Successful CRISPR/Cas9-mediated HDR at individual DNA breakpoints using TFO-based targeted template design

Targeted insertion of the repair template into the genome is a common strategy for high-precision base replacements, however, the main challenge likely remains regarding limited efficiency of homologous-directed repair (HDR). A precise genome cut achieved by CRISPR-Cas9 system combining with a single-stranded oligodeoxynucleotide (ssODN), as the donor template, improves significantly the rate of HDR. It is well-established that the spatial availability of the donor template to the repair system effectively enhances knock-in events in CRISPR-Cas9. PolyPurine Reverse Hoogsteen hairpins (PPRHs), as an alternative repairing strategy, benefits from a Triplex-forming oligonucleotide (TFO) for the repair template providing the ease of access. The main objective of the study was to evaluate the HDR frequency as a result of improvement of the spatial accessibility of the donor template adjacent to the cutting site. Hence, a flanking purine-rich hairpin complementary to the genomic DNA adjacent to the repairing site was fused to the ssODN with the incorporated bases for alteration of EGFP to EBFP. Results from the comparison between the donor templates, ssODN and TFO-tailed ssODN, demonstrated an increased rate of knock-in from 18.2% ± 1.09 to 38.3% ± 4.54, respectively. As another perspective, findings indicated that the targeted Cas9-mediated DNA cleavage improves the efficiency of the repair-PPRH approach by four-fold, as well. Conclusions The present study provides a viewpoint that highlights the significance of the designing of the donor template in terms of the structural features and positional access for the HDR-based repairing CRISPR-Cas9 systems.