Fig 2: Nick location dictates the type of PE3 editing byproducts at ATP1A1.a Schematic representation of the complementary 5' and 3' single-stranded DNA overhangs generated with the PE3-T804N strategy at ATP1A1 exon 17 (PAM-In configuration) and the PE3-Q118R strategy at ATP1A1 exon 4 (PAM-Out configuration), respectively. b PCR-based genotyping of ATP1A1 exon 17 and 4 from single cell-derived MTOR-F2108L/I2017T K562 clones. Single cell-derived clones were isolated in methylcellulose-based semi-solid RPMI media supplemented with 100 µM ouabain and genomic DNA was harvested after co-selection. n = 16 single cell-derived clones from one experiment. c Same as in b with single cell-derived MTOR-F2108L/E2419K K562 clones. n = 16 single cell-derived clones from one experiment. d Schematic representation of pegRNA and nick sgRNA target sites with PAMs facing inwards (PAM-In configuration) and outwards (PAM-Out configuration) at ATP1A1 exon 4. e PCR-based genotyping of ATP1A1 exon 4 from single cell-derived K562 clones targeted with PAM-out and PAM-in configurations using different nick sgRNAs. Single cell-derived clones were isolated in methylcellulose-based semi-solid RPMI media supplemented with 0.5 µM ouabain and genomic DNA was harvested after co-selection. n = 17 single cell-derived clones for each condition from one experiment. Ins, insertion. Del, deletion.

Fig 3: Gi/o-GPCR-mediated ß-cell NKA activity depends on tyrosine kinase signaling.A Representative WT islet SST and dasatinib (Dasa) [Ca2+]i responses (F/Fmin). Insets indicate the percentage of islets exhibiting each type of Dasa [Ca2+]i response. B Percentage of WT islets displaying [Ca2+]i oscillations in response to SST (green) and SST + Dasa (purple; n = 3). C WT islet [Ca2+]i plateau fraction after SST (green) and after SST + Dasa (purple; n = 3). D Relative size of WT islets displaying total (black) or partial (gray) Dasa-mediated inhibition of SST-induced [Ca2+]i oscillations (n = 3). E Representative WT islet SST and NSC 87877 (NSC) [Ca2+]i responses. Insets indicate the percentage of islets exhibiting each type of NSC [Ca2+]i response. F Percentage of WT islets displaying [Ca2+]i oscillations in response to SST (green) and SST + NSC (pink; n = 3). G WT islet [Ca2+]i plateau fraction after SST (green) and after SST + NSC (pink; n = 3). H Relative size of WT islets displaying total (black) or partial (gray) NSC-mediated inhibition of SST-induced [Ca2+]i oscillations (n = 3). I Representative WT islet insulin (Ins) and Dasa [Ca2+]i responses. J Percentage of WT islets displaying [Ca2+]i oscillations in response to Ins (red) and Ins + Dasa (purple; n = 3). K WT islet [Ca2+]i plateau fraction before treatment (white), after Ins (red), and after Ins + Dasa (purple; n = 3). L Representative immunoblots (IBs) of WT islet cell lysates that were treated for 15 min with vehicle (H2O), SST, or Ins at 25 °C. IBs were probed for total NKA (ATP1A1; left) and NKA phosphorylated at Y10 (p-ATP1A1Y10; right). p-ATP1A1Y10 bands were normalized to corresponding ATP1A1 bands; normalized p-ATP1A1Y10 bands from SST-/Ins-treated islets were then normalized to p-ATP1A1Y10 bands from vehicle-treated islets. M Average vehicle-normalized islet p-ATP1A1Y10 relative to total ATP1A1 (n = 4). Statistical analysis was conducted using unpaired two-sided two-sample t tests (B–D, F–H, J) or one-way ANOVA with Šidák’s post-hoc multiple comparisons tests (K, M); *P < 0.05, **P < 0.01, and ***P < 0.001. Source data and exact P values are provided as a Source Data file.

