Extended Figure 6. Extended analyses of engineering with AAV6 homology donors.

(a) Editing rates achieved using sg05 Cas9 RNPs and AAV6-J3 homology donors on Raji and Ramos B cell lines. Representative plots are shown, together with summary data for n = 4–6 replicates. (b) Site-specific insertion of the J3 cassette in primary B cells treated with sg05 RNPs and AAV6-J3 donor was confirmed by in-out PCR followed by Sanger sequencing of the band. The dotted line shows the presumed sg05 cleavage site. Uncropped gel image is provided in Supplementary Fig. 3c. (c) Neutralization of HIV-1 NL4–3, measured by TZM-bl assay, by supernatants of AAV6-J3-edited primary B cells (n = 6). (d-h) Primary human B cells from n = 3–5 donors were edited with sg05 Cas9 RNPs and AAV6-J3 at the indicated MOIs, with BAC activation in XF media. Data from the highest MOI is reproduced here from Fig. 4, for comparison. (d) Editing rates, measured at day 8 by flow cytometry for surface J3-BCR. (e) Editing quantified by in-out ddPCR at day 8 and normalized per cell against a control reaction. (f) The yield of edited cells at day 8 was calculated from 5 × 10⁵ starting B cells. (g) Fold expansion of total cells at day 8. (h) J3 HCAb secretion, measured by gp120-IgG ELISA at day 8. (i) B cell phenotypes of edited cells in BAC (left) or with differentiation (BAC + DP, right) were measured by flow cytometry. Unedited matched control cells were also quantified and subtracted from the frequencies in the edited cells, to highlight any changes in differentiation after editing. (j) Edited primary B cells were differentiated in DP plus IMDM media and RNA extracted at indicated time points. RT-PCR was performed using a J3-specific forward primer and IgG reverse primers specific for the secreted (S) or membrane (M) isoforms. Uncropped gel image is provided in Supplementary Fig. 3d. Error bars show mean ± SEM. Statistics were calculated by 2-way ANVOA (a), 1-way ANOVA (d-h), or 1-sample t-test (i). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.