Newly developed tool helps snip out faulty genes in a safe manner

Scientists from the Massachusetts Institute of Technology (MIT) have come up with a new tool that has the potential to detach faulty genes and exchange them with new ones in a highly safe and effective way. This has been made by building on the CRISPR gene-editing system.

With the availability of this system, the scientists displayed that they could deliver genes as long as 36,000 DNA base pairs to various kinds of human cells, as well as to liver cells in mice. The new method, called PASTE, could turn out to be useful for treating diseases that have been caused by defective genes consisting of a large number of mutations, like cystic fibrosis.

It’s a new genetic way of potentially targeting these really hard to treat diseases. 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.”

Omar Abudayyeh, McGovern Fellow, McGovern Institute for Brain Research, Massachusetts Institute of Technology

The new tool integrates the accurate targeting of CRISPR-Cas9, a set of molecules that have been originally derived from bacterial defense systems, along with enzymes known as integrases. Here, viruses tend to insert their genetic material into a bacterial genome.

Just like CRISPR, these integrases come from the ongoing battle between bacteria and the viruses that infect them. It speaks to how we can keep finding an abundance of interesting and useful new tools from these natural systems.”

Jonathan Gootenberg, McGovern Fellow, Massachusetts Institute of Technology

Gootenberg and Abudayyeh are the study’s senior authors, which appears currently in the journal Nature Biotechnology. The study’s lead authors are MIT technical associates Matthew Yarnall and Rohan Krajeski, former MIT graduate student Eleonora Ioannidi, and MIT graduate student Cian Schmitt-Ulms.

DNA insertion

The CRISPR-Cas9 gene editing system comprises a DNA-cutting enzyme known as Cas9 and a short RNA strand. This helps guide the enzyme to a particular area of the genome, thereby directing Cas9 where to make its split.

When Cas9 as well as the guide RNA targeting a disease gene are sent into cells, a particular cut is made in the genome, and the cells’ DNA repair processes tend to stick to the cut back together. This frequently deletes a genome’s small portion.

Also, if a DNA template is delivered, the cells have the potential to integrate a corrected copy into their genomes at the time of the repair process.

But this process needs cells to create double-stranded breaks in their DNA, which have the potential to cause chromosomal deletions or rearrangements that are detrimental to cells. One more limitation is that it just works in cells that are dividing since nondividing cells do not consist of active DNA repair processes.

The MIT team wished to develop a tool that could reduce a defective gene and substitute it with a new one without provoking any double-stranded DNA breaks. For this aim to be attained, they turned to a family of enzymes known as integrases, which viruses known as bacteriophages utilize to insert themselves into bacterial genomes.

For this study performed, the scientists concentrated on serine integrases, which have the potential to insert huge chunks of DNA, as big as 50,000 base pairs. Such enzymes target specific genome sequences called attachment sites, which function as so-called “landing pads.” When the researchers find the right landing pad in the host genome, they bind to it and combine their DNA payload.

In the previous work performed, researchers have found it hard to come up with such enzymes for human therapy since the landing pads are highly specific. Also, it is hard to reprogram integrases to target other sites.

The MIT group identified that integrating such enzymes with a CRISPR-Cas9 system that inserts the proper landing site would allow simple reprogramming of the strong insertion system.

The newly-developed tool named PASTE (Programmable Addition via Site-specific Targeting Elements) consists of a Cas9 enzyme that reduces at a specific genomic site, directed by a strand of RNA that fixes to that site.

This enables them to target any site present in the genome for insertion of the landing site, consisting of around 46 DNA base pairs. This insertion can be performed without initiating any double-stranded breaks by the addition of one DNA strand first through a fused reverse transcriptase, then its complementary strand.

As soon as the landing site seems to be incorporated, the integrase can come together and insert its much bigger DNA payload into the genome at that specific site.

We think that this is a large step toward achieving the dream of programmable insertion of DNA. It’s a technique that can be easily tailored both to the site that we want to integrate as well as the cargo.”

Jonathan Gootenberg, McGovern Fellow, Massachusetts Institute of Technology

Gene replacement

In this study performed, the scientists displayed that they could make use of PASTE to insert genes into various kinds of human cells, such as T cells, liver cells, and lymphoblasts (immature white blood cells).

The delivery system was tested along with 13 different payload genes, such as a few that could be therapeutically beneficial, and was able to insert them into nine diverse locations in the genome.

In such cells, the scientists were capable of inserting genes with a success rate varying from 5 to 60%. Also, this study provided very few undesired “indels” (insertions or deletions) at the sites of gene integration.

“We see very few indels, and because we’re not making double-stranded breaks, you don’t have to worry about chromosomal rearrangements or large-scale chromosome arm deletions,” stated Abudayyeh.

Also, the scientists illustrated that they could insert genes in so-called “humanized” livers present in mice. Livers in these mice comprise almost 70% human hepatocytes and PASTE successfully combined new genes into nearly 2.5% of these cells.

In this study, the DNA sequences that the scientists inserted were up to 36,000 base pairs long, but they trust even longer sequences could also be utilized. A human gene has the potential to range from a few hundred to more than two million base pairs.

Even though for therapeutic purposes, just the coding sequence of the protein requires to be used, considerably decreasing the DNA segment’s size that requires to be inserted into the genome.

“The ability to site-specifically make large genomic integrations is of huge value to both basic science and biotechnology studies. This toolset will, I anticipate, be very enabling for the research community,” stated Prashant Mali, a professor of bioengineering at the University of California at San Diego, who was not involved in the study.

Currently, scientists are additionally exploring the chance of using this tool as a possible approach to exchange the defective cystic fibrosis gene. Also, this method could be beneficial for treating blood diseases that are caused by faulty genes, like hemophilia and Huntington’s disease or G6PD deficiency, a neurological disorder that has been caused by a defective gene that consists of too many gene repeats.

Also, the scientists have made their genetic constructs available online for other researchers to use.

Gootenberg stated, “One of the fantastic things about engineering these molecular technologies is that people can build on them, develop and apply them in ways that maybe we didn’t think of or hadn't considered. It’s really great to be part of that emerging community.”