CRISPR Cas9 Guide RNA Composition for Eukaryotic Genome Editing
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Solution Overview
Problem
Until now, a genome editing and genotyping method using the RNA-guided endonuclease (RGEN) based on the CRISPR/Cas system has not been developed.
Innovation Solution
A composition for cleaving target DNA in eukaryotic cells or organisms comprising a guide RNA specific for the target DNA and Cas protein-encoding nucleic acid or Cas protein, which induces targeted mutagenesis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If CRISPR/Cas system is used for genome editing, then targeted DNA cleavage is achieved, but the method has not been developed for eukaryotic cells
Solution Approach 1:
The patent introduces a chimeric guide RNA as an intermediary molecule that mediates between the Cas9 protein and target DNA in eukaryotic cells. This guide RNA combines crRNA and tracrRNA functions to enable the CRISPR/Cas system to operate in eukaryotic genomes, resolving the developmental gap in applicability.
Solution Approach 2:
The patent creates a universal CRISPR/Cas system that can function across different eukaryotic cell types and organisms. The chimeric guide RNA design allows the same Cas9 protein to target various DNA sequences in diverse eukaryotic genomes, achieving multi-functionality and broad adaptability.
2Measurement precision
If guide RNA and Cas protein are used for targeted mutagenesis, then high specificity is achieved, but off-target effects may occur
Solution Approach 1:
The patent implements feedback mechanisms through careful design of the guide RNA sequence and Cas9 protein interaction. The system incorporates checks and balances where the guide RNA must perfectly match the target sequence for efficient cleavage, providing inherent feedback that reduces off-target effects while maintaining high specificity.
Solution Approach 2:
The patent applies local quality by designing the guide RNA with specific structural and sequence characteristics tailored to the target site. The chimeric structure combines regions with different properties to achieve precise local binding at the target location while minimizing interactions with off-target sequences.
3Adaptability or versatility
If RNA-guided endonuclease is used for genotyping, then mutation detection is enabled, but the method is newly developed and requires optimization
Solution Approach 1:
The patent segments the genotyping method into distinct components: guide RNA design, Cas9 protein preparation, and analysis protocols. This segmentation allows each component to be optimized independently and facilitates the development and standardization of the genotyping application in eukaryotic cells.
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 with high specificity, allowing for the analysis of naturally-occurring variations and mutations, and avoiding off-target effects.
Implementation Method 1
a genome editing and genotyping method using the RNA-guided endonuclease (RGEN) based on the CRISPR/Cas system
Implementation Method 2
CRISPR functions as a prokaryotic immune system, in that it confers resistance to exogenous genetic elements such as plasmids and phages
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, disclosed herein is a system for inducing targeted disruption of endogenous genes in a eukaryotic cell, the system comprising: an extracellular Cas9/RNA complex; cell-free and a buffer; wherein the extracellular Cas9/RNA complex comprises: a recombinant Cas9; and a guide RNA having a CRISPR RNA (crRNA) and a transactivating crRNA (tracrRNA); wherein the extracellular Cas9/RNA complex is disposed in the cell-free buffer and wherein the extracellular Cas9/RNA complex is complexed prior to being introduced into the eukaryotic cell; and wherein the Cas9/RNA complex functions as an endonuclease that induces targeted disruption of the target DNA upon introduction into the eukaryotic cell.