Barcoded Perturbation Profiling for Genetic Interaction Mapping
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Solution Overview
Problem
Current genomic research faces challenges in achieving a quantitative and predictive understanding of cellular circuits due to the complexity of biological systems, which are not linear, making it difficult to reconstruct and analyze genetic interactions on a genomic scale, especially with the sheer number of perturbations required for higher-order interactions.
Innovation Solution
The development of Massively Parallel Combinatorial Perturbation Profiling (MCPP) methods, including Perturb-seq, which combines single-cell RNA-seq with CRISPR/Cas9-based perturbations using unique barcodes to perform thousands of assays in a single pooled experiment, allowing for the simultaneous assessment of multiple genetic interactions and their effects on cellular networks.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If combinatorial probing of multiple components is performed simultaneously to reconstruct cellular circuits, then the completeness and accuracy of circuit reconstruction is improved, but the complexity and intractability of the experimental system increases due to the staggering size of possible combinatorial spaces
Solution Approach 1:
The patent segments the combinatorial perturbation problem into manageable pieces by using modular barcoded perturbation agents that can be individually tracked. Instead of attempting to control and measure all possible combinations simultaneously, the system divides the genome into targetable segments with associated barcodes, allowing systematic exploration of combinatorial space through sequential or pooled experiments rather than requiring all combinations at once.
Solution Approach 2:
The patent introduces barcoded perturbation agents as intermediaries between the researcher and the genetic circuit under study. These agents carry unique barcodes that serve as mediators for tracking and identifying specific perturbations and their combinatorial effects. The barcodes act as information carriers that bridge the gap between the complex biological system and the measurement apparatus, enabling systematic reconstruction without directly managing the full combinatorial complexity.
2Ease of operation
If individual gene effects are summed to predict cellular circuit function, then the simplicity of analysis is maintained, but the accuracy of prediction deteriorates because biological systems exhibit non-linear interactions
Solution Approach 1:
The patent merges multiple perturbation effects into combinatorial perturbation experiments where multiple genes are perturbed simultaneously in the same cell. By combining perturbations at the experimental level rather than simply summing individual effects mathematically, the system directly captures non-linear interactions. The barcoded agents allow tracking of combined effects, enabling observation of emergent properties that cannot be predicted from individual gene perturbations alone.
Solution Approach 2:
The patent employs partial combinatorial probing rather than attempting to test all possible combinations. By selecting specific subsets of gene combinations based on biological hypotheses or systematic sampling strategies, the approach captures essential non-linear interactions without requiring exhaustive testing of the entire combinatorial space, thus balancing accuracy with feasibility.
3Loss of information
If genome-wide perturbations are tested individually to determine causation, then the depth of mechanistic understanding is improved, but the productivity and scale of genomic research deteriorates due to the time and resources required
Solution Approach 1:
The patent merges multiple individual perturbation experiments into pooled combinatorial experiments. By delivering multiple barcoded perturbation agents simultaneously to cell populations and tracking their combined effects through single-cell RNA sequencing, the system achieves genome-wide coverage with combinatorial depth at a fraction of the time and resources required for sequential individual gene testing.
Solution Approach 2:
The patent uses barcoded perturbation agents as information copies that can be replicated and tracked across many cells. Instead of performing physical experiments on each gene combination in separate cell cultures, the barcoded agents serve as reusable information carriers that encode perturbation identities, allowing parallel assessment of numerous combinations through molecular profiling of cell populations.
Data Source
AI summary
The present disclosure provides compositions, systems, and methods for pooled screening of perturbations correlating to a phenotype. The present disclosure also provides for methods of in vivo perturbation screening. The present disclosure also provides for high-throughput sample multiplexing. The present disclosure also provides for methods to determine the clonal effects associated with millions of genetic perturbation combinations using standard pooled screening reagents.