In utero CRISPR-mediated fetal monkey engineering

The pregnancy after timed mating was assessed periodically by ultrasound examination, and pregnancy was confirmed with ultrasound evidence of fetal heart activity. Pregnant monkeys having fetal monkeys at embryonic day 55 (E55) or gestational day 55 (G55) were selected for in utero injection. After induction of anesthesia of the pregnant monkey with intramuscular injection of Zoletil^®50 at a dose of 5 mg/kg, a 25-G Quincke needle (BectonDickenson) was used to target the LV of the fetal monkey brain closest to the anterior maternal abdomen under continuous ultrasound imaging. Lentiviruses (50 μL lentivirus CHD8 or control gRNA mixed with 100 μL lentiviral Cas9 were injected as a slow bolus to deliver a total dose of 1.0 × 10⁸ vg (lentiviral gRNA) and 2.0 × 10⁸ vg (lentiviral Cas9) in each fetus. The correct delivery would result in ventricular swelling, and the needle was removed immediately. The injected fetus was monitored for another 15 min. We obtained two male and one female newborn monkeys, which were delivered naturally. These newborn monkeys were euthanized with intramuscular injection of Barbiturates with the dose of 100 mg/kg at postnatal day 7 or 8 to isolate their brains for analysis.