Electronic Journal of Biotechnology (Mar 2024)
Successful CRISPR/Cas9-mediated HDR at individual DNA breakpoints using TFO-based targeted template design
Abstract
Background: 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 the 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 the alteration of EGFP to EBFP. Results: 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. From another perspective, findings indicated that the targeted Cas9-mediated DNA cleavage improves the efficiency of the repair-PPRH approach 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.How to cite: Ebrahimi Z, Kazemi B, Salehi M, et al. Successful CRISPR/Cas9-mediated HDR at individual DNA breakpoints using TFO-based targeted template design. Electron J Biotechnol 2024, 68. https://doi.org/10.1016/j.ejbt.2024.01.001.
