Quantifying T-cell Responses to Cas9 Protein

T-cell ELISPOTS were performed as previously described [44 (link)]. Briefly, splenocytes from AAV9-SaCas9-injected mice were isolated and purified using Lympholyte M (Cederlane). 250,000 cells were mixed with either Cas9 protein (Applied Biological Materials) or cell media only as a negative control, and subsequently plated in a 96-well ELISPOT plate (Millipore, MSIPS4510) in 100 µL per well. Stimulation was performed with 0.02 µg/µL of Cas9 protein at 37ºC in a humidified incubator, 7% (v/v) CO₂, for 40 h. Mouse IFN-γ ELISPOT pairs (3321–3-250 and 3321–6-250) and Streptavidin-alkaline phosphatase (3310–10) were purchased from Mabtech. Spots were developed using substrate Sigmafast BCIP/NBT (Sigma, B5655). Plates were shipped to Zellnet Consulting and enumerated using a Zeiss KS ELISPOT system. Full plates images are reported in Extended Data Fig. 4.