New CRISPR-based tool can remove faulty genes and replace them with new ones

Based on the CRISPR gene editing system, MIT researchers have designed a new tool that can cut out defective 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 as long as 36,000 DNA base pairs to several types of human cells as well as to mouse liver cells. The new technique, known as PASTE, may hold promise for treating diseases caused by faulty genes with a large number of mutations, such as cystic fibrosis.

It’s a new genetic way to potentially target 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, a McGovern Fellow at MIT’s McGovern Institute for Brain Research

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 own genetic material into a bacterial genome.

“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 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 Nature biotechnology. The lead authors of the study 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 consists of a DNA-cutting enzyme called Cas9 and a short strand of RNA that directs the enzyme to a specific region of the genome, directing Cas9 where to cut. When Cas9 and the guide RNA targeting a disease gene are delivered into cells, a specific cut is made in the genome, and the cells’ DNA repair processes glue the cut back together, often erasing a small portion of the genome.

If a DNA template is also provided, 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 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 cut out a faulty gene and replace it with a new one without inducing DNA double-strand breaks. To achieve this goal, they turned to a family of enzymes called integrases that viruses called bacteriophages use to insert themselves into bacterial genomes.

For this study, the researchers focused on serine integrases, which can insert huge chunks of DNA as large as 50,000 base pairs. These enzymes target specific genome sequences known as attachment sites, which act as “landing pads”. When they find the correct landing pad in the host genome, they bind to it and integrate their DNA payload.

In previous work, researchers have found it challenging to develop these enzymes for human therapy because the landing pads 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 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 location in the genome for insertion of the landing site, which contains 46 DNA base pairs. This insertion can be performed without introducing double-strand breaks by first adding one DNA strand via a fused reverse transcriptase, then its complementary strand.

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

“We believe this is a big step towards achieving the dream of programmable insertion of DNA,” says Gootenberg. “It’s a technique that can easily be tailored both to the place we want to integrate, as well as to the cargo.”

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 into nine different locations in the genome.

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

“We see very few indels, and because we don’t make double-strand breaks, you don’t have to worry about chromosome rearrangements or large-scale chromosome arm deletions,” says Abudayyeh.

The researchers also showed that they could insert genes into “humanized” mouse livers. Livers in 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 that the researchers inserted in this study were up to 36,000 base pairs long, but they believe that even longer sequences could also 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 must be inserted into the genome.

The researchers are now further investigating the possibility of using this tool as a possible way to replace the defective cystic fibrosis gene. This technique may also be useful for treating blood disorders 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 repeats.

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

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

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

Source:

Massachusetts Institute of Technology

Journal reference:

Yarnall, MTN, et al. (2022) Drag-and-drop genome insertion of large sequences without double-stranded DNA cleavage using CRISPR-directed integrases. Nature biotechnology. doi.org/10.1038/s41587-022-01527-4.

New CRISPR-based tool can remove faulty genes and replace them with new ones

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