Fig. 4: SaCas9.Redi variants show reduced immunogenicity in vivo.

a Experimental workflow to assess adaptive immunogenicity of WT SaCas9 or SaCas9.Redi variants in MHC-I/II-humanized (A2.DR1) mice. b T cell recall (left) and antibody titers (right) against WT epitopes 1–3 and their respective variants. T cell recall was performed by intracellular flow cytometry of IFNγ and IL-2 (Supplementary Fig. 7b) following overnight incubation of splenocytes from WT SaCas9-exposed MHC-I/II humanized mice with the indicated peptides. Anti-SaCas9 IgG levels were measured by ELISA. Absorbance (OD450 nm–570 nm) shown. N = 8 animals. Mean ± SEM shown. Statistical significance was determined by repeated-measures one-way ANOVA followed by Dunnett’s post hoc test for multiple hypothesis correction. c Percent IFNγ reduction by each nuclease variant at d21 post-exposure. Relative proportions of mutated epitopes indicated by stacked bars. Mean ± SEM shown. N = 8 animals per group. d Workflow to assess editing efficiency of Pcsk9 by WT SaCas9 or SaCas9.Redi variants. A2.DR1 mice were treated with 2e11 vg of AAV8-encoded nuclease variants at d0 and d14. Readouts were performed at d21 post-injection. e Quantification of SaCas9 T cell recalls split by epitope and respective variant mutation. IFN-γ ELISpot counts shown. Full counts in Source Data. N = 4 animals per epitope. Mean ± SEM shown. Statistical significance was determined by one-way ANOVA followed by Sidak post hoc test for multiple hypothesis correction. See also Source Data. f Pcsk9 indel rates measured in liver tissue at d21 post-injection. N = 4 biological replicates per condition. Mean with standard deviation is shown. Statistical significance was determined by one-way ANOVA followed by Dunnett test for multiple hypothesis correction. g Serum mouse PCSK9 measurements of treated animals at d21 post-injection by ELISA. Mean ± SEM shown. Statistical significance was determined by one-way ANOVA followed by Sidak post hoc test for multiple hypothesis correction. N = 4 animals per group. h Serum LDL cholesterol measurements of treated animals at d21 post-injection by ELISA. Mean ± SEM shown. Statistical significance was determined by one-way ANOVA followed by Sidak post hoc test for multiple hypothesis correction. N = 4 animals per group. i Percent serum LDL reduction at d21 post-injection by ELISA. Mean ± SEM shown. Statistical significance was determined by one-way ANOVA followed by Sidak post hoc test for multiple hypothesis correction. N = 4 animals per group.