Cell-Stored Barcoded Viral Libraries for Safe Mutation Mapping
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Solution Overview
Problem
Existing deep mutational scanning methods for viral entry proteins are limited by low throughput, biosafety challenges, and inefficiencies in barcode association, particularly for viruses requiring high containment levels, leading to a lack of efficient data collection and analysis.
Innovation Solution
The development of cell-stored barcoded mutational scanning libraries, where virions are produced with a genotype-phenotype link and stored in a non-infective state within cells, allowing for efficient barcode association and sequencing without replication, thus maintaining linkage between barcode and variant sequence.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional deep mutational scanning methods are used to study viral entry proteins, then research on protein mutations can be conducted, but the throughput is low and data collection is inefficient
Solution Approach 1:
The patent segments the viral genome into multiple regions, each tagged with unique barcodes. This allows parallel analysis of multiple mutations simultaneously, dramatically increasing throughput. Each barcode serves as an independent identifier that can be tracked separately, enabling high-throughput sequencing and analysis of many mutant variants in a single experiment.
Solution Approach 2:
The patent performs preliminary actions by pre-tagging viral genomes with barcodes before the mutational scanning experiment. This pre-preparation of barcode-linked viral libraries eliminates the need for time-consuming post-experiment barcode association, allowing direct high-throughput sequencing and analysis of mutation effects.
2Reliability
If viruses requiring high containment levels are studied using traditional methods, then viral protein function can be analyzed, but biosafety challenges arise due to the need for high containment facilities
Solution Approach 1:
The patent creates barcode-tagged copies of viral genomes that can be studied in a safe manner. These barcoded viral libraries allow researchers to track and analyze viral mutations without requiring the handling of live dangerous viruses, as the barcode system enables indirect study of viral protein function through sequencing and computational analysis.
Solution Approach 2:
The barcode sequence serves as an intermediary that links the viral genome to trackable identifiers. This intermediary system allows researchers to study viral mutations and protein function without direct exposure to dangerous viruses, as the barcodes can be sequenced and analyzed independently from the viral particles themselves.
3Loss of information
If barcodes are associated with viral sequences after replication, then viral function can be studied, but the linkage between barcode and variant sequence may be lost due to recombination
Solution Approach 1:
The patent performs barcode association with the viral genome before replication occurs. This preliminary tagging ensures that the barcode remains physically linked to the specific viral variant sequence throughout the replication process, preventing recombination from breaking the association. The barcode is incorporated into the viral DNA at the same time as the variant sequence, maintaining their linkage.
Solution Approach 2:
The patent merges the barcode sequence with the viral genome sequence into a single integrated construct. This combining of barcode and viral sequence ensures they are replicated together as a unit, maintaining their association without requiring separate tracking steps that could lead to loss of linkage.
4Object-affected harmful factors
If viral libraries are stored in a non-infective state within cells, then safe handling is enabled, but the ability to study full viral replication cycles is limited
Solution Approach 1:
The patent creates non-infective copies of viral genomes that are integrated into host cell genomes. These integrated viral sequences can be safely maintained and manipulated without the risks associated with live infectious viruses. The barcoded design allows these safe copies to still be used for studying viral protein function through mutational scanning and sequencing analysis.
Solution Approach 2:
The patent extracts the essential functional information from the viral genome and captures it through barcode tagging and mutational scanning. By taking out the key data elements (mutation effects on protein function) and separating them from the dangerous infectious component, the system enables safe study of viral protein function without requiring full viral replication cycles.
Data Source
AI summary
Cell-stored barcoded viral protein deep mutational scanning libraries are described. The libraries can be used to map resistance mutations to therapeutic treatments. The libraries can be used to predict viruses that become resistant to therapeutic compounds and/or may more easily evolve to infect new species. The libraries can also be used to more safely study dangerous viruses that normally require high safety biocontainment facilities. The libraries include features that allow efficient collection and assessment of informative data, obviating many bottlenecks of previous approaches.