Precise Phage Mutagenesis with NgTET-Assisted CRISPR-Cas Systems

Helene Keuthen; Nadiia Pozhydaieva; Katharina H&#246;fer

doi:10.3791/69022

Mutagénesis precisa de fagos con sistemas CRISPR-Cas asistidos por NgTET

JoVE Journal
Bioengineering

A subscription to JoVE is required to view this content. Sign in or start your free trial.

JoVE Journal Bioengineering

Precise Phage Mutagenesis with NgTET-Assisted CRISPR-Cas Systems

Mutagénesis precisa de fagos con sistemas CRISPR-Cas asistidos por NgTET

Full Text

796 Views

10:52 min

October 14, 2025

DOI: 10.3791/69022-v

Helene Keuthen¹, Nadiia Pozhydaieva^1,2,3, Katharina Höfer^1,4,5

¹Max Planck Institute for Terrestrial Microbiology, ²Kavli Institute of Nanoscience,Delft University of Technology, ³Department of Bionanoscience,Delft University of Technology, ⁴Philipps-Universität Marburg, Department of Chemistry,Center for Synthetic Microbiology (SYNMIKRO), ⁵Institute of Pharmaceutical Biology and Biotechnology, Department of Pharmacy,Marburg University

Please note that some of the translations on this page are AI generated. Click here for the English version.

Overview

This study presents a protocol for reducing DNA modifications in bacteriophages using the NgTET enzyme, which facilitates efficient and scarless CRISPR-Cas mutagenesis. The approach aims to enhance genetic engineering of phages for various applications in biotechnology and phage therapy.

Key Study Components

Area of Science

Phage biology
Genetic engineering
Biotechnology applications

Background

Phage infection is studied at the molecular level.
Understanding phage infection can lead to therapeutic applications.
Recent developments include the discovery of bacterial immune systems against phages.
Phages exhibit clever mechanisms to evade bacterial defenses.

Purpose of Study

To develop a method for efficient CRISPR-Cas mutagenesis in phages.
To enable scarless genetic modifications in bacteriophages.
To explore applications in phage therapy and biotechnology.

Methods Used

Utilization of the NgTET enzyme for DNA modification reduction.
CRISPR-Cas system for targeted mutagenesis.
Experimental protocols for phage engineering.
Analysis of phage-bacterial interactions.

Main Results

Successful reduction of DNA modifications in bacteriophages.
Efficient implementation of scarless CRISPR-Cas mutagenesis.
Enhanced potential for phage engineering applications.
Insights into phage infection mechanisms and bacterial defenses.

Conclusions

The NgTET enzyme is effective for modifying bacteriophage DNA.
This method opens new avenues for phage therapy and biotechnology.
Understanding phage-bacterial interactions is crucial for future research.

Frequently Asked Questions

What is the NgTET enzyme?

The NgTET enzyme is used to reduce DNA modifications in bacteriophages, facilitating genetic engineering.

How does CRISPR-Cas mutagenesis work?

CRISPR-Cas mutagenesis allows for targeted modifications in DNA, enabling precise genetic engineering.

What are the applications of this research?

This research has applications in biotechnology and phage therapy, particularly in treating bacterial infections.

Why is understanding phage infection important?

Understanding phage infection is crucial for developing effective therapies and combating bacterial resistance.

What challenges do phages face from bacterial defenses?

Phages encounter bacterial immune systems that can inhibit their ability to infect and replicate.

Aquí, presentamos un protocolo para reducir las modificaciones del ADN en bacteriófagos utilizando la enzima NgTET, lo que permite una mutagénesis CRISPR-Cas eficiente y sin cicatrices. Este método facilita la ingeniería genética de fagos para aplicaciones en biotecnología y terapia con fagos.

Estudiamos la infección por fagos a nivel molecular. Esta es realmente ciencia fundamental. Sin embargo, no solo estamos haciendo la ciencia fundamental, sino que también estamos interesados en traducir esta ciencia fundamental en aplicaciones en las que usamos este conocimiento para estudiar la infección por fagos y usarlo para la ingeniería de fagos y para aplicaciones terapéuticas.

Uno de los desarrollos más recientes en el campo de la investigación de fagos es el descubrimiento de un sistema inmunológico bacteriano. Este sistema inmunológico está presente en todas las bacterias y ellas identificaron o generan herramientas para luchar realmente contra el fago. Sin embargo, el fago es bastante inteligente.

View the full transcript and gain access to thousands of scientific videos

Explore More Videos

Bioingeniería Número 224 Ingeniería de fagos fago de diseño mutagénesis mediada por CRISPR-Cas TET dioxigenasa glicosilación modificaciones de citosina modificaciones del ADN