Prime editing is a gene-editing technology that functions as a precise “search-and-replace” tool for the genome. First described in 2019, it can correct many genetic mutations responsible for human diseases. Its mechanism allows for targeted insertions, deletions, and all twelve possible base-for-base substitutions. This technology offers high precision and operates without creating double-strand breaks in the DNA, improving its safety profile and broadening the scope of genetic medicine.
Key Molecular Components
At the core of prime editing is the prime editor (PE) protein, a large molecule created by fusing two distinct proteins. One part is a Cas9 “nickase.” Unlike the original CRISPR-Cas9 system that cuts both DNA strands, this engineered version only “nicks,” or cuts, a single strand, which reduces the frequency of unintended mutations.
The second component is a reverse transcriptase (RT), an enzyme that synthesizes DNA using an RNA molecule as a template. The initial prime editor system incorporated the Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase. This RT component is responsible for writing the new genetic information into the genome.
Guidance is provided by the prime editing guide RNA, or pegRNA. The pegRNA is an engineered RNA molecule that directs the entire process. It contains a guide sequence that is complementary to the target DNA sequence, allowing the prime editor protein to locate the precise spot in the genome.
The pegRNA also carries the blueprint for the desired genetic alteration. It includes a primer binding site (PBS) and a reverse transcriptase template (RTT). The PBS allows the nicked DNA strand to attach to the pegRNA, while the RTT contains the new sequence of genetic code that the reverse transcriptase will use as a template.
The Cellular Mechanism
The prime editing process begins with the pegRNA guiding the prime editor protein complex to a designated location in the cell’s nucleus. The guide sequence of the pegRNA hybridizes with the corresponding DNA sequence, ensuring the machinery is positioned correctly. This targeting is the foundational step for the edit to occur at the intended genomic locus.
Once anchored to the target DNA, the Cas9 nickase component makes a precise cut on one of the two DNA strands. This “nick” creates a starting point for the editing process. The free 3′ end of the nicked DNA strand then displaces the corresponding strand and binds to the primer binding site on the pegRNA.
With the nicked DNA strand bound to the pegRNA, the reverse transcriptase enzyme becomes active. It uses the RNA template provided by the pegRNA to synthesize a new segment of DNA that includes the desired genetic edit. This newly synthesized DNA strand is incorporated into the nicked site, creating a heteroduplex with one edited and one original strand.
The final step involves the cell’s DNA mismatch repair (MMR) machinery. This natural cellular process recognizes the mismatch between the edited and unedited strands. The cell’s repair systems then use the newly synthesized, edited strand as the template to repair the other strand, making the change permanent.
Executing a Prime Editing Experiment
A primary stage of a prime editing experiment is designing the pegRNA. Scientists must craft a guide sequence unique enough to direct the editor to a specific genomic location and avoid off-target effects. The reverse transcriptase template must also accurately encode the desired genetic change while promoting efficient reverse transcription.
Once designed and synthesized, the pegRNA and prime editor protein must be delivered into target cells. One technique is transfection, where lipids or nanoparticles carry the editing components across the cell’s membrane. This method is frequently used for cells grown in a laboratory.
Another delivery method is electroporation, which applies an electrical pulse to create temporary pores in cell membranes for the components to enter. After delivery, the cells are incubated to allow the editing process to occur.
Confirming a Successful Edit
After allowing time for the editing process, verification is performed through DNA sequencing. Scientists isolate genomic DNA from the treated cells. They then use sequencing technologies to read the genetic code at the specific DNA region targeted for modification.
Sequencing data confirms if the intended change was incorporated into the DNA. It also allows researchers to assess the efficiency of the edit by revealing the percentage of cells with the desired genetic modification.
Sequencing is also used to evaluate the accuracy of the prime editing event. Scientists analyze the data to check for unintended edits at the target site, such as small insertions or deletions (indels). This analysis of on-target accuracy and the rate of indels measures the experiment’s precision.