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

Researchers at the Massachusetts Institute of Technology (MIT) continue to enhance the CRISPR gene-editing system, creating a new tool that can remove damaged genes and replace them with new genes in an effective and safe method.

In an MIT news release, the Programmable Addition via Site-specific Targeting Elements (PASTE) system is capable of providing genes, up to 36,000 DNA base pairs long, to liver cells in mice and several human cell types.

“It’s a new genetic way of potentially targeting these really hard to treat diseases,” Omar Abudayyeh, a McGovern Fellow at MIT’s McGovern Institute for Brain Research and a senior author of the study, said in the MIT news release. “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.”

According to MIT, the new technique could prove to offer hope for the treatment of illnesses that are the result of defective or mutated genes, including cystic fibrosis. The researchers, according to the release, include former MIT graduate student Eleonora Ioannidi, MIT technical associates Matthew Yarnall and Rohan Krajeski, and MIT graduate student Cian Schmitt-Ulms. They published their findings in Nature Biotechnology, according to the release.

In addition to Abudayyeh, Jonathan Gootenberg, also a McGovern Fellow at the McGovern Institute for Brain Research at MIT, served as a senior author on the study, according to the MIT news release.

Moreover, MIT noted in the news release that PASTE pairs the precise targeting of CRISPR-Cas9 with enzymes that are known as integrases that are used by viruses to insert their genetic material into a bacterial genome. The researchers noted these integrases can be paired off with a CRISPR-Cas9 system that inserts the correct landing site, which can enable scientists to zero in on any location within the genome to insert the landing cells, which are comprised of 46 DNA base pairs.

The researchers, according to the MIT news release, noted that insertion can be completed without sparking double-stranded breaks by placing one DNA strand first by using a fused reverse transcriptase. They then can add a partner strand. After incorporating the landing site, the researchers noted in the news release that the integrase can place its DNA payload into the genome at the landing site.

“We think that this is a large step toward achieving the dream of programmable insertion of DNA,” Gootenberg said in the MIT news release. “It’s a technique that can be easily tailored both to the site that we want to integrate as well as the cargo.”

The system was tested by the researchers, according to MIT, who inserted genes into several human cell types, including T cells, lymphoblasts (immature white blood cells), and liver cells. The release also noted the system was demonstrated with 13 different payload genes, with some that could prove to have therapeutic uses, and researchers inserted them into several sites in the genome.

According to MIT, success varied from 5-to-60 percent when researchers inserted the genes into cells, and the effort also resulted in a couple of unwanted insertions, or deletions, at the integration site of the gene. The researchers also showed how they could insert genes into livers in mice and how PASTE could integrate new genes into about 2.5 percent of these cells.

“We see very few indels, and because we’re not making double-stranded breaks, you don’t have to worry about chromosomal rearrangements or large-scale chromosome arm deletions,” Abudayyeh said, according to MIT.

The researchers concluded that PASTE could be effective in the development of efficient gene therapies and could provide a method of replacing genes, rather than correcting individual mutations, according to the MIT news release. The release went onto note that PASTE could also be used to take aim at diseases that can prove hard to treat with more traditional methods.