Expanding the gene editing toolbox
New method makes room for DNA insertions
Currently, the most versatile method of gene editing is prime editing, a technology based on the CRISPR/Cas9 gene scissors. However, one of the bottlenecks of this method has been the efficient insertion of new, long DNA sequences, as this requires the displacement of the original DNA segments.
Researchers at the Technical University of Munich (TUM) and Helmholtz Munich have now developed a method for the targeted degradation of the original DNA segments to make room for the insertions.
Using CRISPR/Cas9 gene scissors, scientists can edit genes more precisely, quickly, and easily than with previous approaches. Researchers are continually developing the method further, and in 2019, scientists published a successor technique: Prime Editing (PE), the current state-of-the-art in gene editing. With PE, gene modifications such as the insertion, replacement, or deletion of individual DNA building blocks can be carried out with high precision.
Similar to CRISPR/Cas9, PE works by docking a tiny molecular editing machine to a specific site in the genome. It unwinds the double-stranded DNA and cuts the DNA at a specific location. However, unlike CRISPR/Cas9, PE only requires one of the two strands to be cleaved. This makes the system inherently safer.
The editing machine contains an RNA template that encodes the desired modification. The enzyme reverse transcriptase, which is also included in the complex, then transcribes the template directly into the desired DNA at the correct position in the genome.
Advanced machinery degrades original DNA segments
“One of the biggest challenges so far has been the next step: there are now two pieces of DNA, the original and the newly synthesized one containing the desired change,” says Julian Geilenkeuser, one of the study’s first authors. “Only if the original piece of DNA is removed and the new one is inserted in its place will the modification be successful in the long run.”
A team led by Gil Gregor Westmeyer, Professor of Neurobiological Engineering at TUM and Director of the Institute for Synthetic Biomedicine at Helmholtz Munich, has now developed a new approach. The researchers have integrated an additional component into the editing machinery: an exonuclease, a protein that breaks down DNA. It is connected to the machinery via a molecular adapter. The protein specifically degrades the part of the original strand that is no longer required, thus making room for the newly synthesized piece of DNA.
Enhanced editing capabilities with improved machinery
"This new exonuclease-enhanced prime editing strategy, "Exo-PE," is much more efficient than the well-known "PE2" editing strategy when it comes to inserting new pieces of DNA longer than 30 base pairs. This new method makes it possible to insert longer pieces of DNA into cells without the need for a DNA double-strand breaks," says Dr. Dong-Jiunn Jeffery Truong, also first author of the study. The researchers are thus adding an important new element to the gene editing toolbox.
"The Exo-PE method brings us a step closer to systematically generating variants of gene products even in non-dividing cells, such as nerve or heart cells, to better study them in basic research and potentially develop ways to correct longer gene sequences," says Gil Westmeyer.
D.J. J. Truong*, J. Geilenkeuser*, S.V. Wendel, J.C. Heinrich Wilming, N. Armbrust, E. Binder, T. Heinrich Santl, A. Siebenhaar, C. Gruber, T. Phlairaharn, M. Živanić, G.G. Westmeyer: Exonuclease-enhanced prime editors, Nature Methods (2024), DOI: 10.1038/s41592-023-02162-w *Authors contributed equally
This work was supported by the Munich Multiscale Biofabrication Network, which is part of the ONE MUNICH Strategy Forum, in which TUM and LMU identify and promote joint initiatives on major future issues and fields. The ONE MUNICH Strategy Forum is supported by Hightech Agenda Bayern. Funded by the Federal Ministry of Education and Research (BMBF) and the Free State of Bavaria under the Excellence Strategy of the Federal Government and the Länder.