MHC Locus Repetitive Sequence Deletion for Gene Engineering
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Solution Overview
Problem
Current techniques for producing MHC-deficient cells face challenges due to the complexity of the MHC locus, which contains repetitive sequences that are difficult to decipher, and the risk of unpredictable problems from destroying non-MHC molecules like β2 microglobulin.
Innovation Solution
A cell is developed with a large region containing repetitive sequences deleted or replaced from the MHC locus, using a genome engineering method that can efficiently engineer two or more alleles, allowing for specific gene deletion and insertion in the MHC region.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If current sequencing technologies are used to analyze the MHC locus, then the repetitive sequences can be deciphered, but the analysis accuracy is insufficient due to the complexity of the locus
Solution Approach 1:
The patent extracts and removes the problematic repetitive sequence region (approximately 300 kb) from the MHC locus by creating a deletion mutation. This eliminates the sequencing difficulty while preserving the essential MHC gene functions through careful selection of deletion boundaries that spare critical genes.
Solution Approach 2:
The patent segments the MHC locus analysis into manageable parts by defining specific deletion boundaries using flanking unique sequences. This allows precise targeting of the repetitive region while maintaining control over which genes are preserved or removed, making the engineering process systematic and verifiable.
2Reliability
If non-MHC molecules like β2 microglobulin are destroyed to eliminate MHC expression, then MHC-deficient cells can be produced, but unpredictable problems may arise due to unknown functions of the destroyed molecules
Solution Approach 1:
The patent converts the potential harm of destroying unknown molecules into a benefit by using CRISPR/Cas9-mediated deletion to remove only the problematic repetitive sequences while preserving essential MHC genes. This approach eliminates the risk of unintended consequences by providing precise control over what is deleted, transforming a potentially harmful approach into a safe and effective solution.
3Manufacturing precision
If a specific gene at the MHC locus is targeted for deletion, then MHC-deficient cells can be produced, but it cannot be excluded that other parts of the repetitive sequence have been unpredictably engineered
Solution Approach 1:
The patent implements a feedback mechanism by designing the deletion construct with specific flanking sequences that serve as verification markers. After deletion, these flanking sequences allow confirmation that only the intended repetitive region was removed and no unintended modifications occurred elsewhere in the genome, providing quality control feedback.
4Productivity
If large-scale deletions are induced at multiple alleles simultaneously, then efficient genome engineering can be achieved, but the complexity of engineering multiple alleles increases
Solution Approach 1:
The patent merges the engineering of multiple alleles into a single unified process by designing a deletion construct that can simultaneously target and delete the same repetitive sequence region in both alleles. This consolidation maintains high productivity while reducing operational complexity compared to treating each allele separately.
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 resulting cells are suitable for transplantation and further cell engineering applications, reducing the risk of immune rejection and allowing for precise genetic modifications in the MHC region.
Implementation Method 1
a target region targeted by guide RNA undergoes double-strand break by Cas9 nuclease
Implementation Method 2
the DNA that thus has undergone double-strand break is repaired by homologous directed repair (HDR)
Data Source
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AI summary
The present invention provides a cell suitable for gene engineering, cell engineering and cellular medicine, and a method for producing the same. The present invention provides a technique for removing repetitive sequences present in a specific gene region in two or more alleles, thereby facilitating gene targeting or sequencing in the region.