Based on the CRISPR gene editing system, MIT researchers have designed a new tool that can delete defective genes and replace them with new ones, more safely and efficiently.
Using this system, researchers have shown that they can deliver genes of up to 36,000 base pairs of DNA to several types of human cells, as well as liver cells in mice. The new technique, known as PASTE, may hold promise for treating diseases caused by faulty genes with large numbers of mutations, such as cystic fibrosis.
“It’s a new genetic way to potentially target these really hard-to-treat diseases,” says Omar Abudayyeh, a McGovern Fellow at MIT’s McGovern Institute for Brain Research. “We wanted to work on what gene therapy was supposed to do in its original inception, which was to replace genes, not just fix individual mutations.”
The new tool combines the precise targeting of CRISPR-Cas9, a set of molecules originally derived from bacterial defense systems, with enzymes called integrases, which viruses use 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,” says Jonathan Gootenberg, also a McGovern Fellow. “It talks about how we can continue to find an abundance of interesting and useful new tools from these natural systems.”
Gootenberg and Abudayyeh are the senior authors of the new study, which appears today in natural biotechnology. The lead authors of the study are MIT technical colleagues Matthew Yarnall and Rohan Krajeski, former MIT graduate student Eleonora Ioannidi, and MIT graduate student Cian Schmitt-Ulms.
The CRISPR-Cas9 gene editing system consists of a DNA cutting enzyme called Cas9 and a short strand of RNA that guides the enzyme to a specific area of the genome, directing Cas9 where to make its cut. 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 cut together, often eliminating a small portion of the genome.
If a DNA template is also provided, cells can incorporate a correct 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 cells. Another limitation is that it only works in dividing cells, as non-dividing cells do not have active DNA repair processes.
The MIT team wanted to develop a tool that could delete a faulty gene and replace it with a new one without inducing any double-stranded DNA breaks. To achieve this, they turned to a family of enzymes called integrases, which viruses called bacteriophages use to insert themselves into bacterial genomes.
For this study, the researchers focused on serine integrases, which can insert huge pieces of DNA, up to 50,000 base pairs in size. These enzymes target specific sequences in the genome known as attachment sites, which function like “landing plates”. When they find the correct landing path in the host genome, they bind 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 very specific and it is difficult to reprogram the integrases to target other sites. The MIT team realized that combining these enzymes with a CRISPR-Cas9 system that inserts the correct landing site would allow for easy reprogramming of the powerful insertion system.
The new tool, PASTE (Programmable Addition via Site-specific Targeting Elements), includes a Cas9 enzyme that cuts at a specific genomic site, guided by an RNA strand that binds to that site. This allows them to target any site in the genome for landing site insertion, which contains 46 base pairs of DNA. This insertion can be performed without introducing double-strand breaks by first adding a DNA strand via a fused reverse transcriptase, then its complementary strand.
Once the landing site has been incorporated, integrase can arrive and insert its much larger DNA payload into the genome at that site.
“We think this is a big step toward realizing the dream of programmable DNA insertion,” says Gootenberg. “It’s a technique that can be easily adapted to both the site we want to integrate and the load.”
In this study, the researchers demonstrated that they can 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 with 13 different payload genes, including some that could be therapeutically useful, and were able to insert them into nine different locations in the genome.
In these cells, the researchers were able to insert genes with a success rate ranging from 5 to 60%. This approach also resulted in very few unwanted “indels” (insertions or deletions) at gene integration sites.
“We see very few indels, and because we’re not doing double-strand breaks, you don’t have to worry about chromosome rearrangements or large-scale chromosome arm deletions,” Abudayyeh says.
Researchers have also shown that they can insert genes into the ‘humanised’ livers of mice. The livers of these mice are approximately 70% human hepatocytes, and PASTE successfully integrated new genes into approximately 2.5% of these cells.
The DNA sequences that the researchers inserted into this study were up to 36,000 base pairs long, but they believe even longer sequences could also be used. A human gene can vary from a few hundred to more than 2 million base pairs, although for therapeutic purposes only the coding sequence of the protein needs to be used, drastically reducing the size of the DNA segment that needs to be inserted into the genome.
“The ability to perform large genomic integrations in a site-specific manner is of enormous value for both basic science and biotechnology studies. This toolset, I predict, will be very useful to the research community,” says Prashant Mali, a bioengineering professor at the University of California San Diego who was not involved in the study.
The researchers are now further exploring the possibility of using this tool as a possible way to replace the defective cystic fibrosis gene. This technique could also be useful for treating blood disorders caused by faulty genes, such as hemophilia and G6PD deficiency, or Huntington’s disease, a neurological disorder caused by a faulty gene that has too many gene repeats.
The researchers also made their genetic constructs 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 not have thought of or considered,” Gootenberg says. “It’s really great to be a part of that emerging community.”
The research was funded by a Swiss National Science Foundation Postdoc Mobility Fellowship, the US National Institutes of Health, the McGovern Institute Neurotechnology Program, the K. Lisa Yang and Hock E. Tan Center for Molecular Therapeutics in Neuroscience, the G. Harold and Leila Y. Mathers Charitable Foundation, MIT’s John W. Jarve Seed Fund for Science Innovation, Impetus Grants, Cystic Fibrosis Foundation Pioneer Grant, Google Ventures, Fast Grants, Harvey Family Foundation, and McGovern Institute.