Dual inhibition of DNA-PK and Polϴ boosts precision of diverse prime editing systems

Abstract

Prime editing is a genome engineering tool that allows installation of various small edits with high precision. However, prime editing efficiency and purity can vary widely across different edits, genomic targets, and cell types. Prime editing typically offers more precise editing outcomes compared to other genome editing methods such as homology-directed repair. However, it can still result in significant rates of unintended editing outcomes, such as indels or imprecise prime edits. This issue is particularly notable in systems utilizing a second nicking gRNA, such as PE3 and PE5, as well as in dual pegRNA systems and fully active nuclease systems such as PEn, which increase efficiency but compromise precision. In this work, we show that pharmacological inhibition of DNA-PK and Polϴ, two major mediators of mutagenic DNA repair pathways, improves precision of PEn, PE3, PE5, PE7, and dual pegRNA editing systems, including TwinPE, HOPE, and Bi-PE, across multiple genomic loci and edit types. We show that co-inhibition of DNA-PK and Polϴ mitigates both prime editing-unrelated indels and prime editing by-products such as template duplications. Moreover, in the case of PEn, this strategy also substantially improved the off-target editing profile. Together, our data establish small molecule modulation of DNA repair pathways as a general strategy to maximize the precision of diverse prime editing systems.

Fig. 1. Co-inhibition of DNA-PK and Polϴ increases precision of PEn editing.

a Schematic representation of PEn editing depicting molecular mechanisms of four possible PEn editing outcomes and their modulation by DNA repair inhibitors. b Installation of a point mutation in KCNA1 in HEK293T cells. Cells treated with indicated combinations of DNA-PK and Polϴ inhibitors were co-transfected with a pegRNA and indicated gene editing systems. Editing outcomes were analyzed by amplicon-seq and quantified using CRISPResso2 in prime editing mode. Plots show mean ± SD of n  =  3 biological replicates. Source data are provided as a Source Data file. PBS Primer Binding Site, RTT Reverse-Transcriptase Template, alt-EJ alternative End-Joining pathway, NHEJ Non-Homologous End-Joining pathway, indels non-templated insertions and deletions.

Fig. 2. Co-inhibition of DNA-PK and Polϴ allows precise and efficient PEn editing across different loci.

Genome editing with six different prime editing systems to install indicated point mutations or insertions (ins.) using plasmid co-transfection in (a) HEK293T and b HeLa cells. c PEn editing to delete 3 bp at HEK3 and RUNX1 sites in indicated cell line. d Calculated precision score of the six different prime editing systems across all loci tested in (a and b). “Precision score” was calculated as the number of amplicon-seq reads with precise prime edit per the number of amplicon-seq reads with any edit. Plots represent mean ± SD of n = 8 precision scores calculated at target sites in (a and b). P-values were determined using one-way ANOVA (two-tailed, paired). Multiple comparisons were corrected using Tukey’s test. Displayed pairwise comparisons are relative to 2⁺iPEn. Calculated P-values: HEK293T: PE5 vs 2i⁺PEn =0.2371 (ns), uPEn vs 2i⁺PEn =<0.0001 (****), PEn vs 2i⁺PEn = <0.0001 (****), 1iPEn vs 2i⁺PEn = <0.0001 (**), 2iPEn vs 2i⁺PEn = 0.2794 (ns). HeLa: PE5 vs 2i⁺PEn = 0.3556 (ns), uPEn vs 2i⁺PEn = <0.0001 (****), PEn vs 2i⁺PEn = <0.0001 (****), 1iPEn vs 2i⁺PEn = <0.0001 (****), 2iPEn vs 2i⁺PEn = 0.0008 (**). ns not significant. All amplicon sequencing data described above was analyzed using CRISPResso2 in prime editing mode. All bar plots show mean ± SD of n = 3 biological replicates. Drug treatments: 1i = DNA-PKi; 2i = DNA-PKi + PolQi1; 2⁺i = DNA-PKi + PolQi1 + PolQi2. Source data are provided as a Source Data file.

