ORIGINAL RESEARCH article
Front. Genome Ed.
Sec. Genome Editing in Plants
Volume 7 - 2025 | doi: 10.3389/fgeed.2025.1661829
Single-stranded DNA (ssDNA) donor repair templates and CRISPR/Cas9 enable a high high-frequency of targeted insertions in potato
Provisionally accepted
- Swedish University of Agricultural Sciences, Department of Plant Breeding, Box 190, 234 22 Lomma, Sweden
Select one of your emails
You have multiple emails registered with Frontiers:
Notify me on publication
Please enter your email address:
If you already have an account, please login
You don't have a Frontiers account ? You can register here
Homology-directed repair (HDR) holds great promise for plant genetic engineering but remains challenging due to its inherently low efficiency in gene editing applications. While studies in animal systems suggest that the structure of the donor repair template (DRT) influences HDR efficiency, this parameter remains largely unexplored in plants. In this study, we combined protoplast transfection with next-generation sequencing to analyse the impact of DRT structure on HDR efficiency in potato. A highly efficient ribonucleoprotein (RNP) complex targeting the soluble starch synthase 1 (SS1) gene was used in combination with various DRTs, differing in structural factors such as homology arm (HA) length, strandedness (i.e. ssDNA vs dsDNA), and sequence orientation in ssDNA donors. Our results indicate that a ssDNA donor in the target orientation outperformed other configurations, achieving a HDR efficiency of 1.12% of the sequencing reads in the pool of protoplasts. Interestingly, HDR efficiency appeared independent of HA length. Notably, a ssDNA donor with HAs as short as 30 nucleotides led to targeted insertions in up to 24.89% of reads on average, but predominantly via alternative repairimprecise repair pathways, such as microhomology-end joining (MMEJ). This donor structure also consistently yielded the highest HDR and targeted insertion rates at two out of three additional loci tested, offering valuable insights for future genome editing strategies in potato. We further assessed strategies to favour HDR over alternative repair outcomes, including the use of small molecules known to inhibit competing pathways in animal systems, and modifications to DRTs to enhance their availability in the vicinity of the target site. However, these approaches did not improve HDR efficiency. Overall, this study presents an effective platform for rapidly assessing gene editing components in potato and provides insights for achieving high-frequency, targeted insertions of short DNA fragments, especially relevant for efficient knock-in integration in non-coding genomic regions.
Keywords: gene editing, CRISPR, DRT, HDR, Homologous Recombination, protoplast
Received: 08 Jul 2025; Accepted: 03 Sep 2025.
Copyright: © 2025 González, Salaria, Friberg, Liu, Alverup, Andersson and Hofvander. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence: Matías González, Swedish University of Agricultural Sciences, Department of Plant Breeding, Box 190, 234 22 Lomma, Sweden
Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.