Reverse Transcriptase Genome Editing for Long-Sequence Insertion
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Solution Overview
Problem
Existing methods for integrating nucleic acids into genomes suffer from low frequency and lack of site specificity, and existing tools like CRISPR/Cas9 are less effective for inserting longer sequences, while approaches like Cre/loxP require multiple steps.
Innovation Solution
A system comprising a gene modifying polypeptide and a template RNA, which can introduce exogenous genetic elements into a host genome, including a chimeric antigen receptor (CAR) with specific domains for antigen binding, signaling, and insertion into targeted genomic locations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If CRISPR/Cas9 is used for genome editing, then small edits can be made, but integration of longer sequences is less effective
Solution Approach 1:
The patent changes the fundamental mechanism from CRISPR/Cas9's nuclease-based cutting to a reverse transcriptase-based insertion system. This parameter change enables the system to handle longer genetic sequences by using template-directed reverse transcription and integration, rather than relying on double-strand break repair pathways that are less efficient for large inserts.
2Manufacturing precision
If Cre/loxP approach is used, then sequence insertion can be achieved, but multiple steps are required
Solution Approach 1:
The patent merges multiple functions into a single integrated system. The reverse transcriptase enzyme performs both the reverse transcription of the template RNA into DNA and the subsequent integration into the host genome, eliminating the need for separate steps required by Cre/loxP (insertion of loxP sites, expression of Cre recombinase, and recombination). This consolidation reduces procedural complexity while maintaining insertion accuracy.
Solution Approach 2:
The reverse transcriptase enzyme in this system serves multiple functions: it synthesizes cDNA from the template RNA, processes the cDNA for integration, and mediates the actual genomic insertion. This multi-functionality replaces the specialized, sequential enzymes required by Cre/loxP, simplifying the overall process.
3Ease of operation
If nucleic acid integration is performed without specialized proteins, then the process is simple, but integration occurs at low frequency and with little site specificity
Solution Approach 1:
The patent introduces a template RNA molecule as an intermediary that carries the genetic sequence to be integrated. This template RNA binds to the reverse transcriptase enzyme, which then uses it as a template to synthesize cDNA that can be efficiently integrated into the host genome. The template RNA acts as a mediator that bridges the simple mixing approach and the need for high-frequency, site-specific integration.
Solution Approach 2:
The system changes the integration mechanism from passive, low-frequency random integration to an active, enzyme-mediated process. The reverse transcriptase enzyme actively captures the template RNA, synthesizes the corresponding DNA, and facilitates its integration into the host genome, dramatically increasing integration frequency while maintaining relative simplicity.
Data Source
AI summary
Methods and compositions for modulating a target genome are disclosed. For instance, gene modifying systems may be used to insert a heterologous object sequence (e.g., encoding a chimeric antigen receptor) into a target cell. The target cell may be, e.g., a T cell, induced pluripotent stem cell, or respiratory epithelial cell.