Recombinant HVT Genome Insertion Using CRISPR-Cas9
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Solution Overview
Problem
Existing methods for producing recombinant herpesvirus of turkeys (HVT) vaccines are inefficient and time-consuming, particularly in inserting exogenous genes using conventional homologous recombination or bacterial artificial chromosomes (BACs, limiting the production of recombinant viruses.
Innovation Solution
Utilizing CRISPR-Cas9 technology to insert exogenous genes into a specific spacer region between HVT005 and HVT006 regions of the HVT genome, specifically between 8,867 nt and 9,319 nt, with designed sgRNA sequences and donor vector plasmids, enabling rapid and efficient construction of recombinant HVT strains expressing Newcastle disease virus (NDV) F gene, avian influenza virus (AIV) HA gene, or infectious bursal disease virus (IBDV) VP2 gene.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If conventional homologous recombination or BAC methods are used to insert exogenous genes into HVT, then the vaccine can provide multiple disease protections, but the production efficiency is low and the process is time-consuming
Solution Approach 1:
The patent replaces the conventional mechanical recombination methods (homologous recombination in virus-infected cells or BAC recombination) with the CRISPR-Cas9 gene editing system. This substitution enables precise, efficient insertion of exogenous genes into the HVT genome at specific sites (US2, US10, UL45/46, HVT065/066, or TK regions), dramatically improving production efficiency while maintaining the capability to provide multiple disease protections through co-expression of different antigens
Solution Approach 2:
The patent performs preliminary design of the insertion site and donor construct before the actual gene insertion process. By pre-determining the target location in the HVT genome and preparing the donor DNA construct with the desired exogenous genes, the method streamlines the overall process and reduces the time required for producing recombinant HVT vaccines with multiple antigen expressions
2Adaptability or versatility
If conventional homologous recombination or BAC methods are used to insert exogenous genes into HVT, then the vaccine can express multiple antigens, but the construction time is excessive
Solution Approach 1:
The patent replaces the time-consuming conventional recombination methods with the CRISPR-Cas9 system, which enables rapid and precise genome editing. The system allows for straightforward insertion of multiple antigen genes into predetermined sites in the HVT genome, significantly reducing the construction time while maintaining the ability to express multiple antigens for protection against different avian diseases
Solution Approach 2:
The patent changes the fundamental parameter of the gene insertion process by transitioning from low-efficiency homologous recombination to high-efficiency CRISPR-Cas9 mediated insertion. This parameter change in the insertion mechanism directly reduces the construction time from weeks or months to a much shorter period, while preserving the versatility to insert and express multiple different antigen sequences
Data Source
AI summary
The present disclosure provides a recombinant herpesvirus of turkeys (HVT) and a preparation method and use thereof. The present disclosure specifically provides a recombinant HVT, where an exogenous gene is inserted in a spacer region between an HVT005 region and an HVT006 region of an HVT genome; and the exogenous gene is selected from a gene derived from the group consisting of a Newcastle disease virus (NDV), an avian influenza virus (AIV), and an infectious bursal disease virus (IBDV); the spacer region between an HVT005 region and an HVT006 region of an HVT genome is located between 8,867 nt and 9,319 nt of the HVT genome, and has a nucleotide sequence set forth in SEQ ID NO: 1.