Panel A: A schematic diagram shows the design of a single-guide RNA (sgRNA) to reverse a compound heterozygous mutation c.532C>T p.R178*, using SpCas9-ABE. The target nucleotide A7 on the non-coding strand is marked for A to G conversion. Panel B: A to G conversions detected across the protospacer 7 days post-electroporation of SpCas9-ABE messenger RNA (mRNA) and sgRNA to P1 fibroblasts. The reference and next-generation sequencing (NGS) sequences of the non-coding strand are shown with A7 as the target nucleotide. Panel C: A bar graph investigates A to G conversions at nine possible off-target sites in unedited and edited P1 fibroblasts, with genomic DNA isolated and amplicons generated by polymerase chain reaction (PCR) for each site. Panel D: A Western blot image shows XLF expression in unedited and base-edited P1 fibroblasts, with densitometric assessment normalized to a GAPDH housekeeping control. Panel E: A bar graph quantifies double-strand break (DSB) repair over 48 hours following reversal, with P1 unedited and edited fibroblasts, a healthy donor control, and a ligase 4 (LIG4)-deficient control exposed to 3 gray (Gy) ionizing radiation. Panel F: A schematic diagram illustrates the design of a single-guide RNA (sgRNA) to reverse a homozygous mutation c.169C>T p.R57* using SpRY-ABE. The target nucleotide A5 on the non-coding strand is marked for A to G conversion. Panel G: A to G conversions across the protospacer 14 days post-electroporation with SpRY-ABE messenger RNA (mRNA) and sgRNA to P2 fibroblasts, with the A5 target position marked. Panel H: A bar graph investigates A to G conversions at nine predicted off-target sites in unedited and edited P1 fibroblasts using next-generation sequencing (NGS). Panel I: A Western blot image displays XLF expression in unedited and edited P2 fibroblasts, with densitometric analysis normalized to GAPDH and calculated relative to a healthy donor control. Panel J: A bar graph confirms the rescue of double-strand break (DSB) repair for P2 after base editing of fibroblasts, with healthy donor and ligase 4 (LIG4)-deficient controls, by quantification of gamma-H2AX foci.
Base editing reversal to confirm the pathogenicity of NHEJ1 sequence variants identified in two patients with RS-SCID. (A) A bespoke sgRNA was designed to reverse a compound heterozygous mutation (P1) c.532C>T p.R178* (red) with SpCas9-ABE using a canonical distal 5′-NGG-3′ PAM (purple). The variant was restored through A>G conversion (green) of the target nucleotide A7 on the non-coding strand with the PAM located at positions 21–23. (B) A>G conversions detected across the protospacer 7 days after electroporation of SpCas9-ABE mRNA and sgRNA to P1 fibroblasts. Reference and NGS sequences of the non-coding are shown with A7 marked as the target nucleotide. Editing was performed and confirmed by Sanger sequencing in three experiments, with one undergoing additional NGS (GENEWIZ) and bioinformatic analysis (CRISPResso2). (C) A>G conversions were investigated for nine possible “off-target” (OT) sites predicted by Cas-OFFinder, in unedited and edited P1 fibroblasts. Genomic DNA was isolated and amplicons generated by PCR for each site. These were then assessed by NGS (GENEWIZ) and bioinformatic analysis (CRISPResso2), with <5% above controls. (D) XLF (33 kDa) expression with densitometric assessment of western blot (ImageJ) with whole-cell lysate from unedited and base edited P1 fibroblasts, with a healthy donor (HD1) control. XLF levels were normalized to a GAPDH housekeeping control (36 kDa) and calculated relative to healthy donor levels. Representative data from three experiments. (E) Quantification of DSB repairs over a 48 h period following reversal. P1 unedited and edited fibroblasts, as well as a healthy donor control and a ligase 4 (LIG4)-deficient control were exposed to 3 Gy ionizing radiation and cultured. Cells were fixed, permeabilized and stained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) and γ-H2AX antibody, with γ-H2AX foci counted in at least 300 cells in two experiments. (F) A sgRNA was designed to reverse a homozygous mutation (P2) c.169C>T p.R57* (red) in combination with SpRY-ABE utilizing a distal 5′-NRN-3′ PAM (purple). The mutation was restored through A>G conversion (green) of the target nucleotide A5 on the non-coding strand with the PAM located at positions 21–23. (G) A>G conversions across the protospacer 14 days after electroporation with SpRY-ABE mRNA and sgRNA to P2 fibroblasts with the A5 target position marked. Amplicons covering the target nucleotide and PAM were generated and Sanger sequenced in two experiments with one undergoing additional NGS assessments. (H) A>G conversions were investigated for nine predicted “off-target” (OT) sites in unedited and edited P1 fibroblasts using NGS (GENEWIZ) and CRISPResso2, with <5% above controls. (I) Western blot with densitometric analysis of XLF expression (33 kDa) in whole-cell lysate extracted from unedited and edited P2 fibroblasts, normalized to GAPDH (36 kDa) and calculated relative to a healthy donor (HD2) control, and shown in a representative image from two experiments. (J) Rescue of DSB repair for P2 was confirmed after base editing of fibroblasts, with healthy donor and ligase 4 (LIG4)-deficient controls, by quantification of γ-H2AX foci in two experiments. Source data are available for this figure: SourceData F1.