New CRISPR-based tool inserts large sequences of DNA at desired locations in cells

CAMBRIDGE, MA — Building on the CRISPR gene-editing system, MIT researchers have developed a new tool that can excise 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 up to 36,000 base pairs of DNA in length to several types of human cells, as well as to liver cells in mice. The new technique, called PASTE, could show 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 potentially target these really difficult-to-treat diseases,” says Omar Abudayyeh, a McGovern Fellow at MIT’s McGovern Institute for Brain Research. “We wanted to work towards what gene therapy was originally intended to do, which is to replace genes and not just correct individual mutations.”

The new tool combines the precise targeting of CRISPR-Cas9, a series of molecules originally derived from bacterial defense systems, with enzymes called integrases, which viruses use to insert their own 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 speaks to how we can always find a wealth of interesting and useful new tools from these natural systems.”

Gootenberg and Abudayyeh are the lead authors of the new study, which is published today in natural biotechnology. The lead authors of the study are MIT techs Matthew Yarnall and Rohan Krajeski, former MIT graduate student Eleonora Ioannidi, and MIT graduate student Cian Schmitt-Ulms.

DNA insert

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, telling Cas9 where to make its cut. When Cas9 and the guide RNA that targets a disease gene are introduced into cells, a specific cut is made in the genome, and the cells’ DNA repair processes glue the cut back together, often deleting a small portion of the genome.

If a DNA template is also supplied, the cells can incorporate a corrected copy into their genome during the repair process. However, this process requires cells to make double-strand breaks in their DNA, which can result in chromosomal deletions or rearrangements that are harmful to cells. Another limitation is that it only works in dividing cells since non-dividing cells do not have active DNA repair processes.

The MIT team wanted to develop a tool that could cut out a defective gene and replace it with a new one without causing double-stranded DNA breaks. To achieve this goal, they turned to a family of enzymes called integrases that viruses use, called bacteriophages, to insert themselves into bacterial genomes.

For this study, the researchers focused on serine integrases, which can insert huge chunks of DNA of up to 50,000 base pairs. These enzymes target specific genomic sequences known as binding sites, which act as “landing pads”. When they find the right landing spot 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 sites are very specific and it is difficult to reprogram 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 the powerful delivery system to be easily reprogrammed.

The new PASTE (Programmable Addition via Site-specific Targeting Elements) tool contains 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 insertion of the landing site, which contains 46 base pairs of DNA. This insertion can be done without introducing double-strand breaks by first adding a DNA strand via a fused reverse transcriptase and then adding its complementary strand.

Once the landing site is built in, the integrase can come along and insert its much larger DNA payload at that point in the genome.

“We think this is a big step towards realizing the dream of programmable DNA insertion,” says Gootenberg. “It’s a technique that can easily be adapted both to the place we want to integrate and to the load.”

gene replacement

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 with 13 different payload genes, including some that could be therapeutically useful, and were able to insert them at nine different sites in the genome.

The researchers were able to insert genes into these cells with a success rate of 5 to 60 percent. This approach also resulted in very few unwanted “indels” (insertions or deletions) at the sites of gene integration.

“We see very few indels, and since we don’t do double-strand breaks, you don’t have to worry about chromosomal rearrangements or large-scale deletions of chromosome arms,” ​​says Abudayyeh.

The researchers also showed that they could insert genes into “humanized” mouse livers. The livers of these mice are made up of about 70 percent human hepatocytes, and PASTE successfully integrated new genes into about 2.5 percent of these cells.

The DNA sequences the researchers inserted into this study were up to 36,000 base pairs long, but they believe even longer sequences 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 the protein needs to be used, drastically reducing the size of the DNA segment that needs to be inserted into the genome.

The researchers are now further investigating the possibility of using this tool as a potential way to replace the defective cystic fibrosis gene. This technique could also be useful in treating blood disorders caused by faulty genes, such as hemophilia and G6PD deficiency, or Huntington’s disease, a neurological disorder caused by a defective gene with too many gene repeats.

The researchers have also made their genetic constructs available online for other scientists to use.

“One of the fantastic things about constructing these molecular technologies is that people can build on them, develop them, and apply them in ways that we might not have thought of or considered,” says Gootenberg. “It’s really great to be part of this thriving community.”

The research was funded by a postdoctoral mobility grant from the Swiss National Science Foundation, the 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, the MIT John W. Jarve Seed Fund for Science Innovation, Impetus Grants, a Pioneer Grant from the Cystic Fibrosis Foundation, Google Ventures, Fast Grants, and the McGovern Institute.

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