Single-stranded DNA with internal base modifications mediates highly efficient knock-in in primary cells using CRISPR-Cas9

Abstract

Single-stranded DNA (ssDNA) templates along with Cas9 have been used for knocking-in exogenous sequences in the genome but suffer from low efficiency. Here, we show that ssDNA with chemical modifications in 12-19% of internal bases, which we denote as enhanced ssDNA (esDNA), improve knock-in (KI) by 2-3-fold compared to end-modified ssDNA in airway basal stem cells (ABCs), CD34 + hematopoietic cells (CD34 + cells), T-cells and endothelial cells. Over 50% of alleles showed KI in three clinically relevant loci (CFTR, HBB and CCR5) in ABCs using esDNA and up to 70% of alleles showed KI in the HBB locus in CD34 + cells in the presence of a DNA-PKcs inhibitor. This level of correction is therapeutically relevant and is comparable to adeno-associated virus-based templates. The esDNA templates did not improve KI in induced pluripotent stem cells (iPSCs). This may be due to the absence of the nuclease TREX1 in iPSCs. Indeed, knocking out TREX1 in other cells improved KI using unmodified ssDNA. esDNA can be used to modify 20-30 bp regions in primary cells for therapeutic applications and biological modeling. The use of this approach for gene length insertions will require new methods to produce long chemically modified ssDNA in scalable quantities.

Graphical Abstract

Figure 1.

Chemical modification of internal bases in ssDNA improves mutational KI in ABCs. (A) Graphic showing ssDNA with different levels of chemical modifications. (B) Graphic showing the chemical structure of modified bases. (C) In airway cells at the CFTR locus, ssDNA templates with every sixth base modified (41 ± 15%) show significantly higher HDR than end-modified ssDNA (24 ± 6%), which are both significantly higher than unmodified ssDNA (15 ± 5%). Modifying every third base results in the least mutational KI (11 ± 15%). Templates were used at a concentration of 800 nM. Statistical comparison was made using one-way analysis of variance (ANOVA). (D) Chemical modification of every sixth base in ssDNA enables improved mutational KI compared to ssDNA with CTS. (E) ssDNA modified at every sixth base pair is also more efficient than end-modified ssDNA in the HBB locus. The ssDNA templates modified in every sixth base achieved mutational KI in 51 ± 9% alleles at the 800 nM concentration as opposed to 22 ± 4% for end-modified ssDNA. At 400 nM, ssDNA modified at every sixth base achieved 31 ± 6% mutational KI compared to 7 ± 3% mutational KI achieved by end-modified ssDNA. Statistical comparison was made using two-way ANOVA. (F) Every sixth modified ssDNA improves mutational KI compared to end-modified ssDNA in the CCR5 locus at 200 nM (41 ± 5% versus 17 ± 1%) and 400 nM (55 ± 6% versus 40 ± 4%) but not at 800 nM (53 ± 16% versus 51 ± 10%). Statistical comparison was made using two-way ANOVA. (G) In airway cells, ssDNA with every sixth base modified with 2′O-methyl (OM) also shows higher HDR than unmodified ssDNA (55 ± 19% versus 15 ± 8%) (n = 5 biological replicates for ssDNA modified with PS, OM modifications and unmodified ssDNA. n = 3 biological replicates for AAV). (H) Mutational KI of different lengths was attempted in exon 12 of the CFTR locus using ssDNA templates PS modified at every sixth base pair to introduce the G551D mutation. Editing was performed with DNA-PKcs inhibition. Template 1 (T1) which substitutes bases in a 12 bp region achieved ∼80–90% mutational KI while template 2 (T2) which substitutes bases in a 34 bp region achieved ∼60 bp mutational KI. Statistical comparison was made using T-test. For all panels using one-way ANOVA, multiple comparisons were performed using Tukey’s test. Sidak’s test was used for two-way ANOVA. ****, ***, ** and * represent P < 0.0001, P < 0.001, P< 0.01 and P < 0.05 respectively for all panels.

Figure 2.

Correction of F508del mutation restores CFTR function in ABCs from pwCF. (A) Both esDNA-6-PS and esDNA-6-OM (800 nM) corrected ABCs from CF donors (49 ± 14% and 47 ± 14%, corrected alleles respectively) (n = 4 biological replicates). (B) Gene correction of the F508del mutation was confirmed using next-generation sequencing. NGS showed that 57 ± 0% of alleles had perfect HDR in ABCs edited using esDNA-6-PS which was comparable to 65 ± 15% alleles with HDR when editing was performed using AAV. A total of 1–3% alleles had imperfect HDR. INDELs accounted for 27–29% alleles. ABCs treated with esDNA alone only had unmodified alleles. (C) A representative fluorescence-activated cell sorting plot shows that > 98% of ABCs were positive for KRT5 and P63 4 days after editing. There was no reduction in KRT5 and P63 expression in the edited ABCs relative to Mock electroporated controls in experiments from three different donors. (D) Edited samples displayed transepithelial resistances (TEER) similar to unedited controls [wild type (WT) and Mock], indicating that the editing process does not compromise the ability of the ABCs to produce fully differentiated epithelial sheets. (E) Edited ABCs produced epithelial sheets containing basal (KRT5), ciliated (ACT) and secretory cells (MUC5B), further indicating the formation of differentiated epithelial sheets. Scale bar represents 50 μm. (F) Representative traces from Ussing chamber analysis showing a non-CF control, an uncorrected CF sample, esDNA-6-PS- and esDNA-6-OM-corrected CF samples. Uncorrected CF samples show minimal responses to forskolin and CFTR_inh-172 which activate and inhibit CFTR, respectively. All edited samples showed restored responses to forskolin and CFTR_inh-172 indicating restored CFTR function. (G) Percent restoration of CFTR function in corrected CF samples relative to non-CF controls plotted as a function of allelic correction. Edited samples showed 30–60% restoration of CFTR function on average. This change in current was statistically significant when compared using one-way ANOVA followed by Dunnett’s T2 multiple comparisons test (P < 0.05). (H) Raw CFTR_inh-172 short circuit currents observed in CF donor ABCs compared to non-CF controls and uncorrected CF controls (n = 3 biological replicates). Samples corrected using esDNA-6-PS or esDNA-6-OM both show increased response to CFTR_inh-172 relative to uncorrected controls.

