Engineered Retron msDNA Production via Structural Extraction
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Solution Overview
Problem
Existing retron systems face challenges with low efficiency and context-restriction in genome engineering applications due to factors like branched structures, invariant flanking regions, limited total length, and native poly T stretches.
Innovation Solution
Engineered retrons are modified to enhance production of multicopy single-stranded DNA (msDNA) by incorporating a pre-msr sequence, an msr gene, an msd gene, a post-msd sequence with a self-complementary region, and a ret gene encoding reverse transcriptase, thereby improving efficiency and copy numbers.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If native retron structure is used, then reverse transcription can occur, but DNA production efficiency is low due to branched structure and invariant flanking regions
Solution Approach 1:
The patent extracts and removes the problematic branched structure and invariant flanking regions from the native retron. Specifically, the 2'-5' phosphodiester bond linking the 5' end of ssDNA to the 3' end of msr RNA is removed, and the invariant flanking regions are excised to create a linear, simplified retron structure that maintains reverse transcription capability while dramatically improving DNA production efficiency
Solution Approach 2:
The retron is segmented into distinct functional modules: a promoter region, a msr gene encoding the RNA component, an msd gene encoding the DNA component, and a reverse transcriptase gene. This modular segmentation allows independent optimization of each component and facilitates high-throughput screening of variants with improved DNA production properties
2Quantity of substance
If native retron is used, then genome engineering applications are possible, but copy numbers are limited by endogenous structure
Solution Approach 1:
The patent systematically varies key parameters of the retron structure including the length and sequence of the self-complementary region, the poly T stretch length, and the overall retron size. By creating libraries of retrons with different parameter values and screening for high copy number variants, the invention achieves both increased DNA quantity and adaptability to different application contexts
3Length of stationary object
If retron length is increased, then more DNA can be produced, but native poly T stretch acts as transcription terminator
Solution Approach 1:
The patent converts the harmful poly T transcription terminator into a beneficial element by optimizing its length and position. The modified poly T stretch is designed to function as both a transcription terminator (preventing read-through) and a replication origin (enabling high-copy plasmid maintenance), thereby simultaneously achieving long DNA production and high transcription 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 engineered retrons demonstrate enhanced production of msDNA, addressing the efficiency and copy number limitations of native retrons, and enabling effective applications in CRISPR/Cas-mediated genome editing, recombineering, cellular barcoding, and molecular recording.
Implementation Method 1
The self-complementary region is formed by hydrogen bonding between the 3' and 5' ends of the neRNA
Data Source
AI summary
Engineered retrons, modified to enhance production of multicopy single-stranded DNA (msDNA), are provided. In addition, vector systems encoding such engineered retrons and methods of using engineered retrons and vector systems encoding them in various applications such as CRISPR/Cas-mediated genome editing, recombineering, cellular barcoding, and molecular recording are also disclosed.