Optical Barcode mRNA for Single-Cell Genotype Phenotype Correlation
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Solution Overview
Problem
Current methods for genome-wide screening of genetic perturbations are inefficient and costly, particularly when dealing with complex cellular phenotypes that require dynamic, optical assays, as they often necessitate individual synthesis and arraying of gene perturbation reagents and are not regularly conducted at the genomic scale due to logistical and expertise-related challenges.
Innovation Solution
A method involving the use of optical barcodes, where vectors encoding genetic perturbations and nucleic acid sequences are delivered to cells, allowing for the expression of mRNA transcripts that can be detected through sequential hybridization and imaging, enabling the identification of genetic perturbations and their corresponding phenotypes at the single-cell level, thereby correlating genotypes with phenotypes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If individual synthesis and arraying of gene perturbation reagents is performed, then screening accuracy is improved, but device complexity and cost increase
Solution Approach 1:
The optical barcode is divided into multiple segments, each capable of binding to a different probe set with distinct optical labels. This segmentation allows the system to encode multiple pieces of information (gene perturbation identity and phenotypic data) within a single barcode structure, enabling high-accuracy screening without requiring individual synthesis and arraying of each reagent.
Solution Approach 2:
The optical barcode serves multiple functions simultaneously: it identifies the specific gene perturbation, enables pooled screening of multiple genes, and facilitates single-cell resolution analysis. The same barcode structure is used across all reagents in the pool, eliminating the need for individual synthesis and arraying while maintaining screening accuracy through the multi-segment probe binding mechanism.
2Ease of manufacture
If pooled methods are used for genome-wide screening, then cost and labor are reduced, but measurement precision and screening accuracy deteriorate
Solution Approach 1:
Different probe sets are labeled with distinct optical labels (analogous to color changes), allowing simultaneous detection of multiple gene perturbations and phenotypic states in a pooled screen. This optical coding system enables high-accuracy measurement precision to be maintained while using cost-effective pooled methods, as the optical labels provide unambiguous identification of each perturbation type without requiring individual reagent synthesis.
3Productivity
If microplate-based screens are conducted at genomic scale, then productivity increases, but device complexity and expertise requirements worsen
Solution Approach 1:
The system transitions from traditional two-dimensional microplate-based screening to a multi-dimensional approach using optical barcodes with multiple segments that can be read through sequential probe binding and imaging. This dimensional expansion of the barcode structure enables genomic-scale screening by encoding multiple perturbation identities and phenotypic data points within a single compact barcode, thereby increasing productivity while reducing the complexity of automation requirements.
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 enables high-resolution, cost-effective screening of genetic modifications at the single-cell level, allowing for the analysis of complex phenotypes and large-scale genomic perturbations, combining the benefits of pooled and arrayed screens with single-cell resolution, and is suitable for cultured and primary cells, including neural cells.
Implementation Method 1
The optical barcode comprises an ordered series of segments. For each segment there are a set of possible nucleic acid sequences that can be found at that segment. Each of the possible nucleic acid sequences at a segment comprise a unique sequence and are designed to bind to a corresponding probe.
Data Source
AI summary
Embodiments disclosed herein are directed to a new genetic perturbation and screening method that combines advantages of pooled perturbation with imaging assays for complex phenotypes. Specifically, the method may be used to screen pooled genomic perturbations to identify phenotypes and to identify perturbed genes at the single-cell level using optical barcodes. A major advantage offered by this approach is the ability to screen for any cellular phenotype that can be identified by high-resolution microscopy—including live-cell phenotypes, protein localization, or highly multiplexed expression profile and mRNA localization by RNA-FISH—in conjunction with a large array of genetic perturbations applied as a pool in a single test volume.