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Applicable species: mice (P0-P30 are more efficient), rats, guinea pigs, NHP[12-58]. Dose range: mouse 1×10^10-5×10^10 vg/ear; NHP 1×10^11-7×10^11 vg/ear[12-58]. Injection volume: mouse 0.6-2 μL/ear; NHP 10-40 μL/ear (perilymph volume is approximately 26.7 μL); guinea pig 2-10 μL (perilymph 8.66 μL) [12-58]. Needle: mouse borosilicate glass microelectrode (tip 20-40 μm) or 33 G needle; NHP 33 G trans-round window membrane [12-58]. Speed: mouse 0.1-0.5 μL/min; NHP 20-100 nL/s[12-58]. Anesthesia: hypothermic anesthesia for neonatal rats, isoflurane for adults; NHP general anesthesia + postoperative cefazolin/analgesia [12-58]. Key points of the operation: Make an incision behind the ear to expose the auditory bubble, grind open to expose the round window niche (RW niche), puncture the center of the RWM with a glass microelectrode, slowly inject after 5-10 minutes of stable perilymph outflow, and seal the RWM with muscle + tissue glue after injection; full cochlear transduction can be achieved in adult mice by combining posterior semicircular canal fenestration (RWM + CF) [12-58]. Coverage: RWM injection mainly transduces basal hair cells, combined with PSCC windowing, 94.6% apical transduction, 96.8% intermediate transduction, and basal transduction 94.2% of inner hair cells; high-dose almost 100% IHC/OHC transduction; expression peaks in 2-4 weeks [12-58]. Safety: The injection volume does not exceed the perilymph volume (0.62 μL for mice, 26.7 μL for NHP, and 51 μL for humans), and the injection speed must be extremely slow to avoid hearing damage; overdose can overflow into the posterior cranial fossa through the cochlear aqueduct and cause brain transduction [12-58].