Spatial Optical Barcodes for Single-Cell CRISPR Screens
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Solution Overview
Problem
Current methods for pooled CRISPR library screens face challenges in high-throughput, single-cell resolution, and simultaneous phenotypic measurements, often requiring complex imaging and sequencing processes that are technically demanding and limited in throughput.
Innovation Solution
The implementation of spatial optical barcoding using fluorescence-labeled subcellular compartments combined with deep learning for image analysis, allowing for single-round staining and imaging to decode barcodes and correlate perturbations with cellular phenotypes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional pooled CRISPR library screen methods are used, then multiplexed phenotypic measurements can be performed, but throughput is limited and the process requires complex imaging and sequencing steps
Solution Approach 1:
The patent extracts the barcode information from complex sequencing processes and embeds it directly into the CRISPR guide RNA structure as optical barcode regions. This allows barcode reading through simple fluorescence imaging rather than complex sequencing, thereby increasing throughput and reducing process complexity
Solution Approach 2:
The patent replaces the mechanical/chemical sequencing process with an optical imaging system. By incorporating fluorescently detectable barcode regions into the gRNA, the system substitutes complex sequencing machinery with simpler fluorescence microscopy, enabling high-throughput single-cell analysis
2Measurement precision
If single-cell resolution is achieved, then perturbations can be precisely measured, but the process becomes technically demanding and reduces throughput
Solution Approach 1:
The patent merges multiple functions into a single imaging process: perturbation detection, barcode reading, and phenotypic measurement all occur simultaneously in one fluorescence image acquisition step. This consolidation achieves single-cell resolution while maintaining high throughput by eliminating the need for separate sequencing and imaging steps
Solution Approach 2:
The fluorescence imaging system serves multiple purposes: it detects the perturbation phenotype, reads the barcode identity, and provides single-cell spatial information. This multi-functionality allows precise single-cell measurement without sacrificing throughput, as one imaging process accomplishes what would traditionally require multiple separate techniques
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-throughput, single-cell resolution, and multiplexed phenotypic measurements, increasing experimental throughput and accessibility, facilitating applications in drug development and personalized medicine by simplifying the analysis of pooled library screens.
Implementation Method 1
The barcode gRNAs each can comprise a barcode region comprising zero, one, or more optical detection probe binding sites for a plurality of optical detection probes
Implementation Method 2
The implementation of spatial optical barcoding using fluorescence-labeled subcellular compartments combined with deep learning for image analysis
Implementation Method 3
The ΔgRNA can bind to a first dCas9, and the first dCas9 bound to the ΔgRNA can bind to a chromosome sequence of a genome of the single cell
Data Source
AI summary
Disclosed herein include systems, methods, and compositions for determining perturbations in single cells or performing integrated measurements using, for example, plasmids each comprising (i) a perturbation gRNA with a guide region targeting a chromosome sequence and (ii) barcode gRNAs each with a guide region targeting predetermined spatial region of the genome and zero, one, or more optical detection probe binding sites.