Table 4.
Selected designer nuclease approaches in livestock and monkeys
| Designer nuclease | Species | Target gene/gene KO | Deletion | Generation | Overall efficiency* | Method | Off-target effects/comments | Selection of somatic cells | References |
|---|---|---|---|---|---|---|---|---|---|
| ZFN | Rabbit | IgM | NHEJ | F0, F1 | 3.1 % | CPI | Mosaic F0; 14–22 % without mod. | NA | [265] |
| Pig | EGFP | NHEJ, m | F0 | Not given | SCNT | 80 % without mod. | FACS | [209] | |
| Pig | PPAR-γ | NHEJ, m | F0 | 0.15 % | SCNT | 10 % cotransfection | G418 | [210] | |
| Pig | GGTA1 | NHEJ, b | F0 | 1.4 % | SCNT | Resorbed fetuses and neonatal mortality | α-gal− cells counter selection | [186] | |
| Pig | GGTA1 | NHEJ, b | F0 | 1.6 % | SCNT | Resorbed fetuses | α-gal− cells counter selection | [266] | |
| Pig | GGTA/CMAH | NHEJ, b | F0 | 0.8 % | SCNT | Resorbed fetuses | Clonal exp. | [267] | |
| Pig | CMAH | NHEJ, b | F0 | 2.6 % | SCNT | Clonal exp. | [268] | ||
| Pig | CMAH | HDR, b, m | F0 | 0.4 % | SCNT | No integration of ZFN constructs | G418 | [268] | |
| Pig | RELA | NHEJ, b | F0 | 11 % | CPI | NA | [269] | ||
| Pig | IL2rg | NHEJ, b, m | Fetuses | 2 % | SCNT | High neonatal mortality | [270] | ||
| Cattle | BLG | NHEJ, b | F0 | 0.8 % | SCNT | Weak off-target effect | Clonal exp. | [211] | |
| Cattle | Beta-casein | Nick + HR | F0 | <0.5 % | SCNT | High levels of off-target mutations | Clonal exp. | [223] | |
| Rabbit | APOE | NHEJ, b | F1 | 25 % | PNI | No selection | [271] | ||
| TALEN | Cattle | Several | NHEJ, m, b | Preimplant | 50 % | CPI | High deletion/insertion | NA | [187] |
| Cattle | MSTN | NHEJ | F0 | 15 % | CPI | Mosaicism | NA | [233] | |
| Sheep | MSTN | NHEJ | F0 | 3.8 % | CPI | NA | [233] | ||
| Pig | RELA | NHEJ, m, b | Preimplant | 29 % | CPI | High deletion/insertion | NA | [187] | |
| Pig | LDLR | NHEJ, m, b | F0 | Not given | CT | Puromycin | [187] | ||
| Pig | RELA | NHEJ, m, b | F0 | 21 % | CPI | NA | [269] | ||
| Mini-pig | GGTA | NHEJ, b | F0 | 0.16 % | SCNT | High deletion | G418 | [272] | |
| Pig, | DAZL, APC | HDR, NHEJ | F0 | Not given | CT | No selection | [19] | ||
| Monkey | MECP2 | NHEJ | F0 | 1.2 % | CPI | High male fetal mortality, chimeric F0 | NA | [273] | |
| Chicken | OV | NHEJ | Founder, F1 | 8 % | Microinjection of PGC | No off-target effects | FACS | [274] | |
| CRISPR/Cas9 | Pig | vWF | NHEJ, b, m | F0 | 13.2 % | CPI | Small effect on embryo development | NA | [248] |
| Pig | RELA | HDR | F0 | Not given | CT | Off-target cleavage | No selection | [19] | |
| Pig | CD163, CD1D | NHEJ | F0 | 1.2–2.1 % | SCNT | Very low HDR | No selection or G418 | [246] | |
| Pig | CD163, CD1D | NHEJ | F0 | 4.2–3.6 % | CPI | NA | [246] | ||
| Pig | GGTA1, CMAH, iGb3S | NHEJ | F0 | 3.9 % | SCNT | α-gal− cells counter selection | [245] | ||
| Mini-pig | TYR | NHEJ, b | F0 | 0.9 % | SCNT | No off-target effects | G418? | [244] | |
| Mini-pig | PARK2, PINK1 | NHEJ, b | F0 | 1.2 % | SCNT | No off-target effects | G418? | [244] | |
| Goat | Several | NHEJ, b | F0 | 1.1 % | SCNT | No off-target effects | No selection | [247] | |
| Sheep | MSTN | NHEJ | F0 | 5.7 % | CPI | No off-target effects | NA | [249] | |
| Monkey | PPAR-γ, RAG1 | NHEJ | F0 | Not given | CPI | No off-target effects | NA | [275] | |
| Rabbit | TYR | NHEJ | F0, F1 | 3 % | PNI | No off-target effects | No selection | [276] | |
| Rabbit | IL2rg; Tiki1; IL2rg + RAG1 a | NHEJ b | F0 | 7.5–16.6 % | CPI | Low off-target effects | No selection | [238] |
PPAR-γ peroxisome proliferator-activated receptor gamma, GGTA1 α1,3-galactosyltransferase gene, CMAH cytidine monophosphate-N-acetylneuraminic acid hydroxylase, PRNP prion protein, RELA v-rel reticuloendotheliosis viral oncogene homolog A (avian), LDLR LDL receptor, IL2rg interleukin 2 receptor gamma gene, BLG beta-lactoglobulin, MECP2 methyl-CpG-binding protein, vWF Von Willebrand disease gene, MSTN myostatin gene, RAG1 recombination-activating gene 1, DAZL deleted in azoospermia-like, APC adenomatous polyposis coli, CD163 porcine reproductive and respiratory syndrome virus (PRRSV) receptor, CD1D non-classical major histocompatibility complex protein, iGb3S iGb3 synthase candidate glycosyltransferase, TYR tyrosinase gene, PARK2 parkin, RBR E3 ubiquitin protein ligase, PINK1 PTEN-induced putative kinase, OV ovalbumin gene, m mono-allelic, b bi-allelic, NA not applicable
* Transgenic offspring per treated oocytes or embryos
aMultiplex gene targeting