Fluorescent Guide RNA for Direct gRNA Expression Measurement
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Solution Overview
Problem
There is a lack of direct, non-invasive methods for measuring guide RNA (gRNA) expression levels in CRISPR systems, which complicates understanding and engineering of synthetic genetic circuits due to indirect and obfuscated methods currently available.
Innovation Solution
The use of a fluorescent guide RNA (fgRNA) system that includes a fluorophore-binding RNA aptamer, such as the Broccoli aptamer, which fluoresces in the presence of a small molecule like DFHBI-1T, allowing for direct measurement of gRNA expression by monitoring fluorescence, and can be configured for various editing strategies and promoters like RNA Pol II.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If indirect methods (activating or repressing downstream fluorescent genes) are used to measure gRNA expression, then gRNA expression levels can be assessed, but the measurement is obfuscated by complex gRNA/Cas9 and gRNA/DNA interactions and is not direct or non-invasive
Solution Approach 1:
The patent introduces a fluorescent RNA aptamer as an intermediary component embedded within the gRNA structure. This aptamer binds to a non-invasive fluorescent indicator (DFHBI-1T) to produce fluorescence, serving as a direct mediator for measuring gRNA expression levels without requiring downstream gene activation or complex gRNA/Cas9 interactions. The aptamer acts as a built-in reporter that converts gRNA presence into a measurable fluorescent signal.
Solution Approach 2:
The patent merges the gRNA functional sequence with a fluorescent RNA aptamer sequence into a single chimeric molecule. This combination allows the gRNA to simultaneously perform its gene-targeting function and serve as a fluorescent reporter for expression measurement. The merged structure eliminates the need for separate measurement systems and provides direct, real-time monitoring of gRNA expression levels.
2Ease of operation
If direct fluorescence monitoring of gRNA is implemented using fluorescent aptamers, then direct and non-invasive measurement of gRNA expression is achieved, but the gRNA structure must be modified to include aptamer sequences
Solution Approach 1:
The patent designs the fluorescent gRNA to serve multiple functions simultaneously: (1) guiding Cas9 to target DNA sequences through complementary base pairing, (2) binding to fluorescent indicators (DFHBI-1T) through the embedded aptamer for expression monitoring, and (3) maintaining RNA stability and processing signals. This multi-functionality allows a single modified gRNA structure to replace both the traditional guide RNA and separate reporter systems.
Solution Approach 2:
The patent introduces the fluorescent aptamer sequence at specific local positions within the gRNA structure (such as in the trunk or tail regions) without disrupting the critical 20-nucleotide spacer sequence that binds to target DNA. This localized modification approach maintains the essential gene-editing function while adding measurement capability only where it does not interfere with gRNA-Cas9-DNA interactions.
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
Enables direct and non-invasive measurement of gRNA expression, expanding the contexts in which CRISPR technology can be utilized and providing a means to monitor CRISPR-mediated gene repression efficiency.
Implementation Method 1
a fluorescent guide RNA (fgRNA) which fluoresces in the presence of small molecules (e.g., DFHBI-1T)
Data Source
AI summary
Provided herein are tools for understanding and engineering dynamics of synthetic genetic circuits which utilize CRISPR components. More particularly, methods, systems, and compositions for directing Cas9 activity using a fluorescent guide RNA (fgR-NA) which fluoresces in the presence of small molecules (e.g., DFHBI-IT) are described and illustrated in the present provisional application.