Building on the CRISPR gene-editing system, MIT researchers have designed a new tool that can snip out faulty genes and replace them with new ones in a safer and more efficient way.
Using this system, the researchers showed that they could deliver genes of up to 36,000 base pairs of DNA into several types of human cells, as well as into liver cells in mice. The new technology, known as PASTE, could hold promise for treating diseases caused by defective genes with a large number of mutations, such as cystic fibrosis.
It’s a new genetic way to target these really hard-to-treat diseases. We wanted to work toward what gene therapy was supposed to do in its original inception, which is to replace genes, not just correct individual mutations.”
Omar Abudayyeh is a McGovern Fellow at MIT’s McGovern Institute for Brain Research
The new tool combines the precise targeting of CRISPR-Cas9, a group of molecules originally derived from bacterial defense systems, with enzymes called complementarities, which viruses use to insert their genetic material into a bacterial genome.
“Just like CRISPR, these integral phrases come from the ongoing battle between bacteria and the viruses that infect them,” says Jonathan Guttenberg, also a colleague of McGovern’s. “It speaks to how we can continue to find an abundance of interesting and useful new tools from these natural systems.”
Gootenberg and Abudayyeh are the lead authors of the new study, which appears today in Nature Biotechnology. The study’s lead authors are MIT technical assistants Matthew Yarnal and Rohan Krajeski, former MIT graduate student Eleonora Iwanidi, and MIT graduate student Sian Schmidt-Olms.
The CRISPR-Cas9 gene editing system consists of a DNA-cutting enzyme called Cas9 and a short RNA strand that directs the enzyme to a specific region of the genome, and directs Cas9 where to cut it. When Cas9 and guide RNA targeting a disease gene are delivered to cells, a specific cut is made in the genome, and the cells’ DNA repair processes glue the pieces together, often deleting a small portion of the genome.
If a DNA template is also delivered, cells can incorporate a corrected copy into their genomes during the repair process. However, this process requires cells to make double-strand breaks in their DNA, which can cause chromosomal deletions or rearrangements that are harmful to the cells. Another limitation is that it only works in cells that are dividing, as non-dividing cells do not have active DNA repair processes.
The MIT team wanted to develop a tool that could cut out a faulty gene and replace it with a new one without causing any double-strand breaks in the DNA. To achieve this goal, they turned to a family of enzymes called integrases, which viruses called bacteriophages use to insert themselves into the bacterial genome.
In this study, the researchers focused on serine camels, which can insert huge chunks of DNA, up to 50,000 base pairs in size. These enzymes target specific genomic sequences known as attachment sites, which act as ‘landing platforms’. When they find the correct landing pad in the host’s genome, they attach to it and integrate their DNA payload.
In previous work, scientists have found it difficult to develop these enzymes for human therapy because the landing pads are so specific, and it is difficult to reprogram the integrins to target other sites. The MIT team realized that integrating these enzymes with the CRISPR-Cas9 system inserting the correct landing site would enable easy reprogramming of the robust insertion system.
The new tool, PASTE (Programmable Addition via Site-Specific Targeting Elements), involves a Cas9 enzyme that cuts at a specific genetic site, guided by a strand of RNA that binds to that site. This allows them to target any site in the genome to insert the landing site, which contains 46 DNA base pairs. This insertion can be made without introducing any double-stranded breaks by first adding a single DNA strand via an integrated reverse transcriptase, and then its complementary strand.
Once the landing site has been integrated, the integral can come in and insert the much larger DNA payload into the genome at that site.
“We think this is a huge step towards realizing the dream of programmable insertion of DNA,” Guttenberg says. “It’s a technology that can be easily tailored to both the site we want to integrate as well as the merchandise.”
In this study, the researchers showed that they could use PASTE to insert genes into several types of human cells, including liver cells, T cells, and lymphoblasts (immature white blood cells). They tested the delivery system using 13 different payload genes, including some that could be therapeutically useful, and managed to insert them at nine different locations in the genome.
Into these cells, the researchers were able to insert genes with a success rate of 5 to 60 percent. This approach also resulted in very few unwanted ‘indels’ (insertions or deletions) at gene integration sites.
“We see very few indels, and because we don’t do double-stranded breaks, we don’t have to worry about chromosomal rearrangements or extensive chromosome arm deletions,” says Abu Dayyeh.
The researchers also showed that they could insert the genes into “humanized” livers in mice. The livers of these mice consist of about 70 percent of human liver cells, and PASTE has successfully incorporated novel genes into about 2.5 percent of these cells.
The length of the DNA sequence the researchers entered in this study was up to 36,000 base pairs, but they believe that longer sequences could also be used. A human gene can range from a few hundred to more than two million base pairs, although for therapeutic purposes only protein-coding sequences should be used, which greatly reduces the size of the piece of DNA that must be inserted into the genome.
The researchers are now continuing to explore the possibility of using this tool as a possible way to replace the defective cystic fibrosis gene. This technology could also be useful in treating blood diseases caused by defective genes, such as hemophilia and G6PD deficiency, or Huntington’s disease, a neurological disorder caused by a defective gene that has too many redundant genes.
The researchers also made their genetic makeup available online for other scientists to use.
“One of the great things about engineering these molecular technologies is that people can build on them, develop them, and apply them in ways we may or may not have considered,” Guttenberg says. “It’s really cool to be a part of this budding community.”
The research was funded by a Postdoc Mobility Fellowship from the Swiss National Science Foundation, National Institutes of Health, McGovern Institute Neurotechnology Program, the K.Lisa Yang and Hock E. Tan Center for Molecular Therapies in Neuroscience, and G. Harold and Leila Y. Mathers Charitable Foundation, MIT John W. Jarve Seed Fund for Science Innovation, Impetus Grants, Cystic Fibrosis Foundation Pioneer Grant, Google Ventures, Fast Grants, and McGovern Institute.