CRISPR Intron Tagging for Automated Clone Recognition
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Solution Overview
Problem
Current methods for unbiased discovery of biological/pharmacological functions of bioactive compounds and high-content screening approaches are limited by high costs, sample preparation requirements, and the inability to monitor effects on a large scale or multiple drug candidates, with existing tagging technologies facing integration site biases and lack of specificity in targeting specific genes or introns.
Innovation Solution
A CRISPR/Cas9-based intron targeting approach is used to generate diverse pools of cells where each cell has multiple tagged introns, allowing for automated recognition of tagged genes through computational analysis of fluorescent microscopy images without the need for in situ sequencing, enabling the assessment of drug effects on proteome and gene expression levels.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If viral constructs are used for tagging, then gene targeting is achieved, but integration site biases occur and specific gene sets cannot be selected
Solution Approach 1:
The patent replaces the mechanical/viral delivery system with a CRISPR-Cas9 molecular biology system. Instead of using viral vectors that randomly integrate into genomes, the invention uses programmable guide RNAs to direct Cas9 nuclease to specific genomic locations, enabling precise, bias-free gene targeting through sequence-specific DNA binding rather than random viral integration
Solution Approach 2:
The patent changes the targeting mechanism from random viral integration to CRISPR-guided precise editing. By modifying the guide RNA sequence, the system can target any desired gene without integration site biases, allowing flexible selection of specific gene sets through sequence complementarity rather than random insertion
2Ease of operation
If fluorescent tagging approaches are used, then protein monitoring is enabled, but only one or a few selected proteins can be monitored
Solution Approach 1:
The patent creates a universal CRISPR tagging system where a single platform can target and monitor any number of proteins simultaneously. By designing specific guide RNAs for multiple genes, the system enables multiplexed fluorescent tagging and monitoring of numerous proteins in parallel, transforming a single-protein tool into a multi-functional high-throughput analysis platform
Solution Approach 2:
The patent segments the proteome into individually targetable components using specific guide RNAs for each gene. This allows the system to monitor multiple discrete proteins simultaneously through parallel CRISPR tagging events, enabling high-throughput analysis by dividing the complex task of proteome monitoring into manageable, specific gene targets
3Measurement precision
If in situ sequencing is used for clone identification, then tagged intron identity is determined, but the process is time-consuming and reduces throughput
Solution Approach 1:
The patent creates a genetic copy of the guide RNA sequence at the tagging site. Instead of sequencing the intron itself, the system amplifies and sequences the integrated guide RNA copy, which serves as a molecular barcode identifying the tagged gene. This copying approach maintains identification accuracy while enabling faster, more efficient clone recognition through PCR amplification and sequencing of the simpler guide RNA sequence
4Quantity of substance
If multiple tagged introns per cell are generated, then proteome coverage is increased, but automated recognition of tagged genes becomes more difficult
Solution Approach 1:
The patent integrates multiple guide RNA copies into each cell, with each copy serving as a unique molecular barcode identifying a specific tagged intron. This copying strategy allows automated recognition systems to identify multiple tagged genes simultaneously by detecting and sequencing the distinct guide RNA copies, transforming a complex recognition problem into a simpler sequence identification task
Solution Approach 2:
The patent uses different fluorescent tags (analogous to color codes) to mark different introns, with each guide RNA sequence corresponding to a specific fluorescent signal. This color-coding system enables automated recognition by linking fluorescent detection to specific guide RNA sequences, allowing high-throughput identification of multiple tagged introns through fluorescence-activated cell sorting and 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 method enables efficient, scalable, and automated analysis of the proteome, allowing for the identification of drug effects on a large subset of proteins and the recognition of tagged introns with high accuracy, overcoming previous limitations in throughput and specificity.
Implementation Method 1
A CRISPR/Cas9 based intron targeting approach is used to generate diverse pools of cells where each cell has multiple tagged introns
Implementation Method 2
allowing for automated recognition of tagged genes through computational analysis of fluorescent microscopy images
Data Source
AI summary
The present invention relates to a method for obtaining an intron-tagged pool of cells representing and/or comprising tagged introns. Furthermore, the present invention relates to a pool of cells that may be obtainable by said method, in particular an intron-tagged pool of cells. The present invention furthermore provides for a method for automated recognition of the identity of the tagged intron(s) comprised in the genome of an intron-tagged cell within an intron-tagged pool of cells and a method for assessing the effect of a perturbation on the proteome and/or gene expression levels of an intron-tagged cell within an intron-tagged pool of cells.