Figure 3.

esDNA templates (800 nM) improve mutational KI in HSPCs, T-cells and HUVECs but not iPSCs. HDR in CD34 + cells significantly increased with esDNA compared to end-modified ssDNA in the (A) HBB locus in the absence of AZD-7648 and (B) in the presence of AZD-7648. (C) The viability of CD34 + cells was not significantly different between edited CD34 + cells and unedited controls. HDR in CD34 + cells was also increased in the (D) CCR5 locus and (E) CFTR locus. (F) In T-cells, esDNA increased HDR in the CFTR locus. (G) In HUVECs, esDNA increased HDR in the CFTR locus from 4 ± 3% obtained with end-modified ssDNA to 12 ± 5% but the difference was not significant. Mutational KI with esDNA was significantly lower in iPSCs in the CFTR locus than unmodified ssDNA both in the (H) absence (not significant) and (I) presence of AZD-7648. 3 or more biological replicates were tested for each cell type. Groups in A–B and D–G were compared using a paired T-test. Statistical comparisons for panels (C), (H) and (I) were made using one-way ANOVA followed by Tukey’s test. ** and * represent P < 0.01 and P < 0.05, respectively, for all panels.

Figure 4.

Knocking out TREX1 improved mutational KI without chemically modifying ssDNA. (A) Western blot shows TREX1 expression in ABCs and HUVECs but not iPSCs. ABCs with TREX1 KO show reduced TREX1 expression. (B) HDR using unmodified ssDNA templates (800 nM) was improved when TREX1 was knocked out in non-CF ABCs. Knocking out TREX1 did not improve HDR by esDNA templates (n = 4 biological replicates). (C) Mutational KI with ssDNA using combinations of chemical modifications in ABCs. Both esDNA OM (40 ± 21%) and esDNA PS (34 ± 16%) showed improved mutational KI relative to unmodified (3 ± 3%), end-modified (11 ± 6%) and Alt-R HDR modified ssDNA (23 ± 9%). All other combinations did not improve on esDNA OM (40 ± 21%) and esDNA PS (34 (± 16%) (n = 4–6 biological replicates). (D) Modifying the 3′ half of the template alone with PS improved mutational KI compared to end modification (n = 3 biological replicates). Including PS chemical modifications throughout the template improved editing slightly but the improvement was not statistically significant compared to templates in which only the 3′ half was modified. (E) Templates with PS modified internal bases spread throughout the template resulted in more consistent HDR compared to template with PS modified bases in just the last 20 bases (n = 4 biological replicates). (F) Mutational KI in the HBB locus was significantly higher in ABCs using esDNA-6-PS templates (53 ± 14%) compared with Alt-R HDR templates (13 ± 12%) (n = 4 biological replicates). G) Mutational KI in the HBB locus was also significantly higher in CD34 + cells using esDNA-6-PS templates (66 ± 2%) compared with Alt-R HDR templates (17 ± 1%) (n = 4 biological replicates). (H) Modification of every 10th base with PS groups also improved mutational KI compared to unmodified or end-modified ssDNA. Mutational KI was reduced when every fourth base was modified. Statistical comparison was made using one-way ANOVA. Multiple comparisons were performed using Tukey’s test. ***, ** and * represent P < 0.005, P < 0.01, P < and P < 0.05 respectively in all panels. All experiments used 800 nM template.

Figure 5.

Editing using esDNA-6-PS elicits less p21 activation in CD34 + cells. (A) In CD34 + cells, mutational KI by esDNA-6-PS was less than the mutational KI achieved using AAV templates (n = 3–4 biological replicates). (B) There was no significant difference in the viability of edited CD34 + cells when measured using the trypan blue assay on day 2 after editing (n = 3 biological replicates). Statistical comparison was made using ANOVA followed by Tukey’s multiple comparisons test. (C) CD34 + cells edited using AAV templates showed a greater increase in p21 expression when measured by ddPCR. Treatment using AAV alone also resulted in increased p21 expression. Editing with esDNA-6-PS resulted in less p21 activation (n = 3 biological replicates with 2 technical replicates). Statistical comparison was made using one-way ANOVA. Multiple comparisons were performed using Tukey’s test. ** and * represent P < 0.01 and P < 0.05, respectively in all panels.