Abstract: The present disclosure relates to a two-plasmid system for prime editing in yeast, use thereof, and a method for gene prime editing in yeast. The two-plasmid system includes a first plasmid and a second plasmid. The first plasmid includes a sequence encoding for an epegRNA. The epegRNA is an RNA molecule including a motif at a 3?-terminus of a pegRNA, and the motif having a sequence as set forth in SEQ ID NO. 12. The second plasmid includes a sequence encoding for a fusion protein of a nucleic acid nickase nCas9 fused with a reverse transcriptase M-MLV RT. The two-plasmid expression system can be used in gene editing in yeast.

Abstract: Provided herein are methods and compositions for a CRISPR and homology-directed-repair assisted genome-scale engineering that can rapidly output tens of thousands of specific genetic variants in host cells. More than 98% of target sequences can be efficiently edited with a high average frequency.