CRISPR-Cas9 has been editing the human genome for the past decade. Although the types of edits have varied, an unsolved challenge has been the integration of large pieces of DNA without double-stranded DNA breaks. Now, a team from MIT describes a new tool called PASTE (programmable addition via site-specific targeting elements) which delivered genes as long as 36,000 DNA base pairs to several types of human cells (as well as to liver cells in mice). PASTE uses a CRISPR–Cas9 nickase fused to two enzymes—a reverse transcriptase and a serine integrase—for targeted genomic recruitment and integration of DNA.
The research is published in Nature Biotechnology, in the paper, “Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases.”
“It’s a new genetic way of potentially targeting these really hard to treat diseases,” said Omar Abudayyeh, PhD, a McGovern fellow at MIT’s McGovern Institute for Brain Research. “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.”
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. When they find the site in the host genome, they bind to it and integrate their DNA payload.
“Just like CRISPR, these integrases come from the ongoing battle between bacteria and the viruses that infect them,” said Jonathan Gootenberg, PhD, also a McGovern Fellow. “It speaks to how we can keep finding an abundance of interesting and useful new tools from these natural systems.”
In past work, scientists have found it challenging to develop these enzymes for human therapy because the attachment sites are very specific, and it’s 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 site would enable easy reprogramming of the powerful insertion system.
PASTE includes a Cas9 enzyme that cuts at a specific genomic site, guided by a strand of RNA that binds to that site. This allows them to target any site in the genome for insertion of the attachment site, which contains 46 DNA base pairs. This insertion can be done without introducing any double-stranded breaks by adding one DNA strand first via a fused reverse transcriptase, then its complementary strand.
Once the site is incorporated, the integrase can come along and insert its much larger DNA payload into the genome at that site.
“We think that this is a large step toward achieving the dream of programmable insertion of DNA,” Gootenberg said. “It’s a technique that can be easily tailored both to the site that we want to integrate as well as the cargo.”
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. 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 5–60%. This approach also yielded very few unwanted “indels” at the sites of gene integration.
More specifically, the authors noted that PASTE has editing efficiencies “similar to or exceeding those of homology-directed repair and non-homologous end joining-based methods, with activity in nondividing cells and in vivo with fewer detectable off-target events.”
“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.
The researchers also demonstrated that they could insert genes in “humanized” livers in mice. Livers in these mice consist of about 70% human hepatocytes, and PASTE successfully integrated new genes into about 2.5% of these cells.
The DNA sequences that the researchers inserted in this study were up to 36,000 base pairs long, but they believe even longer sequences could also be used. A human gene can range from a few hundred to more than two 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 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 diseases caused by faulty genes, such as hemophilia and G6PD deficiency, or Huntington’s disease, a neurological disorder caused by a defective gene that has too many gene repeats.
“One of the fantastic things about engineering these molecular technologies is that people can build on them, develop and apply them in ways that maybe we didn’t think of or hadn’t considered,” Gootenberg said. “It’s really great to be part of that emerging community.”