CRISPR CD155 Knockout RD Cells for Poliovirus-Resistant Enterovirus Research
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Solution Overview
Problem
Current cell lines used for Enterovirus research, such as the RD cell line, are highly susceptible to poliovirus due to the expression of the poliovirus receptor CD155, posing a risk of accidental poliovirus growth and spread, especially in laboratories handling potentially infectious materials, which complicates biosafety and increases costs for containment measures.
Innovation Solution
Development of a modified poliovirus receptor (PVR/CD155) gene using CRISPR-Cas9 technology to introduce mutations in exons 2, 3, and 4, creating a cell line that is refractory to poliovirus but susceptible to other Enteroviruses, utilizing guide RNAs and Cas9 protein to knock out the CD155 receptor.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If RD cell line is used for Enterovirus research, then susceptibility to Enteroviruses is maintained, but risk of poliovirus growth and spread increases
Solution Approach 1:
The patent extracts and removes the harmful element (CD155 receptor) from the cell line through CRISPR-Cas9 mediated knockout. By specifically eliminating the poliovirus receptor while preserving other cellular functions, the cell line maintains Enterovirus susceptibility through alternative mechanisms while becoming refractory to poliovirus infection.
Solution Approach 2:
The patent applies local quality modification by selectively altering only the specific gene region encoding the CD155 receptor (exons 2, 3, and 4) while leaving the rest of the genome intact. This targeted approach ensures that poliovirus binding is blocked at the specific receptor level without affecting overall cell viability or other viral susceptibilities.
2Reliability
If CRISPR-Cas9 is used to knock out CD155 receptor, then poliovirus refractoriness is achieved, but genetic modification complexity increases
Solution Approach 1:
The patent uses guide RNA as an intermediary molecule that mediates between the CRISPR-Cas9 system and the target CD155 gene. The guide RNA provides sequence-specific targeting to exons 2, 3, and 4 of the CD155 gene, enabling precise knockout without requiring complex direct manipulation of the genome, thereby simplifying the overall process while ensuring reliability.
3Object-affected harmful factors
If poliovirus containment measures are implemented, then biosafety is improved, but operational costs and complexity increase
Solution Approach 1:
The patent implements preliminary action by genetically modifying the cell line beforehand to be inherently resistant to poliovirus infection. This preventive genetic modification eliminates the need for complex containment infrastructure and expensive operational safety measures, as the cell line itself serves as the first line of defense against poliovirus growth.
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 modified cell line effectively prevents poliovirus growth while maintaining susceptibility to other Enteroviruses, ensuring safer handling and reducing biosafety risks and costs associated with containment, aligning with Global Action Plan II guidelines.
Implementation Method 1
Development of a modified poliovirus receptor (PVR/CD155) gene using CRISPR-Cas9 technology to introduce mutations in exons 2, 3, and 4
Implementation Method 2
utilizing guide RNAs and Cas9 protein to knock out the CD155 receptor
Data Source
AI summary
A modified polio virus receptor (PVR/CD155) gene including one or more mutations in exons selected from exons 2, 3 and 4 of poliovirus receptor (PVR/CD155) gene having SEQ ID No.1. More specifically, cell lines including the modified gene and method of producing the same using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR-Cas9) system. The cell line is refractory (non-permissive) to poliovirus and susceptible to most Enteroviruses and many other human viruses. Further, the cell line can be applied in the fields of research, diagnostic and therapy.