100nt) | Mouse and rat zygote | Pronucleus or cytoplasm | - Delivery of large DNA fragments - Stable DNA in cell | - Time-consuming method - Expertise required (less for Cyt-MI) - Poor visualization pronucleus, flexibility of the oolemma and nuclear membranes in rat - Variability in efficiency depending on size, DNA quality or purity - Persistent expression and depending on host transcriptional/transductional machinery | 1st description (Gordon et al., 1980; Palmiter et al., 1982; Mullins et al., 1990) dsDNA-ZFN (Geurts et al., 2009) dsDNA-TALEN (Tesson et al., 2011) dsDNA-Meganuclease (Ménoret et al., 2013) Efficiency (Charreau et al., 1996b; Hirabayashi et al., 2001) Complex/invasive method (Brinster et al., 1985; Charreau et al., 1996b) |
| mRNA encoding gene specific nucleases | Mouse and rat zygote | Pronucleus or cytoplasm | - Moderate efficiency - Transient expression - Cyt-MI more efficient than PN-MI - Off-target reduced - Independent expression dependency of host transcriptional/transductional machinery (mRNA) | - Time-consuming - Expertise required (less for Cyt-MI) - Variation among batches of IVT mRNA - mRNA liable to degradation | mRNA-ZFN (Geurts et al., 2009) mRNA-TALEN (Tesson et al., 2011; Remy et al., 2014) mRNA-CRISPR (Ménoret et al., 2015) Meganucleases (Wang et al., 2014) | |
| Protein (RNP) | Mouse and rat zygote Mouse/ES | Pronucleus or cytoplasm | - Higher efficiency than using DNA or mRNA encoding gene specific nucleases - Short half-life within cells - Less mosaicism - Off-target cleavage reduced | -In vivo stability -Potentially immunogenic | (Ménoret et al., 2015; Wang et al., 2015; Jung C. J. et al., 2017) | |
| Electroporation | DNA donor - dsDNA (linear/plasmid) - ssODN - lssDNA (600–1.5 kb) | Mouse and rat zygote | Uncontrolled cytoplasm (long DNA) Pronucleus (short lsDNA/ssODN) | - Easier delivery than DNA-MI - Processing simultaneously 50–60 zygotes in a short time - Efficient to deliver ssODN or lsDNA (<1 kb) | - Inefficient nuclear transport - Transient nuclear envelop breaking or cell-division required - Inefficient to deliver DNA > 1 kb | ssODN (Hashimoto and Takemoto, 2015; Kaneko and Mashimo, 2015; Qin et al., 2015; Chen et al., 2016; Wang et al., 2016; Remy et al., 2017) lsDNA (Miyasaka et al., 2018) Inefficient delivery dsDNA (Takabayashi et al., 2018) |
| mRNA encoding Cas9 + sgRNA | Mouse and rat zygote | Uncontrolled | - Easier delivery than mRNA-MI | - Embryos are quite sensitive to pulse and toxicity is observed | Rat/mRNA encoding Cas9+sgRNA (Remy et al., 2017) CRISPR/mice/KO/HDR-KI (Qin et al., 2015) Mice/CRISPR/KO (Hashimoto and Takemoto, 2015; Hashimoto et al., 2016) Rat/ZFN/TALEN/Crispr/KO (Kaneko et al., 2014; Kaneko and Nakagawa, 2020) Rat/mice/Crispr/KO/KI (Kaneko and Nakagawa, 2020) | |
| Protein (RNP) | Mouse and rat zygote | Uncontrolled | - Easier delivery than RNP-MI | - High amount of cargo - Uncontrolled delivery amount | Cas9-RNP/mice/indels/large KO/HDR-KI/ssODN-KI (Wang et al., 2016) Cas9-RNP/mice/KO (Hashimoto et al., 2016) | |
| GONAD | DNA - ssODN - lsDNA (<1 kb)/ Cas9 mRNA/sgRNA RNP | Mouse and rat | Oviduct | - Ex vivo embryo handling steps not required - Fewer animals used (e.g., recycling females possible) | - Not yet applicable to deliver long donor DNA (db or long ss DNA) | Cas9mRNA + sgRNA/mice/KO (Takahashi et al., 2015) RNP/lsDNA/mice/KO/ssODN and lsDNA-based KI (Ohtsuka et al., 2018) Rat/ssODN based KI (Kobayashi et al., 2018; Takabayashi et al., 2018) |
| Viral delivery methods | ||||||
| AAV vectors (Non-enveloped, lsDNA) | DNA encoding Cas9/sgRNA (separate AAV or all-in-one AAV) – KI DNA cassette | Mouse and rat zygote (transduction) | Uncontrolled | - minimal immunogenicity - low toxicity - wide-range serotypes - No incorporation into the host genome | Low capacity (<5 Kb) | KO/Mice/separate AAV (Yoon et al., 2018) KO/KI/Mice/Rat/RNP Electroporation/AAVtransduction (Mizuno et al., 2018; Chen et al., 2019) (Edraki et al., 2019) |
| DNA (expression cassette) | Mouse zygote microinjection | Cytoplasmic injection | (Yu et al., 2015) | |||
MI, microinjection; Cyt-MI, cytoplasmic microinjection; PN-MI, pronuclear microinjection; DNA-MI, DNA microinjection; KI, Knockin; ssODN, single-stranded oligonucleotides; lsDNA, long single-stranded DNA; dsDNA, linear double-stranded DNA; HDR-KI, homology directed repair knockin; RNP, ribonucleoprotein complex.