gE-Deleted Pseudorabies Virus Construction via Precise Adenine Base Editing
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Solution Overview
Problem
Existing gene editing technologies for pseudorabies virus (PRV) such as ZFNs, TALENs, and CRISPR/Cas9 suffer from inefficiencies, off-target effects, and structural variations, limiting their effectiveness in silencing the gE virulence gene without affecting immunogenicity.
Innovation Solution
Utilizing an adenine base editor (ABE) to precisely mutate the start codon of the gE gene in PRV, achieving efficient and cost-effective gene silencing by converting adenine to guanine, thereby blocking protein translation and constructing a gE protein-deleted PRV strain.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If CRISPR/Cas9 system is used for gene editing, then gene knockout efficiency is improved, but off-target cuts and structural variations occur in the genome
Solution Approach 1:
The patent changes the fundamental parameter of the gene editing mechanism from double-strand break induction (CRISPR/Cas9) to single base deamination (ABE8e). This parameter change eliminates the harmful effects of off-target cuts and structural variations while maintaining high editing efficiency at the target site.
Solution Approach 2:
The patent replaces the mechanical cutting mechanism of CRISPR/Cas9 with a chemical deamination mechanism using ABE8e. Instead of physically breaking DNA strands, the adenine base editor chemically converts adenine to guanine at the target site, avoiding the harmful mechanical disruptions caused by double-strand breaks.
2Productivity
If ZFNs or TALENs are used for gene editing, then gene knockout can be achieved, but design complexity and cost increase
Solution Approach 1:
The patent employs CRISPR guide RNA technology, which provides universal applicability across different target genes. The sgRNA can be easily redesigned to target any gene sequence, eliminating the need for complex protein design required by ZFNs and TALENs, while maintaining gene knockout capability.
Solution Approach 2:
The patent uses disposable sgRNA molecules that can be easily synthesized and replaced for different targeting needs, rather than investing in complex, expensive protein designs. This approach significantly reduces design complexity and cost while maintaining editing effectiveness.
3Productivity
If CRISPR/Cas9 is used for gene editing, then editing efficiency is improved, but unexpected base mutations and off-target effects occur
Solution Approach 1:
The patent applies local quality modification by using ABE8e to perform site-specific adenine-to-guanine conversion at the precise target location (start codon ATG to GTG). This localized chemical modification achieves high precision without the widespread base mutations caused by CRISPR/Cas9 double-strand break repair.
Solution Approach 2:
The patent substitutes the non-specific mechanical cutting action of CRISPR/Cas9 with a specific chemical deamination reaction catalyzed by ABE8e. This chemical mechanism provides superior base editing precision by converting only the target adenine base while leaving surrounding sequences unchanged.
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 method achieves precise and efficient gene silencing with minimal structural variations and low cost, resulting in a stable gE protein-deleted PRV strain suitable for vaccine development.
Implementation Method 1
The single base editing technology is to fuse a modified Cas protein with no nuclease cleavage activity or single-strand nicking activity and a deaminase, and accurately anchor a resulting fused deaminase to a target site with sgRNA to allow base deamination, thereby achieving precise editing of single bases.
Implementation Method 2
tRNA adenylate deaminase (TadA) is fused with nCas9 to develop a novel single-base conversion system (namely an adenine base editor, ABE) that can accurately convert adenine into guanine.
Data Source
AI summary
Provided is a method for constructing a gE protein-deleted pseudorabies virus (PRV) strain using an adenine base editor (ABE) and use thereof. The method includes: designing an sgRNA sequence using the ABE with a start codon of the gE gene in a PRV as a target site, ligating an enzyme-digested fragment to a double-stranded DNA fragment with sticky ends to obtain a ligation product; and transforming the ligation product into a competent cell to allow plate screening and culture, selecting a resulting positive bacterial strain to allow expanded culture, and extracting a plasmid from a resulting positive bacterial solution; and transferring the plasmid into a target cell to allow the transfection for 24 h, collecting a resulting virus liquid, and centrifuging the virus liquid to collect a resulting supernatant.