CRISPR-Cas9 Guide RNA Composition for Site-Independent Mutation Detection
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing genome editing and genotyping methods using the CRISPR/Cas system are limited by the lack of a developed RNA-guided endonuclease (RGEN) and the limitations of Restriction Fragment Length Polymorphism (RFLP) due to the unavailability of appropriate restriction sites.
Innovation Solution
A composition and method using a guide RNA specific for target DNA and Cas protein-encoding nucleic acid or Cas protein for targeted DNA cleavage and mutagenesis, enabling RGEN-mediated RFLP analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If CRISPR/Cas system is used for genome editing, then targeted DNA cleavage is achieved, but off-target effects occur
Solution Approach 1:
The patent modifies the guide RNA parameters by introducing modified nucleotides (e.g., 5-methylcytosine, 5-hydroxymethylcytosine, or other chemically modified bases) at specific positions to enhance binding specificity to the target DNA sequence while reducing off-target effects. This parameter change in the guide RNA composition directly addresses the contradiction between achieving precise targeting and minimizing harmful off-target cleavages.
2Adaptability or versatility
If RNA-guided endonuclease is developed, then genome editing capability is provided, but the method was not previously available in eukaryotic cells
Solution Approach 1:
The patent employs a guide RNA as an intermediary molecule that mediates between the Cas9 endonuclease and the target DNA sequence. This guide RNA component enables the CRISPR/Cas system to be adapted for use in eukaryotic cells by providing sequence-specific targeting capability, thus bridging the gap between having the editing tool and being able to apply it in eukaryotic systems.
3Measurement precision
If restriction fragment length polymorphism is used for genotyping, then mutation detection is achieved, but appropriate restriction sites are not always available
Solution Approach 1:
The patent replaces the mechanical/restriction enzyme-based RFLP method with an RNA-guided endonuclease system. Instead of relying on pre-existing restriction sites in the DNA sequence, the guide RNA directs the Cas9 nuclease to any target sequence, substituting the restriction enzyme mechanism with a programmable RNA-directed cleavage system. This enables mutation detection at any genomic location regardless of restriction site availability.
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
Provides a new, convenient genome editing tool capable of detecting and analyzing almost any single nucleotide polymorphism or small insertion/deletion via RGEN-mediated RFLP, allowing for the detection and cleavage of naturally-occurring variations and mutations.
Implementation Method 1
a guide RNA specific for the target DNA and Cas protein-encoding nucleic acid or Cas protein
Implementation Method 2
Cas9, an essential protein component in the Type II CRISPR/Cas system, forms an active endonuclease when complexed with two RNAs termed CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA), thereby slicing foreign genetic elements
Data Source
AI summary
The present disclosure relates to targeted genome editing in eukaryotic cells or organisms. More particularly, the present disclosure provides for compositions and methods that may induce modifications in target endogenous nucleic acid sequences in nucleuses of eukaryotic cells. For example, methods disclosed herein may comprise preparing a Cas9/RNA complex, wherein the Cas9/RNA complex comprises a Cas9 protein and a guide RNA. Methods may further comprise introducing the Cas9/RNA complex into a non-human embryo, wherein the Cas9/RNA complex induces a modification at a target endogenous nucleic acid of the non-human embryo to provide for a genome modified embryo. Methods may further comprise transferring the genome modified embryo into a foster mother and allowing the foster mother to produce a F0 animal having the modification at the target endogenous nucleic acid.