Barcoded CRISPR Guide Libraries for Three-Way Synergy Screening
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Solution Overview
Problem
Existing methods struggle to systematically screen high-order genetic and drug combinations due to unanticipated synergy or antagonism, leading to limited discovery of effective drug combinations, especially those comprising three or more drugs, and are prohibitively expensive with microplate arrays and RNA interference systems.
Innovation Solution
A CRISPR-based multi-gene knockout screening system using barcoded high-order combinatorial guide RNA libraries, assembled via CombiGEM-CRISPR v2.0, enables high-throughput screening of three-way synergistic therapeutic target combinations by modifying promoter sequences and gRNA scaffolds to minimize lentiviral vector recombination.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If microplate arrays are coupled to robotics systems to screen large panels of drug combinations, then the screening capacity is improved, but the cost becomes prohibitively expensive as the number of experiments grows exponentially
Solution Approach 1:
The patent uses barcoded gRNAs as informational copies that encode drug combination data. Instead of physically testing every combination in separate wells, the system creates a digital barcode representation of each combination, allowing parallel screening of multiple combinations simultaneously through sequencing rather than manual assay, thereby reducing material costs while maintaining high screening capacity
Solution Approach 2:
The patent transitions from a two-dimensional microplate array format to a multi-dimensional barcoded library format. By encoding combination information in barcodes that can be read through sequencing, the system achieves exponential scaling in combination coverage without linear increase in physical space or cost, effectively adding a digital dimension to the screening approach
2Productivity
If RNA interference and CRISPR-based systems are used to screen drug target pairs, then the screening efficiency is improved, but the ability to simultaneously evaluate more than two targets is limited due to low and variable cleavage efficiency for polycistronic systems
Solution Approach 1:
The patent divides the gRNA expression system into separate monocistronic units, each expressing a single gRNA under its own promoter control. This segmentation allows independent optimization of each gRNA's cleavage efficiency and expression levels, avoiding the variability and low efficiency associated with polycistronic systems, while still enabling simultaneous evaluation of multiple targets through combinatorial assembly
Solution Approach 2:
The patent modifies the promoter sequences and gRNA scaffold structures to optimize cleavage efficiency for each individual gRNA. By adjusting these molecular parameters in the monocistronic system, the patent achieves reliable and consistent cleavage activity across all targets in the combination, overcoming the limitations of polycistronic systems
3Adaptability or versatility
If high-order combination screening is performed to uncover synergistic interactions, then the therapeutic discovery potential is improved, but the experimental complexity and sequencing costs increase
Solution Approach 1:
The patent creates a universal barcoded gRNA library system that can simultaneously screen for pairwise and three-way drug combinations using the same basic platform. The barcoded design allows the same sequencing infrastructure to decode combination information, and the modular gRNA assembly enables flexible configuration for different screening objectives, reducing experimental complexity while maintaining high therapeutic discovery potential
Solution Approach 2:
The patent introduces barcodes as intermediary elements that mediate between the complex biological screening process and data analysis. The barcodes serve as compact information carriers that encode combination data, enabling high-order interactions to be captured and analyzed through sequencing rather than complex manual scoring, thereby reducing experimental complexity while preserving the ability to discover synergistic interactions
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 system efficiently identifies synergistic drug combinations for diseases like cancer and Parkinson's disease, overcoming the limitations of previous methods by reducing sequencing costs and experimental complexity, and validating effective three-way drug regimens.
Implementation Method 1
a lentiviral vector comprising human U6 (hU6) promoter, mouse U6 (mU6) promoter and human H1 (hH1) promoter expressing an array of three or more barcoded guide RNAs
Implementation Method 2
the promoters having a 3' end comprising modified hU6, mU6 and hH1 promoter sequences for paired annealing of the barcoded gRNAs oligo pairs
Data Source
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AI summary
Provided is a system for multiplexed genome editing or a two or three-way combinatorial CRISPR screening. Also provided is high-throughput screening of disease-alleviating genetic combinations to identify two-way and three-way synergistic drug combinations as potential treatment regimens. Also provided is a lentiviral three-way combinatorial guide RNA expression cassette and combinatorial guide RNA libraries.