Barcoded Guide RNA for High-Throughput Genetic Screening
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Solution Overview
Problem
Current high-throughput genetic screening methods are limited in their ability to assess multiple genetic perturbations and phenotypes simultaneously, particularly in diverse cell types, and struggle to detect subtle phenotypic changes that are not easily detectable by existing technologies.
Innovation Solution
A CRISPR-Cas9 system is developed with guide RNAs containing a barcode sequence and a poly(A) tail, enabling high-throughput genetic modification and RNA sequencing to link genetic perturbations to transcriptomic changes in individual cells, allowing for the assessment of thousands of genetic modifications across multiple cell types.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If current high-throughput screening methods are used, then cell growth and survival can be assessed, but multiple genetic perturbations and phenotypes cannot be assessed simultaneously
Solution Approach 1:
The screening system is segmented into distinct functional modules: guide RNA molecules with unique barcodes for genetic perturbation identification, poly(A) sequences for RNA capture, and sequencing domains for phenotypic readout. This segmentation allows simultaneous assessment of multiple genetic perturbations and phenotypes through parallel processing of barcoded RNA molecules
Solution Approach 2:
The guide RNA molecules are designed with multi-functional elements: the spacer sequence provides target-specific genetic perturbation, the barcode sequence enables identification of the perturbation, and the poly(A) sequence facilitates RNA capture and sequencing. This multi-functionality allows a single molecular construct to simultaneously enable genetic perturbation, tracking, and phenotypic assessment across multiple dimensions
2Measurement precision
If existing screening technologies are used, then obvious phenotypes can be detected, but subtle phenotypic changes cannot be detected
Solution Approach 1:
The system replaces traditional mechanical/visual phenotypic assessment methods with molecular sequencing-based detection. By converting phenotypic information into sequencable RNA molecules with unique barcodes, the system achieves high-precision detection of subtle transcriptional changes while maintaining high throughput through parallel sequencing of thousands of barcoded molecules simultaneously
3Loss of information
If guide RNAs without barcode sequences are used, then genetic modification can be performed, but linkage to transcriptomic changes cannot be established
Solution Approach 1:
The guide RNA structure merges multiple functional elements into a single integrated molecule: the spacer sequence for target recognition, the barcode sequence for genetic perturbation identification, the scaffold sequence for Cas9 binding, and the poly(A) sequence for RNA capture. This merging ensures that genotype information (via barcode) and phenotypic information (via transcriptomic changes) remain linked throughout the screening process
4Loss of information
If traditional RNA sequencing methods are used, then gene expression can be measured, but connection to specific genetic perturbations is lost
Solution Approach 1:
The system creates RNA copies of the guide RNA molecules that contain both the barcode sequence (representing the genetic perturbation) and the poly(A) sequence (enabling capture). These copied RNA molecules serve as information carriers that link the specific genetic perturbation (encoded in the barcode) to the transcriptomic phenotype, allowing efficient association of genotype and phenotype data through sequencing
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 comprehensive characterization of genetic modifications and their effects on gene expression, facilitating the identification of disease-related genetic perturbations and therapeutic agents, while improving the efficiency of genetic screening by orders of magnitude.
Implementation Method 1
an RNA capture and sequencing domain that includes a barcode sequence and a primer binding sequence
Data Source
AI summary
A guide RNA comprising: a gRNA spacer sequence at the 5′ end of the guide RNA, wherein the spacer sequence is complementary to a target gene, a scaffold sequence that binds to Cas9, and an RNA capture and sequencing domain comprising: a barcode sequence, and a primer binding sequence; nucleic acids and vectors encoding the guide RNA; cells expressing the guide RNA; and a library comprising a plurality of guide RNAs. Also disclosed are methods of introducing a genetic perturbation into a cell, methods of assessing an effect of at least one genetic perturbation on RNA expression in a cell, methods of identifying nucleic acid sequences associated with a disease state and a method of identifying candidate therapeutic agents.