In the prime editing system, the Cas9 has been modified. Termed a ‘Cas9 nickase’, it doesn’t cause a double-stranded break. Instead, it cuts just one strand of the DNA molecule. The Cas9 is also fused to a reverse transcriptase enzyme, which can synthesise DNA.
To manoeuvre the nickase to the desired location on the genome, the Cas9 binds to a guide RNA sequence
The RNA sequence several parts. One is the guide sequence, which can be made to compliment any area of the genome, and so directs the Cas9 into place. It also has the desired sequence ‘template’, which can be used to add to, or alter, the genome. The guide RNA also includes a primer sequence. This RNA molecule is the prime editing guide or 'pegRNA'.
Once the DNA has been cut by the nickase, the RNA primer in the pegRNA binds to the resulting DNA flap.
The reverse transcriptase then synthesises DNA, based on the RNA template. The edited sequence can be incorporated into the genome by the cell’s natural machinery.
The other, unedited, strand of DNA is repaired by the cell to match the new sequence. Alternatively, the unedited sequence is incorporated back into the genome leaving the site in its original state and the editing process can start again.
The system can be used to change a base of DNA to any other. This is an improvement on previous technologies, which could only make four of the potential 12 base substitutions. Or, it can be used to insert or delete a specific sequence of DNA, of varying length.
Unwanted and off-target effects have been shown to be relatively rare.
Dubbed ‘molecular word processors’, the technology was developed by a team of researchers at the Broad Institute of MIT and Harvard, led by Dr David Liu, who has been working on developing gene-editing technologies for over a decade.