MIT researchers have designed a new tool that can be used to remove faulty genes and replace them with new ones in a safer and more efficient manner.
The researchers were able to deliver genes to several types of human cells, as well as to mice, using this system. The new technique, known as PASTE, could hold promise for treating diseases that are caused by defects in the genes that make up the body.
Abudayyeh is a McGovern Fellow at the McGovern Institute for Brain Research. Gene therapy was supposed to replace genes, not just correct individual genes, and that's what we wanted to do.
The new tool combines the precise targeting of CRISPR-Cas9 with a set of molecules originally derived frombacterial defense systems.
Jonathan Gootenberg is a McGovern Fellow and says that the integrases come from the ongoing battle betweenbacteria andviruses. It speaks to the fact that we can find a lot of useful new tools from these natural systems.
The senior authors of the study are Gootenberg and AbuDayyeh. Matthew Yarnall and Rohan Krajeski are two of the lead authors.
A human's genes are inserted
The CRISPR-Cas9 system uses a short strand ofRNA to guide theCas9 to where it wants to cut. A small portion of the genome is deleted when a specific cut is made in the genome and the cells' DNA repair processes glue the cut together.
A corrected copy of a DNA template can be incorporated into the cell's genomes during the repair process. This process requires cells to make double-stranded breaks in their genes, which can cause chromosomal deletions or rearrangements. One limitation is that it only works in cells that are dividing.
The MIT team wanted to develop a tool that would not cause double-stranded DNA breaks. They turned to a family of enzymes called integrases, which are used by viruses to insert themselves into the genomes ofbacteria.
The researchers focused on serine integrases, which can insert huge chunks of DNA. The attachment sites are known as landing pads. When they find a landing pad in the host genome, they bind it to their own.
The landing pads are very specific and it's difficult to reprogram them to target other sites. The MIT team realized that it would be possible to reprogram the powerful insert system with the combination of the two.
The new tool, PASTE (Programmable addition via site-specific targeting elements), includes a Cas9 enzyme that cuts at a specific genomic site. This gives them the ability to target any site in the genome for the landing site. A fused reverse transcriptase can be used to add one strand of DNA to another strand.
Once the landing site is incorporated, the integrase will be able to insert its larger DNA payload into the genome.
"We think that this is a big step towards achieving the dream of the person who wants to insert their own genes." It can be tailored to the site that we want to integrate as well as the cargo.
Replacement of genes.
The researchers found that they could use PASTE to insert genes into a number of human cells. The delivery system was tested with 13 different genes and they were able to insert them into nine different places in the genome.
The researchers were able to insert genes with a success rate of up to 60 percent. There were very few unwanted "indels" at the sites of genes integration.
Abudayyeh says that they don't see many indels and that they don't have to worry about chromosomal rearrangements.
They were able to insert genes in mice. New genes were integrated into 2.5 percent of the cells in the mice's hepatocytes.
The researchers inserted up to 36,000 base pairs of DNA into the study, but they think even longer strands could be used. A human gene can range from a few hundred to more than 2 million base pairs, although for therapeutic purposes only the coding sequence of theProtein needs to be used.
Researchers are looking into the possibility of using this tool to replace the cystic fibrosis gene. This technique can be used to treat blood diseases caused by faulty genes, such as Huntington's disease, a neurological disorder caused by a faulty gene.
Other scientists can use the genetic constructs the researchers have made online.
One of the great things about engineeringmolecular technologies is that people can build on them, develop and apply them in ways that we didn't think of. Being part of that emerging community is great.
There is more information about Drag-and-drop genome insertion of large sequences without double-strand DNA. The article can be found at www.nature.com/articles/s41587-.
Journal information: Nature Biotechnology
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