Fig. 3. Co-inhibition of DNA-PK and Polϴ decreases PEn off-target editing.

a Amplicon-seq and CRISPResso2 analysis at four on-target and 13 off-target sites for the indicated PEn configurations using plasmid co-transfections in HEK293T cells. Editing is shown as percentages of modified reads in each sample. Mismatches to the on-target gRNA sequence are highlighted in red lower-case letters, the PBS region is underlined. b Computed specificity scores for all off-target data shown in (a). Specificity scores are calculated as 1 minus the ratio of editing frequency at the off-target site to that at the on-target site resulting in a range between 0 and 1. Each dot in the graph represents one individual off-target, horizontal lines represent median and 95% confidence intervals of n = 3 biological replicates. Significance levels of specificity scores were calculated with the non-parametric Friedman test (paired, two-tailed). Multiple comparisons were corrected using Dunn’s test. Calculated P-values: PEn vs. uPEn >0.9999 (ns), PEn vs. 1iPEn = 0.7544 (ns), PEn vs. 2iPEn = 0.0008 (***), PEn vs. 2⁺iPEn <0.0001 (****). ns not significant. Drug treatments: 1i = DNA-PKi; 2i = DNA-PKi + PolQi1; 2⁺i = DNA-PKi + PolQi1 + PolQi2. Source data are provided as a Source Data file. PBS Primer Binding Site (PBS), PAM Protospacer Adjacent Motif.

Fig. 4. Co-inhibition of DNA-PK and Polϴ increases precision of different nickase-based prime editing modalities in different cell lines.

a 11 bp insertion in PCSK9 locus in HEK239T and HeLa cells using PE2, PE3, PE4, PE5. b Installation of indicated edits in HEK293T and HeLa cells with PE3 and PE5. c Calculated precision score across all tested loci in (a and b) and Supplementary Fig. 5a). Plots represent mean ± SD of n = 12 precision scores calculated at target sites in Fig. 2a, b. P-values were determined using one-way ANOVA (two-tailed, paired) with Tukey’s multiple comparisons test. Displayed pairwise comparisons are relative to PE3 or PE5. Calculated P-values: HEK293T: PE3 vs 1iPE3 = 0.9860 (ns); PE3 vs 2iPE3 = 0.2791 (ns); PE3 vs 2⁺iPE3 = 0.0106 (*); PE5 vs 1iPE5 = 0.9946 (ns); PE5 vs 2iPE5 = 0.4350 (ns); PE5 vs 2⁺iPE5 = 0.0277 (*). HeLa: PE3 vs 1iPE3 = 0.0655 (ns); PE3 vs 2iPE3 < 0.0001 (****); PE3 vs 2⁺iPE3 < 0.0001 (****); PE5 vs 1iPE5 = 0.2166 (ns); PE5 vs 2iPE5 < 0.0001 (****); PE5 vs 2⁺iPE5 < 0.0001 (****). d 11 bp insertion of PCSK9 locus in HEK239T using PE2, PE7, and PE7 with ngRNA. e +4T>G substitution of FANCF locus in iPSCs, PHH, HepG2, and Huh7 cell lines using PE3 (mRNA) and synthetic pegRNA/ngRNA. f +5G>A substitution of HBB locus in K562 cells using PEn (mRNA) and synthetic pegRNA/ngRNA. Editing outcomes and precision scores were analyzed as described above. P-values were determined using one-way ANOVA (two-tailed, paired) with Tukey’s multiple comparisons test. Displayed pairwise comparisons are relative to 2i⁺PEn or PE5. Calculated P-values = PEn vs. 2⁺iPEn = 0.0109 (*), 1iPEn vs. 2⁺iPEn = 0.0215 (*). All amplicon sequencing data described above was analyzed using CRISPResso2 in prime editing mode. All bar plots show mean ± SD of n = 3 biological replicates. Drug treatments: 1i = DNA-PKi; 2i = DNA-PKi + PolQi1; 2⁺i = DNA-PKi + PolQi1 + PolQi2. Source data are provided as a Source Data file.

Fig. 5. Co-inhibition of DNA-PK and Polϴ increases precision of dual pegRNA editing systems TwinPE, HOPE, Bi-PE, and TwinPEn.

a Schematic representation of TwinPE-mediated replacement with possible editing outcomes. b TwinPE mediated attP (50 bp) or attB site (38 bp) installation at three different sites (AAVS1, ACTB, CCR5) with indicated drug treatments in HEK293T cells. c Schematic representation of HOPE and Bi-PE editing. d Editing outcomes of HOPE (insertion) and Bi-PE2 and Bi-PE3 (five individual substitutions) at indicated target site subjected to indicated drug treatments in HEK293T cells. e Schematic representation of TwinPEn-mediated replacement and possible editing outcomes. f TwinPEn mediated attP (50 bp) or attB site (38 bp) installation at three different sites (AAVS1, ACTB, CCR5) with indicated drug treatments in HEK293T cells. All amplicon sequencing data described above was analyzed using CRISPResso2 in HDR mode. All bar plots show mean ± SD of n = 3 biological replicates. Drug treatments: 1i = DNA-PKi; 2⁺i = DNA-PKi + PolQi1 + PolQi2. Source data are provided as a Source Data file. RTT Reverse-Transcriptase Template, bp base pairs, del. deletion, ins. insertion.