Single-Stranded DNA with Internal Base Modifications Mediates Highly Efficient Gene Insertion in Primary Cells

Abstract

Single-stranded DNA (ssDNA) templates along with Cas9 have been used for gene insertion but suffer from low efficiency. Here, we show that ssDNA with chemical modifications in 10-17% of internal bases (eDNA) is compatible with the homologous recombination machinery. Moreover, eDNA templates improve gene insertion by 2-3 fold compared to unmodified and end-modified ssDNA in airway basal stem cells (ABCs), hematopoietic stem and progenitor cells (HSPCs), T-cells and endothelial cells. Over 50% of alleles showed gene insertion in three clinically relevant loci (CFTR, HBB, and CCR5) in ABCs using eDNA and up to 70% of alleles showed gene insertion in the HBB locus in HSPCs. This level of correction is therapeutically relevant and is comparable to adeno-associated virus-based templates. Knocking out TREX1 nuclease improved gene insertion using unmodified ssDNA but not eDNA suggesting that chemical modifications inhibit TREX1. This approach can be used for therapeutic applications and biological modeling.

Figure 1.

Extensive Chemical Modifications Improve Gene Insertion in Airway Cells. A) Graphic showing the chemical modifications of ssDNA and predicted nuclease degradation activity and inactivity. 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 HR than end-modified ssDNA (24 ± 6%), which are both significantly higher than unmodified ssDNA (15 ± 5%). Modifying every third base results in the least gene insertion (11 ± 15%). Statistical comparison was made using one-way ANOVA. D) Chemical modification of every sixth base in ssDNA enables improved gene insertion compared to ssDNA with CTS. E) ssDNA modified at every sixth base pair (esDNA) is also more efficient than end-modified ssDNA in the HBB locus. The esDNA templates achieved gene insertion in 51 ± 9% alleles at the 800nM concentration as opposed to 22 ± 4% for end-modified ssDNA. At 400 nM, esDNA achieved 31 ± 6% gene insertion compared to 7 ± 3% gene insertion achieved by end-modified ssDNA. Statistical comparison was made using two-way ANOVA. F) Using esDNA improves gene insertion compared to end-modified ssDNA in the CCR5 locus at 200 nM (41 ± 5% vs 17 ± 1%) and 400 nM (55 ± 6% vs 40 ± 4%) but not at 800 nM (53 ± 16% vs 51 ± 10%). Statistical comparison was made using two-way ANOVA. G) In airway cells, esDNA with OM modification also shows higher HR than unmodified ssDNA (55 ± 19% vs 15 ± 8%) which is not significantly different from HR obtained using AAV templates (82 ± 15%). (n = 5 biological replicates for esDNA with PS, OM modifications and ssDNA. n = 3 biological replicates for AAV). H) Gene insertion of different lengths was attempted in exon 12 of the CFTR locus using esDNA templates to introduce the G551D mutation. Editing was performed with DNA-PKcs inhibition. Template 1 (T1) which replaces 12 bp achieved ~80–90% gene insertion while template 2 (T2) which replaces 34 bp achieved only ~60 bp gene insertion. Statistical comparison was made using one-way ANOVA. For all panels using one-way ANOVA, multiple comparisons was 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 OM and esDNA PS corrected ABCs from CF donors (49 ± 14% and 47 ± 14%, respectively). (n=4 biological replicates). B) Gene correction of the F508del mutation was confirmed using next-generation sequencing. NGS showed 41.8% alleles with perfect HR and ~5% alleles with imperfect HR. Unintended substitutions were found in only 0.6% of alleles. The sgRNA sequence is highlighted in blue. C) A representative FACS 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 (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. F) Representative traces from Ussing chamber analysis showing a non-CF control, an uncorrected CF sample, esDNA PS and esDNA 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. 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 show increased response to CFTR_inh-172 relative to uncorrected controls. This change in current was statistically significant when compared using one-way analysis of variance (ANOVA) followed by multiple comparisons test (p < 0.05).

Figure 3.

esDNA templates improve gene insertion in HSPCs, T-cells and HUVECs but not iPSCs. HR in HSPCs significantly increased with esDNA compared to end-modified ssDNA in the A) HBB locus (47 ± 22% vs 18 ± 13%), B) CFTR locus (20 ± 6% vs. 5 ± 5%), and C) CCR5 locus (38 ± 26% vs. 15 ± 3%). D) In HUVECs, esDNA increased HR in the CFTR locus from 4 ± 3% obtained with end-modified ssDNA to 12 ± 5% (p = 0.0531). E) In T-cells, esDNA increased HR in the CFTR locus from 13 ± 6% obtained with end-modified ssDNA to 30 ± 10%. F) Gene insertion with esDNA (30 ± 12%) was significantly lower in iPSCs in the CFTR locus than unmodified ssDNA (71 ± 17%). 3 biological replicates were tested for each cell type. Groups in A-E were compared using a paired T-test. ** and * represent p <0.01, p < and p < 0.05 respectively for all panels.

Figure 4.

Knocking out TREX1 improved Gene Insertion without chemically modifying ssDNA. A) Percentage of alleles with TREX1 knocked out in non-CF ABCs samples (n=3 biological replicates). B) HR using unmodified ssDNA templates was improved when TREX1 was knocked out in non-CF ABCs. Knocking out TREX1 did not improve HR by esDNA templates (n = 4 biological replicates). C) Graphic showing additional chemical modification combinations including commercially available proprietary end modifications. D) Gene insertion with ssDNA using combinations of chemical modifications in ABCs. Both esDNA OM (40 ± 21%) and esDNA PS (34 ± 16%) showed improved gene insertion relative to unmodified (3 ± 3%), end-modified (11 ± 6%) and end-blocked ssDNA (Alt-HDR) (23 ± 9%). All other combinations did not improve on esDNA OM (40 ± 21%) and esDNA PS (34 ± 16%) (n=6 biological replicates). E) Modification of every 10^th base with PS groups also improved gene insertion compared to unmodified or end-modified ssDNA. Gene insertion was reduced when every fourth base was modified. ** and * represent p <0.01, p < and p < 0.05 respectively in all panels.