CRISPR-Cas3 Eukaryotic Cell Editing With Pre-crRNA and Nuclear Targeting
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Solution Overview
Problem
Establishing a functional CRISPR-Cas3 system in eukaryotic cells has been challenging, as previous attempts in eukaryotic cells were unsuccessful, and efficient genomic editing was not achieved using mature crRNA, unlike the widely used CRISPR-Cas9 system.
Innovation Solution
The CRISPR-Cas3 system is established in eukaryotic cells by using a pre-crRNA and adding a nuclear localization signal, particularly a bipartite nuclear localization signal, to the Cas3 protein, enabling efficient genomic editing and large deletions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If a CRISPR-Cas3 system is introduced into eukaryotic cells using mature crRNA, then the system can be established, but efficient genomic editing is not achieved
Solution Approach 1:
The patent changes the crRNA parameter from mature crRNA to pre-crRNA, which fundamentally alters the system's functionality in eukaryotic cells. This parameter change enables both reliable system establishment and efficient genomic editing by allowing proper nuclear import and processing of the crRNA component.
2Ease of operation
If conventional CRISPR methods are used, then DNA editing can be performed, but large deletions including regions inaccessible to conventional methods cannot be achieved
Solution Approach 1:
The patent employs multiple parameter changes: using pre-crRNA instead of mature crRNA, adding bipartite nuclear localization signals to Cas3, and utilizing the CRISPR-Cas3 system architecture. These changes collectively enable the system to access and edit previously inaccessible genomic regions while maintaining ease of operation.
Solution Approach 2:
The bipartite nuclear localization signal acts as an intermediary that facilitates the nuclear import of the Cas3 protein, enabling the system to reach and edit genomic regions that were previously inaccessible to conventional CRISPR methods.
3Reliability
If Cas3 is used without nuclear localization signal, then the system can be introduced, but efficient genomic editing in eukaryotic cells is not achieved
Solution Approach 1:
The bipartite nuclear localization signal serves as a mediator that enables efficient nuclear import of the Cas3 protein. This intermediary element is crucial for achieving high genomic editing efficiency while maintaining reliable system introduction into eukaryotic cells.
Solution Approach 2:
The patent modifies the Cas3 protein by adding a bipartite nuclear localization signal, which changes its cellular localization parameter. This modification enables the protein to efficiently reach the nucleus where genomic editing occurs, thereby dramatically improving editing efficiency.
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 CRISPR-Cas3 system achieves accurate and extensive DNA editing in eukaryotic cells, allowing for the recognition of target sequences and causing deletions ranging from one hundred to several thousand bases, including regions inaccessible to conventional methods.
Implementation Method 1
the systems cleave and eliminate the foreign genomes by using the complementarity of the information introduced in the self genome and the genome sequence
Implementation Method 2
the CRISPR-Cas3 systems degrade target DNA in E. coli by helicase activity and exonuclease activity of Cas3
Implementation Method 3
the CRISPR-Cas3 systems degrade target DNA in E. coli by helicase activity and exonuclease activity of Cas3
Implementation Method 4
addition of a nuclear localization signal, particularly a bipartite nuclear localization signal to Cas3 made it possible to further improve the genome editing efficiency
Data Source
AI summary
A CRISPR-Cas3 system was successfully established in a eukaryotic cell.