Homologous recombination-mediated targeted integration in monkey embryos using TALE nucleases

Area of Science:

• Biomedical Research
• Genetics
• Non-human Primate Models

Background:

• Non-human primates (NHPs) closely mimic human physiology, making them valuable for biomedical research.
• Genome editing technologies like transcription activator-like effector nucleases (TALENs) and CRISPR/Cas9 enable precise genetic modifications in embryos.
• Previous work established gene editing in macaques and a methyl CpG binding protein 2 (MECP2)-mutant cynomolgus monkey model for Rett syndrome.

Purpose of the Study:

• To assess the feasibility of homologous recombination (HR)-mediated gene replacement in monkeys using TALENs.
• To generate preimplantation embryos with a specific gene knock-in, incorporating an EmGFP fluorescent reporter into the OCT4 gene.

Main Methods:

• Assembled TALENs targeting the first exon of the OCT4 gene.
• Constructed a donor vector with homology arms and an EmGFP cassette.
• Co-injected TALENs and donor plasmids into 122 monkey zygotes post-fertilization.

Main Results:

• Successfully generated OCT4-EmGFP knock-in alleles in monkey embryos via TALENs-mediated HR.
• Achieved a knock-in efficiency of 11.3% (7/62) and 11.1% (1/9) as confirmed by sequencing and immunofluorescence.
• Demonstrated successful gene replacement using TALEN-assisted homologous recombination.

Conclusions:

• Successfully produced OCT4-EmGFP knock-in monkey embryos using TALEN-mediated HR, a first for this approach.
• Validated TALEN-assisted HR as an effective strategy for precise genetic modification in non-human primates.
• This method advances the development of NHP models for studying genetic diseases.