Case study

HSV-1 Mutagenesis Using Hyperactive MuA Transposase

Transposon-based approaches are powerful tools for functional genomics, but applying them to large and complex genomes remains challenging. This case study demonstrates how the hyperactive v2 MuA transposase variant was successfully applied in a research setting to enable efficient mutagenesis of the large HSV-1 genome, turning a technically demanding concept into a functional experimental platform.

Overview

A research team led by Prof. Dr. med. Lars Dölken at Hannover Medical School, together with PhD student Lena Wolpert, set out to build a random mutagenesis library of Herpes Simplex Virus 1 (HSV-1), a large DNA virus with a complex genome.

Their objective was to create a robust platform for functional genomics studies. However, while transposon-based methods are highly useful for this type of work, extending them to a large viral genome such as HSV-1 required both efficient enzyme performance and a carefully optimized workflow.

The Challenge

Scaling the system to HSV-1 presented several technical and experimental challenges that had to be solved before a reliable mutagenesis library could be generated.

• Low initial transposition efficiency

• Need for optimization of in vitro transposition conditions

• Handling large-scale DNA preparation and processing

The Approach

Strategic Consultation and Experimental Design

Domus Biotechnologies provided early guidance on reaction optimization, including DNA amounts, incubation times, appropriate control plasmids, and quantitative workflows for accurate yield estimation. This helped ensure that the first experiments would produce reliable and interpretable data.

Custom Transposon Preparation and Purification

To streamline the workflow and improve input quality, Domus implemented a service-based solution that included chromatographic purification of the transposon and functional validation using both wild-type MuA and the hyperactive v2 MuA variant.

This step ensured delivery of high-quality, validated transposon DNA suitable for the mutagenesis workflow.

The Results

Following optimization and implementation, the project achieved a major technical milestone and demonstrated that MuA-based transposition can be effectively extended to a large viral genome.

• Approximately 50% transposition efficiency in HSV-1 using the hyperactive v2 MuA transposase variant

• A mutant library of approximately 1000 clones

• A robust and reproducible workflow for viral mutagenesis

Scaling and Future Directions

Library Expansion

• Scaling up via parallel transposition reactions

• Increasing library complexity and genome coverage

Quantitative Analysis

• Mapping insertion sites using NGS

• Determining the number of unique mutants

• Analyzing genome-wide insertion distribution

Platform Extension

• Applying the method to other herpesviruses

• Testing additional engineered transposon variants

Key Takeaways

• Hyperactive MuA variants can unlock new applications in complex systems

• Small sequence variations, such as terminal base pairs, can matter but may be tolerated with proper validation

• Workflow optimization is just as important as molecular design

• Collaborative iteration helps accelerate success in new application areas

Conclusion

By combining expertise in MuA transposition technology with hands-on experimental support, Domus Biotechnologies enabled the successful development of a novel HSV-1 mutagenesis platform.

This case highlights how high-performance MuA variants such as v2 MuA can be applied in research to overcome technical bottlenecks and enable experiments that would otherwise be difficult to achieve. The core value lies in integrating optimized molecular tools into practical workflows for complex systems such as large viral genomes.