Targeted CD163 Gene Inactivation for Precise PRRSv Resistance
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for preventing Porcine Reproductive and Respiratory Syndrome Virus (PRRSv) in pigs are ineffective due to genetic diversity within the virus, leading to significant economic losses and the need for costly herd depopulation, with existing genome editing techniques lacking precision and specificity for CD163 gene editing.
Innovation Solution
Targeted inactivation of the CD163 gene in pigs using specific guide RNAs (gRNAs) to excise exon 7 and introduce repaired genomic sequences, creating resistance to PRRSv through CRISPR-Cas9 technology, resulting in edited CD163 proteins with reduced virus uptake.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If existing genome editing techniques are used to target CD163 gene, then some level of gene editing can be achieved, but precision and specificity are insufficient leading to unintended edits
Solution Approach 1:
The patent divides the guide RNA into two separate functional components: a crRNA (guide sequence) that provides target specificity and a tracrRNA (scaffold sequence) that provides structural stability and Cas9 binding. This segmentation allows independent optimization of each component - the crRNA can be designed for maximum specificity to the CD163 exon 7 target site while the tracrRNA ensures proper Cas9 complex formation and stability, thereby improving editing precision and reducing off-target effects
Solution Approach 2:
The patent optimizes specific parameters of the guide RNA sequences, including the length and composition of the crRNA spacer region (20 nucleotides complementary to exon 7), the PAM sequence requirement (NGG motif), and the structural features of tracrRNA. These parameter changes enhance the specificity and efficiency of Cas9 binding to the intended target while minimizing unintended genomic modifications
2Reliability
If herd depopulation is used to eliminate PRRSv, then the virus can be eliminated from infected herds, but significant economic loss occurs
Solution Approach 1:
The patent applies preliminary action by genetically modifying pigs before PRRSv infection occurs. Through CRISPR-Cas9 mediated knockout of the CD163 receptor gene, pigs are pre-equipped with resistance to PRRSv, eliminating the need for costly depopulation and repopulation cycles. This preventive genetic modification approach ensures virus elimination capability is built into the herd rather than requiring reactive herd replacement
Solution Approach 2:
The patent converts the harmful CD163 receptor protein, which enables PRRSv entry into cells, into a benefit by precisely knocking out exon 7 of the CD163 gene. This converts the vulnerability into resistance, allowing the herd to maintain full productivity without depopulation while achieving effective virus elimination. The genetic modification transforms the very mechanism the virus uses to cause harm into a protective feature
3Reliability
If CD163 gene is targeted for editing to confer PRRSv resistance, then resistance can be achieved, but existing guide RNAs lack sufficient activity and specificity
Solution Approach 1:
The patent segments the guide RNA into crRNA and tracrRNA components with distinct optimized functions. The crRNA contains a 20-nucleotide spacer sequence specifically complementary to exon 7 of CD163, ensuring precise target recognition. The tracrRNA provides structural scaffolding and Cas9 interaction. This segmentation enables each component to be independently optimized for its specific function, achieving both high resistance reliability and high guide RNA specificity
Solution Approach 2:
The patent optimizes critical parameters including the crRNA spacer sequence (20 nucleotides matching exon 7), the PAM sequence (NGG), and the structural characteristics of tracrRNA. These parameter optimizations ensure the guide RNA complex binds with high specificity to the intended CD163 exon 7 target site while maintaining efficient Cas9 activation, thereby achieving reliable PRRSv resistance through precise genetic editing
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 confers robust resistance to PRRSv by precisely editing the CD163 gene, reducing virus uptake and minimizing unintended edits, thereby improving herd health and reducing economic losses.
Implementation Method 1
Targeted inactivation of the CD163 gene in pigs using specific guide RNAs (gRNAs) to excise exon 7 and introduce repaired genomic sequences, creating resistance to PRRSv through CRISPR-Cas9 technology
Data Source
AI summary
The present disclosure relates to methods and compositions useful for prevention of porcine reproductive and respiratory syndrome virus (PRRSv) in animals, including animals of the species Sus scrofa. The present teachings relate to swine wherein at least one allele of a CD163 gene has been inactivated, and to specific methods and nucleic acid sequences used in gene editing to inactivate the CD163 gene. Swine wherein both alleles of the CD163 gene are inactivated are resistant to porcine reproductive and respiratory syndrome virus (PRRSv). Elite lines comprising homozygous CD163-edited genes retain their superior properties.