CRISPR Plasmids: Prime Edit
Prime editing is a “search and replace” gene editing method in which Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) is fused to the C-terminus of Cas9 H840A nickase. The fusion enzyme is capable of installing targeted insertions, deletions, and all possible base-to-base conversions using a prime editing guide RNA (pegRNA). As with a typical gRNA, the pegRNA directs the nickase to the target site by homology to a genomic DNA locus. The longer pegRNA also encodes a primer binding site (PBS) and the desired edits on a RT template.
In the initial version of this tool, PE1 (depicted in the image below), the pegRNA directs the Cas9 nickase to the target sequence where it nicks the non-target strand and generates a 3’ flap. The 3’ flap binds to the primer binding site (PBS) of the pegRNA and the desired edit is incorporated into the DNA by reverse transcription. The edited DNA strand displaces the unedited 5’ flap and the resulting heteroduplex is resolved by the cell’s mismatch repair (MMR) system. Alternatively, the edited 3’ flap may be excised and the target sequence will remain unchanged but available as a substrate for another round of prime editing.
Further improvements engineered into the prime editing components help to optimize and improve the efficiency of this method. In the PE2 system, five mutations were introduced into the RT enzyme (D200N/L603W/T330P/T306K/W313F) to increase activity, enhance binding between the template and PBS, increase processivity, and improve thermostability. PE3 uses the PE2 Cas9 nickase-pentamutant RT fusion enzyme and pegRNA plus an additional simple sgRNA, which directs the Cas9 nickase to nick the unedited strand at a nearby site. The newly edited strand is then favored as the template for repair during heteroduplex resolution. The process of double nicking, however, increases indel formation slightly. Designing the sgRNA with a spacer that only binds the edited strand, as in the PE3b system, guides nicking of the unedited strand only after the edit has occurred.
Note: PE2, PE3, and PE3b systems all use the PE2 enzyme. In the PE3 and PE3b systems an sgRNA targeting the opposite strand is added.
Plasmids are available for expression in mammalian systems, bacteria, plants, and Drosophila.