GFP-Based CRISPR Reporter Systems for Sensitive, Specific Editing Detection
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Solution Overview
Problem
Current CRISPR-mediated gene editing systems using mTomato-containing cassettes suffer from insufficient sensitivity, high off-target effects, and poor reporter gene expression in specific tissues, necessitating improved reporter systems for effective gene editing.
Innovation Solution
Development of novel guide RNAs (gRNAs) with specific targeting sequences and Cas endonucleases, formulated into RNPs and compositions, which include lipid-based transfection competent vesicles (TCVs) for enhanced delivery and efficiency in CRISPR-mediated gene editing systems.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If mTomato-containing cassettes are used for CRISPR-mediated gene editing, then reporter gene expression can be achieved, but sensitivity is insufficient and off-target effects are high
Solution Approach 1:
The patent changes the reporter gene parameters by replacing mTomato with GFP (green fluorescent protein) and optimizing the cassette structure. This parameter change improves sensitivity for detecting gene editing events while reducing off-target effects through better signal-to-noise ratio and more specific detection capabilities.
Solution Approach 2:
The patent substitutes the optical detection system by using GFP fluorescence instead of mTomato red fluorescence, enabling different excitation and emission wavelengths that improve detection sensitivity. This substitution allows for better differentiation between on-target and off-target editing events through enhanced fluorescent signal characteristics.
2Reliability
If mTomato-containing cassettes are used for CRISPR-mediated gene editing, then reporter gene expression can be achieved, but reporter gene expression is poor in specific tissues
Solution Approach 1:
The patent changes the reporter gene parameter from mTomato to GFP, which has different expression characteristics and tissue compatibility. GFP demonstrates superior expression levels and reliability in specific tissues compared to mTomato, thereby improving overall reporter gene expression reliability across different tissue types.
3Measurement precision
If novel gRNAs with specific targeting sequences are developed, then sensitivity and specificity are improved, but device complexity increases
Solution Approach 1:
The patent applies local quality by designing gRNAs with specific targeting sequences that are optimized for particular genomic loci. Each gRNA is tailored with precise nucleotide sequences (e.g., 20-nucleotide targeting regions) that provide high sensitivity and specificity for their intended targets, while the overall system maintains manageable complexity through modular gRNA design.
Data Source
AI summary
The present disclosure provides: reporter systems for CRISPR-mediated gene editing; gRNAs, RNPs, and compositions for the reporter systems; and methods of testing CRISPR-mediated gene editing. The present disclosure further provides compositions and methods for knocking out a DNA segment of interest; and methods of effecting CRISPR-mediated gene editing in the eye. The present disclosure also provides compositions and methods for effecting base editing using base editors.