Engineered Retron Activity Assessment by Double-Stranded RT-DNA Sequencing
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Solution Overview
Problem
Existing methods for introducing exogenous DNA into cells for genome editing are inefficient and often result in low abundance and imprecise editing due to issues with retron DNA synthesis and delivery, leading to poor replication fidelity and efficiency.
Innovation Solution
A method involving debranching retron nucleic acids, extending 3' ends of reverse transcribed DNA (RT-DNA) with deoxynucleotide triphosphates, generating a second strand complement using DNA polymerase, attaching adapters, and sequencing the double-stranded RT-DNA to analyze and identify retron systems with improved replication fidelity and efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If retron DNA is synthesized in high amounts using retron reverse transcriptase, then the abundance of template DNA increases, but replication fidelity and precision deteriorate due to errors in DNA synthesis
Solution Approach 1:
The patent applies preliminary action by performing debranching of retron nucleic acids before generating reverse transcribed DNA, and by extending 3' ends of RT-DNA with deoxynucleotide triphosphates before generating the second strand. These preparatory steps ensure proper structure and fidelity before the main DNA synthesis process, preventing errors that would otherwise occur during high-volume replication.
Solution Approach 2:
The patent uses debranching enzymes as intermediaries to process retron nucleic acids before they are used as templates. The debranching enzyme acts as a mediator that resolves the branched structure of retron nucleic acids into linear forms suitable for accurate reverse transcription, thereby maintaining replication fidelity while enabling high-yield DNA production.
2Reliability
If exogenous DNA is delivered to target cells for genome editing, then genome editing capability is achieved, but delivery efficiency is low resulting in poor editing rates
Solution Approach 1:
The patent applies self-service by enabling host cells to autonomously produce retron reverse transcribed DNA through introduced retron components. Once the retron system is delivered to target cells, the cells themselves perform the DNA synthesis and production functions, eliminating the need for continuous external delivery and achieving both high delivery efficiency and reliable genome editing capability.
Solution Approach 2:
The patent delivers retron components (nucleic acids and reverse transcriptase) to target cells as preliminary action, establishing a self-sustaining DNA production system before genome editing is performed. This preliminary establishment of the retron system enables efficient subsequent DNA production for genome editing without requiring repeated delivery operations.
3Measurement precision
If retron systems are evaluated for DNA production efficiency, then identification of useful retron systems is enabled, but the evaluation process itself introduces errors and reduces accuracy
Solution Approach 1:
The patent applies taking out by extracting and sequencing only the 3' ends of reverse transcribed DNA after debranching and adapter attachment. This extraction of specific regions for sequencing minimizes the impact of evaluation processes on overall DNA fidelity, as the sequencing targets predetermined regions that do not interfere with the functional integrity of the retron system.
Solution Approach 2:
The patent uses copying by generating a second strand complement of the 3'-end extended RT-DNA to create double-stranded RT-DNA for sequencing. This copying process creates a stable, sequencable form of the DNA without altering the original single-stranded retron DNA that remains available for functional use, thereby maintaining DNA production fidelity while enabling accurate evaluation.
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 allows for the efficient identification and quantification of retron systems that can produce precise genome editing, enhancing the production of retron reverse transcribed DNA in host cells.
Implementation Method 1
debranching retron nucleic acids and generating reverse transcribed DNA (RT-DNA) from retron RNA in the retron nucleic acids
Implementation Method 2
generating a second strand complement to the 3′-end extended RT-DNA using a DNA polymerase and a primer
Data Source
AI summary
Methods and compositions are described herein that are useful for analyzing and identifying retron systems with improved replication fidelity and efficiency. The methods and compositions described herein facilitate the engineering, quantification, and identification of retron elements for precise genome engineering.