Stitch-Seq CRISPR Screening Linking gRNAs to Transcripts
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current CRISPR screening methods are limited in their ability to scale and report complex transcriptomic consequences, particularly in identifying individual gRNA-associated transcriptional perturbations across large populations of cells, such as tens of thousands to millions of cells, due to throughput limitations.
Innovation Solution
The development of a method called Stitch-Seq, which involves physically linking exogenous nucleic acids to target transcripts in single cells through overlap extension amplification, enabling the sequencing of both and thereby associating exogenous nucleic acid perturbations with gene expression levels, allowing for higher throughput by stitching mRNA transcripts of interest to cognate gRNAs within individual cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of information
If traditional CRISPR screening methods are used, then the assay can report guide RNA enrichment, but the ability to report complex transcriptomic consequences is limited
Solution Approach 1:
The patent combines CRISPR perturbation screening with single-cell RNA sequencing to simultaneously capture both guide RNA identity and transcriptomic consequences in the same cell population, eliminating the information loss inherent in separate assays
Solution Approach 2:
The invention introduces an intermediary molecular linkage system where guide RNAs are physically connected to cellular transcripts through co-encapsulation and PCR amplification, enabling indirect detection of transcriptomic changes while maintaining association with the original perturbation
2Loss of information
If CROP-seq is used to sequence expressed gRNAs in single-cell gene expression workflows, then transcriptomic consequences can be reported, but the method is substantially hampered by its inability to efficiently scale
Solution Approach 1:
The patent segments the complex CROP-seq workflow into distinct modular steps: droplet encapsulation, lysis, targeted PCR amplification, and sequencing, allowing each step to be optimized independently and processed in parallel across millions of cells
Solution Approach 2:
The invention changes key parameters including droplet volume (to nanoliter scale), PCR cycle number (optimized for droplet efficiency), and target enrichment strategies, enabling scalable high-throughput processing while maintaining data quality
3Loss of information
If bead barcode is used for linkage of polynucleotide expression to gene expression, then association can be established, but the throughput is substantially reduced
Solution Approach 1:
The patent extracts and eliminates the bead barcode component from the linkage system, replacing it with a direct molecular association method where guide RNA and transcript information are co-amplified and sequenced together without requiring physical beads as intermediaries
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 significantly enhances the throughput of CRISPR screens, enabling the analysis of tens of thousands to millions of cells in a single experiment, allowing for robust transcriptional-based CRISPR-screens and assessment of protein variants' impact on intracellular signaling.
Implementation Method 1
the expressed mRNA transcripts of interest in such cells are stitched to the cell's cognate gRNA via overlap extension RT-PCR
Implementation Method 2
overlap extension RT-PCR
Data Source
AI summary
The present disclosure relates to methods and compositions for enhanced assessment of exogenous polynucleotide and/or polypeptide-mediated transcriptional perturbations at high throughput and single cell/droplet levels of resolution. In embodiments, nucleic acid fusions of exogenous polynucleotide(s) and associated target transcript(s) are produced within individually sequestered or discretely identifiable cells/lysates and analyzed for exogenous polynucleotide mediated perturbations across a vast population of droplets/cells within individual reactions. Kits for performance of the methods are also provided.