Recently, Crop Precision Breeding Technology Innovation Team of the Institute of Crop Sciences of the Chinese Academy of Agricultural Sciences (ICS-CAAS) has successfully developed efficient surrogate prime editors PE3-HS, PE3-AS, and PE3-DS, realizing multiplex precision gene editing in rice for the first time. This study further expanded the application of the prime editing (PE) system in multigene pyramiding crop breeding. The research was published online in Molecular Plant.

According to the corresponding author, Professor Xia Lanqin, the ability to manipulate multiple genomic sites in a predefined manner is of great significance in pyramiding beneficial alleles in one variety for crop improvement. The PE system enables precise installation or replacement of short indels in a specific gene without requiring double-strand DNA breaks and donor repair templates. It provides an effective tool for precise genome editing and will greatly improve the efficiency of crop breeding. So far, only accurate modification of a single gene by prime editing has been reported in various species including crop plants at variable efficiencies. Multiplex precise gene editing by prime editing has not been documented yet.

In this study, the Team further optimized the PE3 system on the basis of previous research and developed three surrogate prime editors, including hygromycinY46*-based (based on hygromycin resistance gene), OsALSS627I-based (based on herbicide resistance gene), and a combined double surrogate system (based on both hygromycin resistance gene and herbicide resistance gene), respectively, for prime editing of endogenous OsSPL14, OsDHDPS, and OsNR2 genes with substantially improved editing efficiency in rice stable lines. While the hygromycinY46*-based and OsALSS627I-based surrogate prime editors could increase the editing efficiencies by ~2-14-fold, the double surrogate system could stimulate the prime editing efficiencies by up to ~50-fold. Furthermore, the Team precisely edited several endogenous genes simultaneously and obtained stable lines by using this double surrogate system. Together, the developed surrogate prime editors demonstrated substantially improved editing efficiency and enabled multiplex precise gene editing, thereby greatly expanding the potential of prime editing in the concurrent improvement of multiple traits in crop plants.

Li Huiyuan and Zhu Ziwei are the co-first authors, and Professors Xia Lanqin and Ma Youzhi are the corresponding authors of this work. This work is partly funded by the National Natural Science Foundation of China (32188102), Hainan Yazhou Bay Seed Lab (B21HJ0215), National Key Research and Development Program of China (2020YFE0202300), the Central Public-interest Scientific Institution-Based Research Fund (S2022ZD03) and National Engineering Research Centre of Crop Molecular Breeding.

Reference resource: https://doi.org/10.1016/j.molp.2022.05.009

  By Xia Lanqin (xialanqin@caas.cn)