sgRNA-Cas9 Composition for Targeted Genome Editing in Eukaryotic Cells
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Solution Overview
Problem
Existing genome editing methods using the CRISPR/Cas system have not been developed, and RFLP analysis is limited by the availability of restriction sites, making it difficult to detect engineered nuclease-induced mutations effectively.
Innovation Solution
A composition and method using a guide RNA specific for target DNA and Cas protein-encoding nucleic acid or protein for targeted DNA cleavage and mutagenesis, enabling programmable RNA-guided endonucleases (RGENs) for genome editing and genotyping.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If CRISPR/Cas system is used for genome editing, then targeted DNA cleavage and mutagenesis is achieved, but the method has not been developed for eukaryotic cells
Solution Approach 1:
The patent introduces a guide RNA as an intermediary molecule that mediates between the Cas9 endonuclease and the target DNA. The guide RNA binds to the target DNA sequence and directs Cas9 to the precise location, enabling the CRISPR/Cas system to function in eukaryotic cells by providing the necessary specificity and targeting capability that was previously missing.
Solution Approach 2:
The patent segments the CRISPR/Cas system into distinct functional components: the Cas9 endonuclease protein, the guide RNA molecule, and the target DNA sequence. This segmentation allows each component to be independently optimized and delivered to eukaryotic cells through different mechanisms (protein transfection, RNA transfection, or plasmid delivery), thereby achieving reliable genome editing application.
2Measurement precision
If RFLP method is used for genotyping, then detection of mutations is achieved, but appropriate restriction sites may not be available
Solution Approach 1:
The patent changes the fundamental parameter of site recognition from enzyme-based (restriction enzymes requiring specific palindromic sequences) to RNA-based (guide RNAs requiring complementary DNA sequences). This parameter change allows the system to target any DNA sequence regardless of whether traditional restriction sites are present, thereby maintaining detection precision while dramatically improving adaptability to various genomic locations.
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
RGENs allow for targeted DNA cleavage and mutagenesis, enabling detection of almost any single nucleotide polymorphism or small insertion/deletion via RFLP, providing a new genome editing tool for analyzing 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 that may induce modifications in target endogenous nucleic acid sequences in nucleuses of eukaryotic cells. The composition may comprise a single-chain guide RNA (sgRNA) and a Streptococcus pyogenes Cas9 protein. In some embodiments, the sgRNA and the Cas9 protein may be present in a molar ratio ranging from 29:14.0 to 29:1.4.