Fig. 1: Preexisting Cas9 antibodies are prevalent in human serum but not vitreous fluid.

a Human retinal CRISPR–Cas9 gene editing is administered by subretinal or intravitreal injections. All eye tissues besides the lens contain antibodies (in red). b Estimated vitreous fluid immunoglobulin concentrations (n = 26) from isotyping detected all subclasses except possibly IgM, showing approximately 143-fold fewer total antibodies in aggregate compared to serum guideline ranges (from manufacturer). Bar heights represent mean average concentrations (mg/dL) and error bars represent range for each immunoglobulin subclass. c ELISA measurements from paired serum and D vitreous fluid samples (1:50 dilutions, n = 13) showed high α-Cas9 prevalence in the serum but not in vitreous fluid. Dotted red line indicates positivity cutoff value: mean α-hemoglobin A450 plus three standard deviations. Red symbols = positive, black symbols = negative. S. pyogenes or S. aureus-derived Cas9 denoted SpCas9 and SaCas9, respectively. e Analysis of paired samples showed that higher serum antibody levels generally corresponded to higher vitreous fluid antibody levels for each particular antibody. f ELISA measurements of vitreous fluid validation samples (1:50 dilutions, n = 36) confirmed very low α-Cas9 prevalence, though vitreous fluid from a patient with S. pyogenes intraocular infection (endophthalmitis) tested positive for SpCas9. For c, d, f error bars represent standard deviation. Anderson-Darling tests were performed to test for normal distribution of data. Statistical comparisons between normally distributed samples (α-SaCas9, α-SpCas9) used parametric unpaired two-tailed Student’s t-tests, while similar comparisons between non-normally distributed samples (α-Tetanus) used non-parametric two-tailed Mann–Whitney U-tests. P-values < 0.05 were considered significant. Asterisks indicate p-value size: **** indicates p-value ≤ 0.0001. Source data are provided as a Source data file.