Cas9/gRNA Complex Stability via Differential Scanning Fluorimetry

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Solution Overview

Problem

Current methods for evaluating the quality of Cas9 molecule/gRNA complexes are inadequate, as they do not effectively quantify productive complex formation necessary for gene editing activity, despite the importance of precise conditions for Cas9 protein and gRNA interaction.

Innovation Solution

The use of differential scanning fluorimetry (DSF) to detect melting temperature (Tm) values of Cas9 molecule/gRNA complexes, allowing for the selection of stable complexes based on Tm comparisons or thresholds, which correlates with complex stability and gene editing efficiency.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If dye-binding assays (Bradford or riboquant) are used to quantify protein and RNA amounts, then the quantity of Cas9 and gRNA can be measured, but the productivity of productive RNP complex formation cannot be quantitated

Engineering Contradiction:

Improvequantitation of productive RNP complexVSAvoidinformation on complex formation quality

Core Design Contradiction:

Measurement precisionVSLoss of information

Solution Approach 1:

The patent introduces a fluorescent dye as an intermediary substance that binds to the RNP complex and provides a measurable signal. This dye acts as a mediator between the RNP complex formation and the detection system, enabling indirect measurement of complex quality and productivity through fluorescence intensity or thermal shift signals.

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The patent replaces traditional mechanical/chemical assay methods (Bradford dye binding, riboquant) with a fluorescent detection system based on thermal melt curves. This substitution transitions from conventional biochemical assays to a physics-based optical detection method that provides more informative measurements of complex formation.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

2Productivity

If thermal melt curves and DSF are used to characterize Cas9/gRNA complexes, then the stability and binding affinity can be measured with high throughput, but additional equipment and methodology complexity are introduced

Engineering Contradiction:

Improvehigh throughput assessment of RNP complexesVSAvoidbiophysical characterization equipment

Core Design Contradiction:

ProductivityVSDevice complexity

Solution Approach 1:

The patent employs a fluorescent dye that serves multiple functions: it binds to the RNP complex for detection, reports on complex stability through thermal melt curves, and enables high-throughput screening when combined with plate reader technology. This multi-functional approach allows a single detection system to provide multiple types of information about the RNP complexes.

Inventive Principle:

Principle #6Universality (Multi-functionality)

3Reliability

If precise conditions are used for Cas9 protein and gRNA complex formation, then complete and productive complex formation is achieved, but the optimization process becomes time-consuming and complex

Engineering Contradiction:

Improvecomplete and productive complex formationVSAvoidoptimization time for complex conditions

Core Design Contradiction:

ReliabilityVSLoss of time

Solution Approach 1:

The patent performs preliminary thermal melt curve analysis to characterize the expected stability and binding properties of the Cas9/gRNA complexes before actual gene editing experiments. This preliminary characterization establishes reference conditions and expectations, allowing researchers to quickly assess whether complex formation conditions are appropriate without extensive trial-and-optimization during the main experiment.

Inventive Principle:

Principle #10Preliminary action

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 the identification and selection of stable Cas9/gRNA complexes with enhanced thermostability, improving the efficiency and precision of gene editing by ensuring optimal complex formation.

Implementation Method 1

Differential Scanning Fluorimetry (DSF) is a biophysical technique where the change in fluorescence of a small molecule dye, e.g., SYPRO® orange, is used to monitor the thermal denaturation of a protein

Methodology Applied

Scientific EffectFluorescence: Fluorescence

Implementation Method 2

the thermal denaturation of a protein and to determine its thermal melting temperature (Tm)

Methodology Applied

Scientific EffectThermal denaturation: Melting

Implementation Method 3

Binding of ligands to the protein tend to stabilize the protein to differing extents and change its Tm

Methodology Applied

Scientific EffectProtein-ligand binding stabilization:

Data Source

PatentUS20220325326A1Evaluation of cas9 molecule/guide RNA molecule complexes

Publication Date: 2022.10.13 EDITAS MEDICINE INC

AI summary

Disclosed herein are methods for evaluation, selection, optimization, and design of Cas9 molecule/gRNA molecule complexes.