Artificial DNA Replisome for In Vivo Targeted Mutagenesis

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Solution Overview

Problem

Existing directed evolution methods are inefficient in generating a large variety of DNA variants due to limitations in transferring DNA into microbes, restricting the exploration of molecular functions to a small fraction of possibilities.

Innovation Solution

A recombinant polypeptide comprising a T5 DNA polymerase amino acid sequence operably linked to a DNA helicase amino acid sequence, specifically with mutations like I308V, is used to create a DNA replisome complex that replicates and mutagenizes target DNA sequences in vivo, enhancing mutagenesis efficiency.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If in vitro manipulations are used to create DNA libraries for directed evolution, then the process can be controlled and optimized, but the transfer efficiency into microbes is limited to about a million variants, which is only a small fraction of possibilities

Engineering Contradiction:

Improvevariant generation efficiencyVSAvoidDNA transfer efficiency

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The patent replaces the mechanical DNA transfer process (transformation, transfection, or conjugation) with an in vivo mutagenesis system that uses a mutagenic DNA polymerase to generate variants directly within the microbial cell. This substitution eliminates the efficiency bottleneck of external DNA transfer while maintaining controlled evolution through selective pressure.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

Solution Approach 2:

The system employs a mutagenic DNA polymerase that is expressed within the host cell and utilizes the cell's own replication machinery to generate and propagate mutations. The polymerase self-assembles with host proteins to form a functional replisome, enabling the cell to perform its own mutagenesis without external intervention, thereby achieving high variant generation efficiency.

Inventive Principle:

Principle #25Self-service

2Productivity

If a mutagenic DNA polymerase is used to replicate target DNA in vivo, then mutagenesis efficiency is enhanced up to 2.3×10^5-fold, but off-target mutations may occur

Engineering Contradiction:

Improvemutagenesis efficiencyVSAvoidoff-target mutations

Core Design Contradiction:

ProductivityVSObject-affected harmful factors

Solution Approach 1:

The patent employs a mutagenic DNA polymerase that is selectively expressed only in the presence of a specific inducer (e.g., IPTG), creating a localized mutagenesis effect at the target locus. The polymerase is recruited to the target site through specific promoter sequences and replication origins, ensuring that mutagenesis occurs only where intended while minimizing off-target effects throughout the genome.

Inventive Principle:

Principle #3Local quality

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 DNA replisome complex achieves up to 2.3×105-fold enhanced mutagenesis of target genes with minimal off-target mutations, enabling extensive exploration of protein sequence space for improved molecular functions.

Implementation Method 1

a T5 DNA polymerase amino acid sequence operably linked to a DNA helicase amino acid sequence... capable of replicating the nicked dsDNA

Methodology Applied

Scientific EffectDNA replication:

Implementation Method 2

a DNA helicase amino acid sequence... capable of replicating the nicked dsDNA

Methodology Applied

Scientific EffectDNA unwinding:

Data Source

PatentUS20250333714A1Artificial DNA replisome and methods of use thereof

Publication Date: 2025.10.30 REGENTS OF THE UNIVERSITY OF MINNESOTA

AI summary

Certain embodiments of the invention provide a recombinant polypeptide comprising a T5 DNA polymerase amino acid sequence operably linked to a DNA helicase amino acid sequence, as well as methods of using such a recombinant polypeptide for DNA replication and/or mutagenesis. Certain embodiments of the invention provide a targeted artificial DNA replisome complex. Certain embodiments of the invention provide a targeted DNA mutagenesis system.