Characterizing and controlling CRISPR repair outcomes in nondividing human cells

Abstract

Genome editing is poised to revolutionize treatment of genetic diseases, but poor understanding and control of DNA repair outcomes hinders its therapeutic potential. DNA repair is especially understudied in nondividing cells like neurons, limiting the efficiency and precision of genome editing in many clinically relevant tissues. Here, we address this barrier by using induced pluripotent stem cells (iPSCs) and iPSC-derived neurons to examine how postmitotic human neurons repair Cas9-induced DNA damage. CRISPR editing outcomes differ dramatically in neurons compared to genetically identical dividing cells: neurons take longer to fully resolve this damage, and upregulate non-canonical DNA repair factors in the process. Manipulating this response with chemical or genetic perturbations allows us to direct DNA repair toward desired editing outcomes in nondividing human neurons, cardiomyocytes, and primary T cells. By studying DNA repair in clinically relevant cells, we reveal unforeseen challenges and opportunities for precise therapeutic editing.

Fig. 1. Modeling CRISPR repair outcomes in postmitotic human neurons.

a Schematic: Genome editing proteins can perturb DNA, but cellular DNA repair determines the editing outcome. b Timeline of differentiating iPSCs (blue) into neurons (green). After at least 2 weeks of differentiation/maturation, postmitotic neurons are treated with VLPs delivering Cas9 protein (yellow) and sgRNA (orange). c Cas9 VLPs induce DSBs in human iPSC-derived neurons. Representative ICC images of neurons 3 days post-transduction with B2Mg1 VLPs, and age-matched untransduced neurons. Scale bar is 20 µm. Arrows denote examples of DSB foci: yellow puncta co-labeled by γH2AX (red) and 53BP1 (green). Dose: 1 µL VLP (FMLV) per 100 µL media. Additional images shown in Supplementary Fig. 5, with similar results replicated independently in Supplementary Fig. 12. d Genome editing outcomes differ between iPSCs and isogenic neurons. CRISPResso2 analysis of amplicon-NGS, from cells 4 days post-transduction with B2Mg1 VLPs. Dose: 2 µL VLP (HIV) per 100 µL media. Data are averaged across 6 replicate wells per cell type transduced in parallel, and expressed as a percentage of total reads. Thick blue background bars are from iPSCs; thin green foreground bars are from neurons.

Fig. 2. Cas9-induced indels accumulate over a prolonged time span in neurons.

a Cas9-induced indels accumulate more slowly in neurons than in genetically identical iPSCs. Dose: 2 µL B2Mg1 VLP (HIV) per 100 µL media. For (a, b) 6 replicate wells per condition transduced in parallel (some obscured by overlap); curves pass through the mean at each timepoint. CRISPResso2 analysis of amplicon-NGS. b Several sgRNAs show weeks-long accumulation of indels in neurons. Dose: 1 µL VLP (FMLV) per 100 µL media. c–e Cas9-induced DSB foci (γH2AX+) remain detectable in neurons for at least 7 days post-transduction. Quantified in (c) by manual counting across n = 3 wells per condition; center points show means and error bars show SD. Representative ICC images of neurons 1 day (d) and 7 days (e) post-transduction, with age-matched untransduced neurons. Dose: 2 µL VLP (FMLV) per 100 µL media. See Supplementary Fig. 12 for unmerged/uncropped panels and full time course. ICC time course was conducted once; representative images chosen from 3 replicate wells per condition. f MRE11 is bound near the cut site in neurons for at least 8 days post-transduction. Dose: 2 µL B2Mg1 VLP (FMLV) per 100 µL media. Binding quantified by ChIP-qPCR, normalized for amplification efficiency and input chromatin. Average of 3 replicate reactions, normalized to untransduced control for each amplicon. Error bars show SD, centered at the mean. g Schematic: prolonged indel accumulation in neurons could be caused by neurons repairing DSBs more slowly, and/or by neurons undergoing more cycles of indel-free repair and re-cutting before edits arise. Our results do not rule out either model, but the early presence of post-repair products (Supplementary Fig. 13) and the surprising longevity of Cas9 protein in neurons (Supplementary Fig. 14) more strongly support the second model.

