The CRISPR-Cas9 gene editing system can be used to remove, or even replace, specific portions of a genome using the DNA-cutting enzyme Cas9 along with guide RNA. However, inserting large DNA sequences to “correct” a faulty gene requires cells to make double-stranded breaks in their DNA, which can cause chromosomal deletions or rearrangements that are harmful to the cells. MIT researchers have designed a new CRISPR-Cas9-based tool that can remove defective genes and replace them with large sequences – up to 36,000 base pairs – without the need to induce any double-stranded DNA breaks, offering a safer and more efficient approach to potentially treating genetic diseases.
The new system, called Programmable Addition via Site-specific Targeting Elements (PASTE), combines the precise targeting of CRISPR-Cas9 with the genetic payload integration abilities of serine integrase, an enzyme used by bacteriophages to insert their own genetic material into bacterial genomes. Serine integrases are capable of inserting sections of DNA up to 50,000 base pairs in nature, and target specific genome sequences known as attachment sites to bind to before inserting their payload. While integrases are difficult to reprogram to target sites other than their natural attachment sites, by combining these enzymes with a CRISPR-Cas9 system, the researchers were able to insert correct attachment sites into human genomes. The system can insert the attachment site – which contains 46 DNA base pairs – to any site in the genome without introducing double-stranded breaks, by adding one DNA strand first via a fused reverse transcriptase, and then its complementary strand.
The team showed they could use PASTE to insert genes into several human cell types including liver cells, T cells and lymphoblasts. Thirteen different payload genes, including some with potential therapeutic use, were tested, and the researchers were able to insert these payloads into nine different locations in the genome. In these cells, PASTE integrated genes with a success rate ranging from 5 to 60% while yielding very few unwanted insertions or deletions (indels) at the sites of integration. The system was also used to insert genes into “humanized” livers in mice, containing about 70% human hepatocytes, in which PASTE successfully integrated new genes in about 2.5% of cells. The payloads integrated in this study ranged in size up to 36,000 base pairs, but the researchers believe that even larger sequences could be inserted using PASTE. This research was published in Nature Biotechnology.
“We think that this is a large step toward achieving the dream of programmable insertion of DNA,” said co-senior author Jonathan Gootenberg. “It’s a technique that can be easily tailored both to the site that we want to integrate as well as the cargo.”
The research team is now further exploring the potential use of this CRISPR-based tool as a way to replace the defective gene that causes cystic fibrosis. PASTE could also be useful to treat genetic blood diseases, like hemophilia and G6PD deficiency, or Huntington’s disease, which is caused by a gene with too many gene repeats. The researchers have made their genetic constructs available online for other scientists to use.
“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,” said Gootenberg. “It’s really a great way to be part of that emerging community.”