CRISPR Library Screening via Dummy Guide Integration

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Solution Overview

Problem

Current gene regulatory techniques are limited in characterizing elements necessary for gene expression and cannot determine the relative importance of each gene regulatory element on native gene expression levels, as they focus on activation in heterologous contexts and lack a high-throughput approach to analyze native gene expression.

Innovation Solution

A CRISPR/Cas system-based library screen method that integrates dummy guide RNAs into the genome, allowing for the introduction of variable guide sequences to target specific genomic regions, enabling mutation analysis and eliminating the need for molecular cloning, thereby controlling the number of functional guide RNAs and facilitating high-throughput screening.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If reporter assays are used to identify gene regulatory elements, then elements sufficient to activate gene expression in heterologous contexts can be identified, but elements necessary but not sufficient for gene expression and elements whose activity does not transfer to non-native contexts cannot be characterized

Engineering Contradiction:

Improveaccuracy of identifying essential regulatory elementsVSAvoidapplicability to native gene expression contexts

Core Design Contradiction:

ReliabilityVSAdaptability or versatility

Solution Approach 1:

Instead of asking what elements are sufficient to activate gene expression in heterologous contexts (reporter assay approach), the invention inverts the question by using CRISPR/Cas9 to systematically disrupt regulatory elements in their native contexts and observe the consequences on endogenous gene expression. This inversion allows identification of necessary elements rather than sufficient ones, directly addressing the limitation of reporter assays.

Inventive Principle:

Principle #13The other way round (Inversion)

Solution Approach 2:

The invention uses the cell's own endogenous gene expression system as the readout mechanism rather than requiring external reporter genes. By measuring changes in native gene expression levels following CRISPR-mediated disruption of regulatory elements, the system leverages the cell's inherent biological machinery to provide information about regulatory element function in its natural context.

Inventive Principle:

Principle #25Self-service

2Measurement precision

If traditional methods are used to determine the relative importance of each gene regulatory element, then detailed analysis of individual elements is possible, but high-throughput analysis of multiple elements on native gene expression is not feasible

Engineering Contradiction:

Improvedetermination of relative importance of regulatory elementsVSAvoidthroughput of regulatory element analysis

Core Design Contradiction:

Measurement precisionVSProductivity

Solution Approach 1:

The invention segments the genome into individual regulatory elements that can be independently targeted by specific CRISPR guide RNAs. By designing libraries of guide RNAs that each target a specific regulatory element, the system enables systematic, high-throughput disruption and analysis of multiple elements simultaneously, maintaining precision while achieving high throughput.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The invention changes the experimental parameter from low-throughput individual element analysis to high-throughput parallel analysis by using pooled CRISPR libraries. Multiple regulatory elements are disrupted in parallel across large populations of cells, with readout through sequencing or expression analysis, thereby increasing productivity while maintaining measurement precision through quantitative comparison.

Inventive Principle:

Principle #35Parameter changes

3Manufacturing precision

If molecular cloning is used to prepare guide RNA libraries, then precise control of guide sequences is achieved, but the process is time-consuming and complex

Engineering Contradiction:

Improveprecision of guide sequence integrationVSAvoidtime required for library preparation

Core Design Contradiction:

Manufacturing precisionVSLoss of time

Solution Approach 1:

The invention performs preliminary action by pre-integrating dummy guide RNA sequences into the genome before introducing the CRISPR library. These dummy sequences serve as predetermined integration sites that facilitate efficient homologous recombination with the library guide RNAs, eliminating the need for time-consuming molecular cloning steps while maintaining precise control over guide sequence integration.

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The dummy guide RNA sequences integrated in the genome serve as intermediary elements that mediate between the introduced CRISPR library and the cellular machinery. These intermediaries facilitate efficient homologous recombination by providing predefined homology regions, thereby simplifying the integration process and reducing the time required compared to traditional molecular cloning approaches.

Inventive Principle:

Principle #24Intermediary (Mediator)

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 allows for the identification of essential genomic regions and elements involved in transcription regulation, providing a high-throughput approach to analyze the effect of mutations on gene expression, including non-coding regions, and elucidating the function of regulatory elements at single-base resolution.

Implementation Method 1

The method exploits the ability of the CRISPR/Cas system to cleave specific genomic sites in order to introduce exogenous guide sequences of interest into the cell

Methodology Applied

Scientific EffectCRISPR/Cas system nuclease cleavage: