New CRISPR-based tool inserts large DNA sequences into cells at desired locations | MIT News

Building on the CRISPR gene-editing system, MIT researchers have designed a new tool that can safely and efficiently delete faulty genes and replace them with new ones.

Using this system, the researchers demonstrated that they could 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 defective genes with multiple mutations, such as cystic fibrosis.

“It’s a new genetic way to target these diseases that are very difficult to treat,” said Omar Abudaih, a McGovern Fellow at MIT’s McGovern Institute for Brain Research. “We wanted to do what gene therapy was originally supposed to do, which was to replace genes, not correct individual mutations.”

The new tool targets CRISPR-Cas9, a collection of molecules originally derived from the bacterial immune system, along with enzymes called integrases, which viruses use to insert their own genetic material into bacterial genomes.

“Like CRISPR, these combinations come from the ongoing battle between bacteria and viruses,” says McGovern’s Jonathan Gottenberg. It discusses how we can derive a wealth of interesting and useful new tools from these natural systems.

Gothenburg and Abudayeh are senior authors of the new study, which appears today in Nature Biotechnology. The study’s lead authors are MIT Technical Associates Matthew Yarnall and Rohan Krajeski, former MIT graduate student Eleora Ioannidi, and MIT graduate student Cian Schmitt-Ulms.

DNA insertion

The CRISPR-Cas9 gene editing system uses a DNA-cutting enzyme called Cas9 and a short RNA strand that directs the enzyme to a specific location in the genome, directing where Cas9 will make the cut. CAS9 and the gene that targets the disease gene is a specific piece of the genome that is made by splicing and is often spliced ​​together, usually a part of the genome.

If the DNA template is also provided, the cells can incorporate the corrected copy into the genome through the repair process. But this process requires cells to make double-strand breaks in their DNA, causing chromosomal deletions or rearrangements that are harmful to cells. Another limitation is that it only works in dividing cells, because non-dividing cells do not have active DNA repair processes.

The MIT team wanted to create a device that could cut out a defective gene and replace it with a new one without causing any double-stranded DNA breaks. To achieve this goal, viruses called bacteriophages turn to a family of enzymes called to insert themselves into the bacterial genome.

For this study, the researchers focused on serine integrases, which integrate large stretches of DNA of up to 50,000 base pairs. These enzymes target specific genomic sequences known as attachment sites, which act as “landing pads”. When they find the right landing pad in the host’s genome, they bind to it and integrate their DNA cargo.

In previous work, scientists have found it challenging to develop these enzymes for human therapy because the binding sites are very different and it is difficult to reengineer combinations to target other sites. The MIT team realized that by combining these enzymes with the CRISPR-Cas9 system, it could simplify the powerful insertion system if it inserted the correct landing site.

The new tool involves PASTE (Programmed Addition of Site-Specific Targeting Elements) that cuts at a specific genomic site, guided by the RNA that binds to the site, with the Cas9 enzyme. This allows them to target any site in the genome to insert the landing site, which contains 46 DNA base pairs. A single DNA strand can be inserted without introducing any double-strand breaks by first adding it through the reverse transcriptase.

Once the landing site is integrated, the integrator can come along and insert a much larger DNA load into the genome at that location.

“We think this is a big step toward realizing the dream of programming DNA,” Gutenberg said. It’s a method that easily adapts to both the site and the load we want to integrate.

Gene replacement

In this study, the researchers used PASTE to insert genes into a variety of human cells and showed that they increased liver cells, T cells, and lymphoblasts (immature white blood cells). They tested the delivery system with 13 different loading genes, including some potentially useful for medicine, 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 5 to 60 percent. This approach resulted in very few unwanted “indels” (insertions or deletions) at 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 chromosomal rearrangements or large-scale chromosomal manual deletions,” Abudai says.

The researchers have shown that they can insert genes into “artificial” livers in mice. The livers in these mice contained 70 percent of human hepatocytes, and PASTE successfully integrated new genes into 2.5 percent of these cells.

Although the DNA sequences used in this study were up to 36,000 base pairs long, the researchers believe that longer sequences could be used. The human genome can range from a few hundred to more than 2 million base pairs, although for therapeutic purposes only protein sequencing must be used, greatly reducing the amount of DNA that enters the genome.

“The ability to perform site-specific large-scale genomic synthesis is of great value to basic science and biotechnology studies,” said Prashant Mali, a professor of bioengineering at the University of California, San Diego, who was not involved in the study.

The researchers are further studying the possibility of using this device to replace the defective cystic fibrosis gene. This approach could be useful in treating blood disorders caused by faulty genes, such as hemophilia and G6PD deficiency, or Huntington’s disease, a neurological disorder characterized by multiple gene duplications.

The researchers also presented their genetic constructs Online for other scientists to use.

“One of the amazing things about these molecular technologies is that people can build on them, develop them, and use them in ways that we didn’t think of or think about,” Gutenberg said. “It’s 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, K. Lisa Yang and Hock E. Tan in Molecular Therapeutics in Neuroscience, 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, Rapid Grant, Harvey Family Foundation, and the McGovern Institute.