CRISPR/Cas has been developed for targeted mutagenesis in diverse species, including plants. However, precise genome editing via homology-directed repair (HDR) is inefficient in plants, limiting our ability to make large deletions or insertions in the plant genomes. Prime editing increases the control over the desired editing and allows the precise introduction of all types of mutations, including insertion, deletions, and all possible base conversions, albeit at low efficiencies. Here, we designed a dual prime editing system to generate large deletions and precise insertions of sequences by repairing template complementarity. We coupled dual pegRNA with Cas9 nickase (nCas9) to generate deletions and insertions. In another modality, we used dual pegRNA with wild-type Cas9 to generate double-stranded breaks to improve the editing at the targeted sites. We tested dual pegRNAs to delete the last exon in OsCCD7, delete the microRNA targeted sequence in OsIPA, and insert the T7 promoter in the 3'UTR of OsALS. Our results showed a high frequency of targeted insertion of the T7 promoter sequence in the 3'UTR of OsALS with wtCas9 and nCas9. Sanger sequencing analysis showed partial deletions at the targeted locus. Further improvements in the designs of pegRNAs will increase the precise genome insertions and deletions in plants.
| Identifer | oai:union.ndltd.org:kaust.edu.sa/oai:repository.kaust.edu.sa:10754/680478 |
| Date | 08 1900 |
| Creators | Moreno-Ramírez, Jose Luis |
| Contributors | Mahfouz, Magdy M., Biological and Environmental Science and Engineering (BESE) Division, Blilou, Ikram, Lauersen, Kyle J. |
| Source Sets | King Abdullah University of Science and Technology |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Rights | 2023-08-23, At the time of archiving, the student author of this thesis opted to temporarily restrict access to it. The full text of this thesis will become available to the public after the expiration of the embargo on 2023-08-23. |
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