RNA Ribozyme Nucleic Acid Cleavage Without Cas Protein Burden
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Solution Overview
Problem
Current CRISPR-Cas gene editing technologies face issues such as off-target effects, large protein sizes affecting transfection efficiency, and potential immune responses due to bacterial origins, limiting their application in nucleic acid manipulation.
Innovation Solution
An RNA ribozyme-based DEAR nucleic acid manipulation system derived from bacterial Group IIC introns, comprising domains I-VI, with a target recognition site for sequence-specific targeting and catalytic activity, enabling targeted cleavage of DNA and RNA.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If CRISPR-Cas protein-based system is used for nucleic acid manipulation, then gene editing capability is achieved, but protein size exceeds 1300 amino acids causing poor transfection efficiency
Solution Approach 1:
The patent replaces the protein-based CRISPR-Cas system with an RNA-based ribozyme system. The ribozyme is an RNA molecule that performs catalytic cleavage of nucleic acids without requiring large protein structures, thereby eliminating the transfection efficiency problem associated with large proteins while maintaining gene editing capability.
Solution Approach 2:
The patent changes the fundamental molecular parameter from protein (amino acid-based) to RNA (nucleotide-based). This parameter change reduces the molecular size and improves transfection efficiency while preserving the ability to perform sequence-specific nucleic acid cleavage and gene editing.
2Reliability
If CRISPR-Cas system is used for nucleic acid manipulation, then targeting capability is achieved, but off-target effects cause uncontrollable deleterious variations
Solution Approach 1:
The patent substitutes the protein-based CRISPR-Cas targeting mechanism with an RNA ribozyme system that uses base-pairing complementarity for target recognition. This substitution provides more precise targeting through RNA-DNA hybridization, reducing off-target effects and deleterious variations while maintaining sequencing capability.
3Reliability
If CRISPR-Cas system is used for nucleic acid manipulation, then editing function is achieved, but large molecular weight affects transfection efficiency
Solution Approach 1:
The patent replaces the high molecular weight protein-based CRISPR-Cas system with an RNA-based ribozyme system. The ribozyme has significantly lower molecular weight than the Cas proteins, improving transfection efficiency while maintaining the editing function through RNA-catalyzed nucleic acid cleavage.
4Reliability
If CRISPR-Cas system is used for nucleic acid manipulation, then gene editing capability is achieved, but immune responses are triggered due to bacterial origins
Solution Approach 1:
The patent substitutes the bacterial protein-based CRISPR-Cas system with an RNA ribozyme system. The ribozyme is derived from bacterial Group IIC introns but functions as an RNA catalyst rather than a protein, reducing immunogenicity while maintaining gene editing capability through RNA-mediated nucleic acid cleavage.
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
The DEAR system overcomes the limitations of CRISPR-Cas by providing efficient and specific nucleic acid cleavage in both E. coli and mammalian cells without triggering immune responses, while avoiding the size and efficiency issues of protein-based systems.
Implementation Method 1
the RNA molecule comprises a target recognition site that hybridizes to a target sequence in a target nucleic acid
Implementation Method 2
RNA ribozyme-based DEAR nucleic acid manipulation system... with catalytic activity, enabling targeted cleavage of DNA and RNA
Data Source
Figure 1A~1C
Figure 1D~1E
Figure 1F~1G
AI summary
Disclosed in the present disclosure are an RNA enzyme-based DEAR nucleic acid manipulation system and a use thereof. The present disclosure provides a DEAR nucleic acid manipulation system, wherein the DEAR nucleic acid manipulation system comprises an RNA molecule derived from a bacterial group II intron of type C, and the RNA molecule contains a substrate recognition region hybridized with a target sequence in a target nucleic acid. The RNA enzyme-based DEAR nucleic acid manipulation system provided by the present disclosure avoids the problem that the transfection efficiency is affected by large protein molecules in a CRISPR-Cas system, the problem of the potential immunogenicity caused by Cas proteins, etc. The RNA enzyme-based DEAR nucleic acid manipulation system provided by the present disclosure can realize cutting of DNA and RNA.