| Title: |
Prime editing of a candidate rice gene for broad-spectrum resistance against fungal pathogens |
| Authors: |
Balahadia, Christian Paolo; Atienza-Grande, Genelou; Micouin, Cédric; Yanoria, Mary Jeanie; Slamet-Loedin, Inez; Oliva, Ricardo; Schepler-Luu, Van |
| Publisher Information: |
Zenodo |
| Publication Year: |
2023 |
| Collection: |
Zenodo |
| Description: |
Rice has been devastated by various fungal pathogens resulting in significant yield losses and posing a threat to global food security. Breeding for disease resistance in wheat and rice has resulted in the identification of various susceptibility genes that shows to offer broad-spectrum resistance against fungal pathogens. Studies in wheat showed that a simple glycine to arginine (G142R) mutation of the wheat STP13 gene conferred resistance against wheat fungal pathogens. The rice OsMST4 gene was found to be an ortholog of STP13 and bears the putative susceptible G142 allele. This study aims to make use of a precise gene editing technique that would mimic the G142R mutation found in resistant STP13 allele into OsMST4 gene, to potentially confer broad-spectrum resistance against rice fungal pathogens. Prime editing offered a new approach to precise gene editing by combining the Cas9 nuclease with an engineered reverse transcriptase that allows the direct rewriting of the target DNA sequence. In this study, we made use of the recent advances in the prime editing system to introduce the G142R mutation into the OsMST4 gene of rice. We made use of the enhanced Plant Prime Editor (ePPE) which removed the RNAse activity of the reverse transcriptase, added viral nucleotide-binding domain, and improved the linker sequence between the two enzymes to increase the efficiency and precision of prime editing in rice. We also employed the epegRNA design strategy which makes use of rice RNA polymerase III promoters to express the guide and template RNA sequence together with the truncated evopreq1 3' terminal knot to also help increase editing efficiency. The final ePPE construct contained three epegRNAs driven by OsU3, OsU6.1, and OsU6.2 promoters, targeting specific regions of the OsMST4 gene with the same repair template that would convert G142 to R142. Using the improved ePPE system, we successfully edited the OsMST4 gene in rice, introducing the G142R mutation with very high editing efficiency. Further characterization of ... |
| Document Type: |
conference object |
| Language: |
unknown |
| Relation: |
https://zenodo.org/communities/irc2023/; https://zenodo.org/records/10397977; oai:zenodo.org:10397977; https://doi.org/10.5281/zenodo.10397977 |
| DOI: |
10.5281/zenodo.10397977 |
| Availability: |
https://doi.org/10.5281/zenodo.10397977; https://zenodo.org/records/10397977 |
| Rights: |
Creative Commons Attribution 4.0 International ; cc-by-4.0 ; https://creativecommons.org/licenses/by/4.0/legalcode |
| Accession Number: |
edsbas.9D7B9FD9 |
| Database: |
BASE |