RT-DNA Retron Genome Editing for Fidelity and Frequency Optimization

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Solution Overview

Problem

Existing methods for introducing exogenous DNA into cells for genomic editing are limited in quantity and variability, with unclear optimal editing sites and DNA types, leading to inconsistent editing fidelity and frequency.

Innovation Solution

A method involving transforming host cells with a library of expression cassettes encoding modified retrons, each with a promoter linked to a nucleic acid segment for a barcode, guide RNA, and donor DNA, followed by sequencing genomic sites to evaluate editing fidelity and frequency, using modified retron non-coding RNAs and CRISPR nucleases to optimize genomic editing systems.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Quantity of substance

If exogenous DNA is introduced into cells for genomic editing, then genomic editing can be performed, but the amounts of exogenous DNA that can be introduced are limited and not all cells will be transformed

Engineering Contradiction:

Improveamount of exogenous DNAVSAvoidtransformation efficiency

Core Design Contradiction:

Quantity of substanceVSReliability

Solution Approach 1:

The retron system enables cells to self-produce abundant donor DNA internally through reverse transcription of retron RNA by the retron's own reverse transcriptase, eliminating the need for external DNA introduction and achieving both high quantity and high transformation efficiency

Inventive Principle:

Principle #25Self-service

Solution Approach 2:

The retron ncRNA serves multiple functions simultaneously: it acts as a template for reverse transcription to generate donor DNA, encodes guide RNA for CRISPR targeting, and includes barcode sequences for tracking, making it a multi-functional editing system

Inventive Principle:

Principle #6Universality (Multi-functionality)

2Productivity

If different retron structures and sequences are used for genomic editing, then editing frequency may vary, but it remains unclear which type optimally edits a genomic site

Engineering Contradiction:

Improveediting frequencyVSAvoidoptimality information

Core Design Contradiction:

ProductivityVSLoss of information

Solution Approach 1:

The barcode sequences in the retron donor DNA are sequenced from genomic DNA to provide feedback information about which retron structures and sequences successfully edited which genomic sites, enabling identification of optimal combinations

Inventive Principle:

Principle #23Feedback

Solution Approach 2:

The retron ncRNA is divided into functional segments including barcode regions, guide RNA regions, and donor DNA regions, allowing systematic evaluation of different segments and their contributions to editing efficiency

Inventive Principle:

Principle #1Segmentation

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

Enables systematic evaluation and optimization of genomic editing variables, such as ncRNA chasses, gRNA sequences, and CRISPR nucleases, improving editing fidelity and frequency by identifying optimal combinations for specific genomic sites.

Implementation Method 1

retron DNA can be made abundantly in vivo by reverse transcription from retron RNA using the retron's own reverse transcriptase

Methodology Applied

Scientific EffectReverse transcription:

Implementation Method 2

the sequences of genomic edits made by the guide RNA and cas nuclease

Methodology Applied

Scientific EffectCRISPR-Cas nuclease activity:

Data Source

PatentUS20250215422A1RT-DNA Fidelity and Retron Genome Editing

Publication Date: 2025.07.03 THE J DAVID GLADSTONE INSTITUTES

AI summary

Described herein are compositions and methods that can analyze optimal systems for improved fidelity and frequency of genomic editing. The compositions and methods involve use of modified retrons.