Fig. 3. Neuronal response to Cas9 reveals unexpected factors that influence editing outcomes in nondividing cells.

Neurons (b), but not iPSCs (a), dramatically upregulate transcription of DNA repair factors upon Cas9-VLP transduction. For a–d: dashed lines show cutoffs for significance (p_adj< 0.05) and effect size (fold-change > 2 or < 0.5). Statistical tests detailed in “Methods” section under “Bulk RNAseq”. For a–c, dose: 1 µL HIV VLP per 20,000 cells. c The most significantly altered DEGs in Cas9-VLP-treated neurons are highly enriched for factors canonically associated with DNA replication/repair. d RRM2 is more strongly upregulated in neurons treated with Cas9-VLP compared to dCas9-VLP. Dose: 2 µL FMLV VLP per 20,000 cells. e Inhibiting RRM2 yields a ~50% increase in neuron editing efficiency, within 4 days post-transduction. Dose: 1 µL B2Mg1 VLP (FMLV) per 20,000 cells in 100 µL media. Error bars show SEM. One-factor ANOVA, **p < 0.005. For e, f: n = 3 replicate wells transduced. For e-i: CRISPResso2 analysis of amplicon-NGS. f RRM2 inhibition shifts neuron indels from insertions toward deletions, and triples the frequency of 1-base deletions at 4 days post-transduction. Thick gray bars are DMSO condition; thin green bars are 3AP. g Inhibition of RRM2 or RRM1 consistently shifts neuron indels from insertions toward deletions. Dose: 2 µL B2Mg1 VLP (FMLV) per 20,000 cells in 100 µL media. n = 3 (top) or 6 (bottom) replicate wells transduced. h RRM2 inhibition also shifts indels from insertions toward deletions in resting (nondividing) primary human T cells. For h, i: n = 5 replicate electroporations with 6.25 pmol Cas9 RNP (B2Mg1), harvested 4 days post-electroporation. i RRM2 inhibition boosts total indel efficiency in resting primary human T cells from two independent donors. For g–i: Error bars show SEM. One-factor ANOVA with Tukey’s multiple comparison test, *p < 0.05, ***p < 0.0005, ns not significant.

Fig. 4. All-in-one particles deliver Cas9 and sgRNA while simultaneously manipulating DNA repair factors.

a LNP formulation F16-15 effectively delivers Cas9 mRNA and sgRNA to human iPSC-derived neurons. “F0” is DLin-MC3-DMA. n = 3 replicate wells transfected. For a–f: 125 ng total RNA per 20,000 cells in 100 µL media, targeting B2Mg1. For a: Synthego ICEv2 analysis, 2 weeks post-transfection. For c–f: CRISPResso2 analysis of amplicon-NGS, averaged across n = 5 (c), 8 (d), 6 (e), or 8 (f) replicate wells per condition transfected in parallel. b Schematic illustrating all-in-one LNPs that encapsulate Cas9 mRNA (yellow) and sgRNA (orange), along with siRNAs (blue) against a repair gene of interest. c All-in-one LNPs reveal additional targets that increase editing efficiency at 4 days post-transfection. One-Factor ANOVA with Tukey’s multiple comparison test, for each condition vs siNT: *p < 0.05, ns not significant. In both neurons (d) and cardiomyocytes (e), all-in-one LNPs knocking down RRM1 or POLL each phenocopy drug inhibition of RNR: shifting indels strongly from insertions toward deletions. Inhibiting XRCC5 boosts both insertions and deletions (see Supplementary Fig. 24). One-factor ANOVA with Tukey’s multiple comparison test. Each condition vs siNT, *p < 0.05, **p < 0.005, ***p < 0.0005, ns not significant. f Knockdown of XRCC5, POLL, or RRM1 significantly increases total editing efficiency in neurons at 2 weeks post-transfection. One-factor ANOVA with Tukey’s multiple comparison test. Each condition vs siNT, ***p < 0.0005. Error bars show SEM.