Chip-Based Biosensor for Genome Editing Agent Selection
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Solution Overview
Problem
Current methods for selecting targeted genome manipulating agents, such as CRISPR-Cas9, face challenges in achieving precise on-target recognition and avoiding off-target sites, leading to inefficient use of resources and potential for unintended genetic modifications.
Innovation Solution
The development of a chip-based biosensor system that measures response signals from nucleic acid samples with and without a blocking agent, allowing for the comparison of binding interactions to determine genome manipulating efficiency parameters and identify off-target sites, using functionalized capture surfaces with CRISPR-associated proteins like Cas9 and guide RNAs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional screening methods are used to select genome manipulating agents, then the selection process is simple and low-cost, but the precision in identifying off-target sites is poor and the efficiency is low
Solution Approach 1:
The screening process is segmented into two distinct measurement stages: first measuring binding interactions without blocking agents to identify potential off-target sites, then measuring with blocking agents to confirm off-target binding. This segmentation allows each measurement to serve a specific purpose, improving overall precision while keeping the system manageable through modular chip-based biosensors.
Solution Approach 2:
Blocking agents serve as intermediaries that specifically bind to off-target sites and prevent genome manipulating agents from binding there. By introducing this intermediary element, the system can distinguish between on-target and off-target binding events, significantly improving measurement precision without requiring overly complex analytical instruments.
2Reliability
If comprehensive screening for off-target sites is performed, then the reliability of genome editing is improved, but the time and resources required increase
Solution Approach 1:
The system performs preliminary screening by first measuring binding interactions without blocking agents, which quickly identifies potential off-target sites. This preliminary action filters out candidates that don't show off-target binding, allowing subsequent confirmation measurements to focus only on promising candidates, thus reducing overall screening time while maintaining reliability.
Solution Approach 2:
The two-measurement approach uses partial action by not requiring exhaustive testing of all possible off-target sites with blocking agents. Instead, it performs a quick initial screen followed by selective confirmation only for sites showing potential off-target binding, optimizing the balance between comprehensive screening and time efficiency.
3Manufacturing precision
If blocking agents are used to prevent off-target binding, then the specificity of genome manipulating agents is improved, but the complexity of the assay increases
Solution Approach 1:
Blocking agents are applied locally and specifically to certain chip surfaces or regions where off-target binding is suspected, rather than uniformly to all measurements. This localized application allows the assay to maintain simplicity in most measurements while adding specificity only where needed, improving agent specificity without proportionally increasing overall assay complexity.
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
This approach enhances the precision and efficiency of targeted genome editing by reducing off-target binding, improving the selection of effective genome manipulating agents and reducing the time and cost associated with screening and validation processes.
Implementation Method 1
sensing surfaces configured to detect biomolecular binding interactions between a nucleic acid sample and one or more capture surfaces functionalized with a targeted genome manipulating agent
Implementation Method 2
a blocking agent configured to bind to a sequence overlapping an on-target sequence of the nucleic acid sample
Data Source
AI summary
For enhanced selection of efficient targeted genome manipulating agents, an apparatus includes first and second chip-based biosensors having one or more sensing surfaces configured to detect biomolecular binding interactions between a nucleic acid sample and a targeted genome manipulating agent functionalized to a capture surface within a sensing range of the one or more sensing surfaces. The first chip-based biosensor uses a nucleic acid sample incubated with a blocking agent that blocks on-target binding and the second chip-based biosensor holds a nucleic acid sample that omits the blocking agent. A measurement apparatus measures first and second sets of response signals produced in response to the biomolecular binding interactions occurring between the nucleic acid sample and the targeted genome manipulating agent. An analysis module determines the genome manipulating efficiency parameters of the targeted genome manipulating agent. A system and a method perform the functions of the apparatus.