Retron Reverse Transcriptase Genome Editing
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Solution Overview
Problem
Current methods for precise genome editing in mammalian cells, such as those using CRISPR nucleases, face inefficiencies due to low abundance of template DNA and context-restriction issues, particularly stemming from native retron structures and poly T stretches.
Innovation Solution
The use of engineered retron ncRNAs combined with programmable nucleases and retron reverse transcriptase to generate repair donors inside cells, allowing for precise editing by targeting specific genomic sites.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If exogenous DNA is introduced into cells as a template for genome editing, then precise editing can be achieved, but the delivery efficiency is low and template DNA abundance is insufficient
Solution Approach 1:
The retron system enables cells to self-produce the repair template DNA internally through reverse transcription of retron ncRNA, eliminating the need for external DNA delivery. The cell's own reverse transcriptase converts the retron ncRNA into msDNA that serves as the repair template, making the system self-sufficient and overcoming delivery efficiency limitations.
Solution Approach 2:
The retron ncRNA acts as an intermediary molecule that is easily deliverable into cells. Instead of delivering complex exogenous DNA templates directly, the system delivers compact retron ncRNA sequences that serve as precursors, which are then converted into functional DNA templates by the cell's own reverse transcriptase machinery.
2Productivity
If native retron structures with poly T stretches are used, then reverse transcription can occur, but transcription termination is triggered reducing template availability
Solution Approach 1:
The invention extracts and removes the problematic poly T stretch element from the retron ncRNA sequence. By designing retron ncRNAs without poly T stretches, the system eliminates the transcription termination signal that would otherwise prevent full-length template production, while retaining the essential reverse transcription function.
3Manufacturing precision
If in vitro-produced DNA templates are used for genome editing, then precise editing is possible, but the abundance of template DNA is low reducing editing rates
Solution Approach 1:
The system enables cells to self-amplify and produce abundant repair template DNA internally. Once retron ncRNA is delivered and reverse transcriptase is expressed, the cell continuously produces msDNA templates through reverse transcription, generating high local concentrations of template DNA right at the editing site without requiring continuous external supply.
Solution Approach 2:
The retron ncRNA and reverse transcriptase are delivered and positioned in advance within the cell before the actual editing event. This preliminary setup allows the cell to generate template DNA on-demand when needed, ensuring high template availability at the precise moment and location of DNA repair, thereby increasing editing rates.
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
This approach enables precise editing of mammalian cells, tissues, and organs, including human cells, with improved efficiency and flexibility under various conditions (in vitro, in vivo, ex vivo).
Implementation Method 1
a retron reverse transcriptase to carry out the synthesis of the retron repair donors (i.e., by way of the RT-catalyzed conversion of the ncRNA molecule to its cognate msDNA (multicopy single-stranded DNA
Data Source
AI summary
Described herein are compositions and methods that provide precise editing of cells, including mammalian cells and human cells. The compositions and methods utilize retrons as repair donors, retron reverse transcriptases to make those retron repair donors, retron-encoded guide RNAs, and CRISPR nucleases.