“Drag-and-Drop” Gene Editing Holds Potential for Treating Genetic Diseases

In a promising development for treating genetic disease, scientists have built upon gene editing technology, creating a novel technique called PASTE to edit large genes safely and more effectively. The study is published in Nature Biotechnology.

Improving gene editing technology

CRISPR-Cas9 is a gene editing system adapted from the processes used by bacteria as an immune defence. In CRISPR-Cas9, the DNA-cutting enzyme Cas9 works alongside a small strand of RNA which guides Cas9 to where it needs to make a cut in the genome – for example, cutting out a faulty disease-causing gene. A DNA template of the correct gene can then be inserted in its place by the cell’s own DNA repair machinery.

However, successful insertion of the new gene requires the cell to be actively dividing as the DNA repair process is not active in non-dividing cells. This process also requires breaks to be made in both strands of the DNA double helix, which can potentially create more problems for the cell by rearranging or even deleting chromosomes.

The researchers in the current study aimed to develop and improve CRISPR-Cas9 so that defective genes could be replaced without the need for potentially harmful double-stranded breaks.

Copy and PASTE functioning genes

To do this, the researchers turned to integrases, a family of enzymes that are used by some viruses to insert their DNA into the genomes of bacteria. In particular, the researchers focused on serine integrases as these can insert very large DNA molecules, potentially up to 50,000 base pairs in length.

Serine integrases target specific DNA sequences in the genome called attachment sites, which function as “landing sites” for integrases to insert their DNA payload. However, previous work shows that targeting such landing sites in humans is especially challenging as they are incredibly specific, and the integrases are difficult to reprogram. To create the desired sites, researchers in the current study combined the use of integrases with a CRISPR-Cas9 system.

This newly developed technique, named Programmable Addition via Site-specific Targeting Elements (PASTE) enables any site in the genome to be targeted for insertion of integrase landing sites using the appropriate guide RNA. Integrases then insert their DNA payloads at these sites, eliminating the need for double-stand breaks.

With this technique, DNA molecules up to around 36,000 base pairs can be inserted into cells – but the researchers think that eventually, this could work with even longer sequences.

“We think that this is a large step toward achieving the dream of programmable insertion of DNA,” says Dr. Jonathan Gootenberg, a McGovern Fellow at the McGovern Institute for Brain Research and co-senior author of the study. “It’s a technique that can be easily tailored both to the site that we want to integrate as well as the cargo.”

Gootenberg and colleagues demonstrated the efficacy of PASTE by incorporating 13 different genes into several human cell types – including liver and immune cells – across 9 different locations in the genome. Success rates varied between 5–60%. Additionally, PASTE was used to insert genes into the “humanized” livers of mice, comprised of around 70% human liver cells. They were able to successfully edit about 2.5% of these cells.

“It’s a new genetic way of potentially targeting these really hard-to-treat diseases,” explained Dr. Omar Abudayyeh, also a McGovern Fellow and co-senior author. “We wanted to work toward what gene therapy was supposed to do at its original inception, which is to replace genes, not just correct individual mutations.”

Advancing gene therapies

In the future, the researchers plan to explore the possibility of using this tool to replace the defective genes that cause conditions such as cystic fibrosis, hemophilia and Huntington’s disease.

Details of the genetic constructs used in the study have been made available online, in the hope that other scientists can further develop them and apply them in other novel ways.

Reference: Yarnall MTN, Ioannidi EI, Schmitt-Ulms C, et al. Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases. Nat Biotechnol. 2022:1-13. doi: 10.1038/s41587-022-01527-4

This article is a rework of a press release issued by Massachusetts Institute of Technology. Material has been edited for length and content.