Fig 4: Assessing the impact of complementary-strand nick locations on prime editing outcomes.a Schematic representation of the EBFP to EGFP reporter system using different nick sgRNAs with PAMs facing outwards (PAM-Out) or inwards (PAM-In) towards the pegRNA. b Schematic of the FACS-based quantification of EBFP to EGFP conversion via PE. In K562 cells homozygous for EBFP integration at ATP1A1 (triallelic), FACS-based analysis reports PE outcomes indirectly; (i) EBFP(+)/EGFP(+) cells result from monoallelic or biallelic PE, (ii) EBFP(-)/EGFP(+) cells are generated by either triallelic PE or combinations of mono- and biallelic PE along with indels*, (iii) EBFP(-)/EGFP(-) occur from indels on the three copies of the reporter. Indels are defined broadly in this context as any edits that inactivates the reporter. c FACS-based quantification of EBFP to EGFP conversion via PE after co-selection. K562 cells stably expressing the EBFP reporter from the ATP1A1 locus were transfected with PE3max vectors targeting ATP1A1 exon 4 (pegRNA-Q118R_v1) and EBFP (epegRNA). Cells were treated with 100 µM ouabain starting 3 days post-transfection until all non-resistant cells were eliminated. n = 3 independent biological replicates performed at different times with equivalent results (see Supplementary Figs. 22,23). d PCR-based genotyping of EGFP(+) single cell-derived K562 clones targeted with the PE2 or PE3b strategy. Single cell-derived clones were isolated in methylcellulose-based semi-solid RPMI media supplemented with 100 µM ouabain and genomic DNA from EGFP(+) clones was harvested after co-selection. The number of precise prime edited alleles was determined from BEAT Sanger sequencing trace analysis and small indels were analysed with DECODR. n = 17 single cell-derived clones for each condition from one experiment. e PCR-based genotyping of EBFP from single K562 cell-derived clones after sorting for EBFP(-)/EGFP(+) cells. The number of precise prime edited alleles was determined from BEAT Sanger sequencing trace analysis and small indels were analysed with DECODR. Larger insertions and deletions are indicated, and homozygous single cell-derived clones are highlighted in bold and green. PE alleles harboring pegRNA scaffold incorporation are indicated (si). n = 17 and 16 single cell-derived clones from one experiment for the PE3-G3 and PE3-G6 conditions, respectively. Ins, insertion. Del, deletion.

Fig 5: Co-selection for the installation of clinically relevant mutations at the MTOR locus.a Schematic of mScarlet-I mTOR Signaling Indicator (mSc-TOSI) reporter degradation under mTORC1 signaling. b PE and small indels quantification as determined by BEAT and TIDE analysis from Sanger sequencing. K562 cells stably expressing the mSc-TOSI reporter were transfected with PE3 vectors targeting ATP1A1 exon 4 (Q118R) and MTOR. Genomic DNA was harvested 3 days post-transfection (before selection) and cells were treated (ouabain) or not (untreated) with 100 µM ouabain until all non-resistant cells were eliminated. n = 2 independent biological replicates performed at different times. c Histogram plot of mSc-TOSI intensity in homozygous single cell-derived K562 clones harboring MTOR hyperactivating mutations. d Same as in c with a homozygous clone harboring the MTOR-F2108L rapamycin resistance mutation. e Same as in c with a homozygous clone harboring the MTOR-F2108L rapamycin resistance mutation and the MTOR-E2419K hyperactivating mutation. f Same as in c with a homozygous clone harboring the MTOR-F2108L rapamycin resistance mutation and the MTOR-I2017T hyperactivating mutation. Where indicated, cells were treated for 24 h with 50 nM rapamycin or 50 nM AZD8055 before FACS analysis. Representative images are from one of two independent biological replicates performed at different times with equivalent results (see Supplementary Figs. 15 and 16). Source data are provided in the Source Data file.