Prime Editing
A new CRISPR genome-editing approach was developed recently by a team of scientists from the Broad Institute of MIT and Harvard. The revised system called ‘prime editing’ could correct up to 89% of known disease-causing genes.
The prime editing system combines a couple of the most important proteins in molecular biology — the CRISPR-Cas9 and reverse transcriptase. It’s capable of precisely editing human cells efficiently and flexibly. The research has been published in the journal Nature.
David Liu, the study’s senior author, said:
A major aspiration in the molecular life sciences is the ability to precisely make any change to the genome in any location. We think prime editing brings us closer to that goal. We’re not aware of another editing technology in mammalian cells that offers this level of versatility and precision with so few byproducts.
The First CRISPR
The first CRISPR system was made to edit the human genome. It worked using an enzyme called Cas9. A guide RNA would direct the Cas9 tool to the chosen section of the genome that needs editing. The Cas9 then cuts both strands of DNA in two places. In so doing, it removes a segment of DNA to replace the void with something else.
In this system, the Cas9 is guided to the problem zone to cut out troublesome genes that cause disease and replace them with more benign or beneficial bits of DNA. Sounds like the perfect plan, right? Well, it is, and it isn’t. There are risks involved in completely cutting out sections of DNA. Mistakes can easily happen when introducing the new replacement sections. Some studies suggest the modification can even lead to cancer. Or, the Cas9 edits the wrong section of DNA (an off-target edit), which can cause problems.
The Revised System
With the new prime editing system, the RNA still guides the Cas9 to the desired location. However, rather than cutting both strands of DNA, it cuts just one. The RNA in this system then guides the Cas9 as to what to insert into the genome. The Cas9 is coupled with a protein called reverse transcriptase. That protein translates the instructions from the RNA.
The scientists explain in a press release:
A new type of engineered guide RNA, called a pegRNA, directs the prime editor to its target site, where a modified Cas9 cuts one strand of the DNA. The pegRNA also contains additional RNA nucleotides encoding the new edited sequence. To transfer this information, the reverse transcriptase element reads the RNA extension and writes the corresponding DNA nucleotides into the target spot.
The result is a fully edited section of DNA, executed by a more precise edit and with less chance of off-target mutations.
Testing
The researchers demonstrated the prime editing’s ability to precisely correct gene variants with DNA strands of sickle-cell anemia and Tay-Sachs disease. With sickle-cell anemia, the system had to change one specific T in the DNA code to an A. With Tay-Sachs disease it had to remove four letters from a particular location. Both procedures were successful.
Liu said:
With prime editing, we can now directly correct the sickle-cell anemia mutation back to the normal sequence and remove the four extra DNA bases that cause Tay-Sachs disease, without cutting DNA entirely or needing DNA templates. The beauty of this system is that there are few restrictions on the edited sequence. Since the added nucleotides are specified by the pegRNA, they can be sequences that differ from the original strand by only one letter, that have additional or fewer letters, or that are various combinations of these changes.
Then, the team used the prime editing system to substitute all 12 different DNA letters individually. When that worked out, they used it to insert new DNA segments up to 44 letters long and delete portions up to 80 letters long. Those edit types also worked out.
Andrew Anzalone, first author of the study, said:
The versatility of prime editing quickly became apparent as we developed this technology. The fact that we could directly copy new genetic information into a target site was a revelation. We were really excited.
Now the team is testing the technique with the intention of optimizing the prime editing system. They plan to maximize the system’s efficiency in a variety of cell types, animals, and humans. The ultimate goal is to get it ready for clinical trials in humans to eventually use it for therapeutic applications.
