Development of a highly efficient prime editor system in mice and rabbits

Abstract

Abstract The recent prime editing (PE) technique is more precise and permits base-to-base conversion, replacement, and insertion and deletion in the genome. However, previous reports show that the efficiency of prime editing is insufficient for producing genome edited animals. In fact, prime-guide RNA (pegRNA) designs have been the drawback in achieving favorable editing efficiency. Here, we designed prime binding sites (PBS) with a melting temperature (Tm) of 42 ℃ leads to optimal performance in cells, and found the optimal Tm was affected by the culture temperature. In addition, the ePE3max system was developed by updating the PE architecture to PEmax and expressing engineered pegRNA (epegRNA) based on the original PE3 system. The updated ePE3max system can efficiently induce gene editing in mouse and rabbit embryos. Furthermore, we successfully generated Hoxd13 (c. 671 G > T) mutation in mice and Tyr (c. 572 del) mutation in rabbits by ePE3max. Collectively, the editing efficiency of modified ePE3max systems is superior to original PE3 system in producing genome editing animals, which can serve as an effective and versatile genome editing tool for precise genome modification in animal models.