Yeast Prime Editing via Segmented Two-Plasmid System
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Solution Overview
Problem
Current gene editing techniques for Saccharomyces cerevisiae, such as those based on recombinases and homologous recombination, are inadequate for large-scale modification of engineered strains, necessitating a more efficient and precise gene editing tool.
Innovation Solution
A two-plasmid system for prime editing in yeast, comprising a first plasmid encoding an engineered pegRNA with a 3′-terminus motif and a second plasmid encoding a nucleic acid nickase nCas9 fused with a reverse transcriptase M-MLV RT, including specific mutations for improved stability and efficiency, is developed.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If recombinase and homologous recombination techniques are used for gene editing in yeast, then gene editing can be achieved, but the efficiency and precision are insufficient for large-scale modification of engineered strains
Solution Approach 1:
The prime editing system is divided into two separate plasmids: one expressing the nCas9-RT fusion protein and the other expressing the pegRNA. This segmentation allows independent optimization of each component's expression and function, enabling high-efficiency gene editing while maintaining precision through controlled co-delivery and expression timing.
Solution Approach 2:
The pegRNA serves as an intermediary molecule that guides the nCas9-RT fusion protein to the target DNA sequence. The pegRNA contains both the guide sequence for target recognition and the reverse transcription template for precise editing, mediating between the editing machinery and the target genome with high precision and efficiency.
2Device complexity
If the pegRNA 3′-terminus is left without structural RNA motifs, then the pegRNA structure is simpler, but the pegRNA is prone to degradation which disturbs editing efficiency
Solution Approach 1:
Structural RNA motifs are added specifically at the 3′-terminus of the pegRNA, while the rest of the pegRNA structure remains relatively simple. This localized enhancement provides the necessary stability at the vulnerable 3′ end without significantly increasing the overall complexity of the pegRNA structure, thereby improving reliability with minimal complexity penalty.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The two-plasmid system achieves high editing efficiency and accuracy, with successful mutation of the GGT codon to a stop codon in the ADE2 gene of yeast strains, demonstrating its potential for cost-effective translation of engineered strains and genome editing.
Implementation Method 1
The M-MLV RT protein can prime reverse transcription along the RTT sequence, transferring the information required for editing from the pegRNA to the DNA
Implementation Method 2
a nucleic acid nickase Cas9-nickase (nCas9)... breaks one of the DNA strands which contains a protospacer adjacent motif (PAM) target site
Implementation Method 3
This would form a dynamic equilibrium between 3′-flap and 5′-flap structures at the nick of the DNA strand... the 3′-flap with the information required for editing can be, through competing, integrated into the DNA double strands
Implementation Method 4
the 5′-flap without the information required for editing is prone to be recognized and cleaved by an exonuclease
Data Source
AI summary
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.