Extended Data Fig. 2: bNAb genomic integration, sera titers and neutralization as a function of immunizations and co-injection of the CRISPR-Cas9 vector.
From: In vivo engineered B cells secrete high titers of broadly neutralizing anti-HIV antibodies in mice
a. Area under the Curve (AUC) of Fig. 2d for YU2.DG (left) and JRFL (right) **; pv = 0.0036 (YU2.DG) and pv = 0.005 (JRFL) for unpaired t-test for CMV-Cas9gRNA + donor to PBS comparison and ##; pv = 0.0072 (YU2.DG) and pv = 0.0063 (JRFL) for one-sample t-test for Naïve to PBS comparison. n = 3 for CMV-Cas9gRNA + donor and PBS. Naïve sample is from a single, non-immunized, non-AAV-injected mouse. Mean values ± SD are indicated. b. Area under the curve (AUC) of Fig. 2c. From top to bottom, *; pv = 0.0185 and pv = 0.0103, **; pv = 0.0036 for two-sided unpaired t-test. n = 3 biologically independent animals. Mean values ± SEM are indicated. c. RT-PCR on RNA from sorted, 3BNC117+, CD19+, CD4− blood lymphocytes from day 37. Here, we used a reverse primer in a membranal exon of either IgHCμ or IgHCγ (all subtypes) and a forward primer on the VH of the coded 3BNC117. Numbers indicate different mice, injected with either a) PBS, b) the donor vector and the CMV-Cas9gRNA vector, or c) the donor vector only, as indicated above the gels. Control sample (C+) comes from in-vitro engineered primary mouse splenic lymphocytes, as described previously7. Ladder sizes are indicated on the left. Arrow indicates the expected amplicon size. For each group, experiment was reproduced 3 times with independent samples, as indicated by the numbers. Molecular weight markers (M) and their respective size in base pairs (MW) are indicated. d. Total DNA from the previous reaction as in (C) was purified and a semi-nested PCR with the same forward primer and a reverse primer on the CH1 of the respective constant domains. Ladder sizes are indicated on the left. Arrow indicates the expected amplicon size. For each group, experiments were reproduced 3 times with independent samples. Molecular weight markers (M) and their respective size in base pairs (MW) are indicated. e. Sanger sequencing alignment and chromatogram of the purified amplicon from the previous step. Reference sequences are indicated above. For the IgHCγ, each subtype reference is indicated. Sequencing of the IgHCμ amplicon of donor 3 has failed. f. Experimental design for (G-I). Splenic lymphocytes were activated with LPS and IL-4 and engineered, ex vivo, by AAV transduction and Cas9 electroporation with or without a gRNA. h. Flow cytometry of engineered splenic lymphocytes two days following treatment. Pregated on live, singlets. FcR block was used in the staining. Engineering parameters are indicated above each plot. h. EtBr gel electrophoresis showing products of an RT-PCR reaction with RNA from cells two days following treatment as in (F). For each sample, a control (C) reaction was performed amplifying the endogenous IgHG1 cDNA. Ladder sizes are indicated on the left. Arrow indicates the expected amplicon size. The experiment was reproduced once, with similar results. Molecular weight markers (M) and their respective size in base pairs (MW) are indicated. i. Sanger sequencing of the previous amplicons, confirming the integration.
