Prime editing in plain language

Prime editing installs a short new sequence that is written on the guide RNA, then copied into the genome by a reverse transcriptase after a nick. There is no separate double-stranded donor in the design that gave the method its name. This page is a research overview of that architecture: what the pegRNA contains, what PE2 and PE3 add, and which results are still only a hope. Nuclease Cas9 is the comparison point in how CRISPR-Cas9 editing works in research. Base editors, which substitute inside a deaminase window and do not write an arbitrary template, are a different concept in Cas9 nickases and base editing concepts.

Enzymes, guide RNA and verification reagents are catalogue classes in the molecular biology catalogue. The design specification goes with the quote request.

The molecule that carries the edit

A prime-editing guide RNA, the pegRNA, is an ordinary Cas9 guide with an extra 3-prime extension. In order along the RNA, the functional parts are the spacer, the scaffold the nickase binds, the reverse-transcription template and the primer-binding site at the 3-prime end. The spacer still has to match a site next to a PAM the nickase accepts. The extension is what nuclease Cas9 guides do not have. If a synthesis order loses the extension, you have ordered a nick, not an edit.

The editor protein in the original method is a Cas9 nickase fused to a reverse transcriptase. The nickase commonly used is the H840A class, which cuts the PAM-containing strand and leaves the other strand intact. That is the opposite nick from the D10A nickase used in many base editors. Swapping the mutant because both are called nickase-Cas9 points the reverse transcriptase at the wrong strand.

After the nick, the primer-binding site anneals to the freed end of the PAM strand. The reverse transcriptase copies the reverse-transcription template onto that end, producing a short DNA flap that contains the new sequence. The cell then has to keep that flap and discard the competing unedited flap. The edit is only real when that resolution goes the way you drew it. Much of the time, at many loci, it does not.

PE2, PE3 and the decision to nick twice

The first published fusion was later improved by mutations in the reverse transcriptase. That improved fusion, programmed by a pegRNA and without a second guide, is the PE2 arrangement. It is the cleaner starting comparison when you want to know what the pegRNA itself can do.

PE3 keeps PE2 and adds a simple guide RNA that nicks the non-edited strand at a nearby position. The second nick biases the cell toward using the edited strand as information. In practice it often raises the fraction of intended alleles and raises indels, because two nicks can be processed as a break. A common planning distance for that second nick is on the order of about 40 to 100 base pairs from the first nick, and the method paper or the design tool for your editor version is the authority for the interval you actually use. PE3b places the second guide so that it matches the edited sequence rather than the original one, which means the second nick is more likely after the edit exists. That is a concept for lowering the indel cost. It is not a guarantee, and it requires the second spacer to be designed against the new bases.

Later arrangements inhibit mismatch repair or change the pegRNA structure. They are further method classes. Do not import their efficiencies into a PE2 experiment because the word prime is shared. Record the fusion name, the pegRNA and whether a second nick was present.

No step in this design requires a donor plasmid or a single-stranded donor oligonucleotide. If you add one, you have left the method. Donor classes for true knock-ins that do use a template DNA are discussed in donor templates for a knock-in.

Lengths you may plan with, and then replace

The primer-binding site is often started in a range of roughly 8 to 15 nucleotides. The reverse-transcription template for a point substitution is often started long enough to encode the new base plus a short additional flap, commonly discussed around 10 to 16 nucleotides beyond the nick for a minimal change. An insertion lengthens the template by the insertion itself. These figures are planning ranges from the method literature so that a first design is not arbitrary. They are not an optimised protocol for your locus. Melting behaviour, sequence composition and the editor version move the useful lengths. Follow the paper or the design software that names your editor, and keep several pegRNA variants when the locus matters. One drawing is a hypothesis in the same sense as a nuclease guide.

The spacer is still about 20 nucleotides for a standard SpCas9 scaffold, and the PAM still has to sit so the nick falls at the position that editor expects, typically a few bases upstream of the PAM as for ordinary Cas9. A beautiful template attached to a spacer the protein will not bind does nothing. Design the spacer on a named assembly from Ensembl or the UCSC Genome Browser, and check the parent line if variants are plausible.

Mismatch repair is the quiet opponent. The cell can treat the newly written flap as the mistake and restore the original base. Some substitution types are more vulnerable than others. Strategies that add silent changes to evade that repair, or that dampen the repair pathway, belong to specific papers. Use them as published, with the controls those papers use, not as an informal extra mutation you forget to mention in the genotype.

What to do when the control locus stays silent

Run an unedited sister culture beside every prime-edited sample. If you have a pegRNA already shown to edit a control site in these cells, it tells you the editor and the delivery can work. It does not tell you the experimental pegRNA is folded correctly. PegRNAs are long. Their extensions can pair with the spacer or the scaffold and spoil both binding and priming. A failure at a new locus with success at a control locus is a reason to redesign the extension, not to increase dose without a limit.

If indels appear without the intended bases, look at the second nick first. A PE3 guide that cuts, while the reverse transcriptase does little, is a nickase experiment. If the intended bases appear and so do scars, report both. Checking whether a genome edit worked applies directly: a bulk trace that splits is a mixture, and a clone is the unit that can carry a single genotype claim.

If the editor construct is a plasmid that lingers, cutting and nicking continue. Transient delivery of protein and RNA changes that exposure. The delivery choice does not prove the sequence. It changes how long the machinery is present.

Failure modes that look like a bad sequencer

A pegRNA that mismatches the parent genome in the primer-binding site will prime poorly or prime in the wrong place. Check the parent sequence, not only the reference. A template that encodes the edit far from the nick asks the flap to do more work, and efficiency usually falls. Very long insertions move you toward other prime-editing variants or toward a donor method. Forcing them on a minimal PE2 design produces a low intended rate and a notebook full of explanations.

Unintended mutations inside the templated interval are a known class of product, because the reverse transcriptase and the subsequent repair are not a photocopy. Read the whole interval you templated, not only the one base you care about. Indels at the nick, especially under PE3, can sit on alleles that lack the intended edit. Counting intended reads and ignoring scar reads overstates success.

Contamination with a synthetic DNA version of the edit, if you used one as a control oligo, will impersonate prime editing in a PCR. Keep that control physically away from the genotyping, as with any edited amplicon. The discipline is the same as in PCR controls and contamination control.

Safety as an institutional decision

Prime editing changes genomic sequence. The nick is limited compared with a constitutive nuclease, and it is still an editing experiment under the biosafety and ethics approval for that cell type and that delivery. This page is not a therapeutic protocol and does not describe editing of human embryos or the germline. Reverse transcriptase fusions are research materials. Handling them, especially with viral delivery, is an institutional classification, not a detail this article can settle. A methods repository such as protocols.io can hold a laboratory's own written method. It does not replace that classification.

Long RNA in a warm, humid laboratory

PegRNAs are longer than standard guides and more prone to degradation and to being mislabelled as "the Cas9 guide". Store RNA as its own sheet requires, on ice while you handle it, and do not leave it in a warm room while a plasmid prep is finished. Humidity loosens labels on small tubes. The extension sequence should be in the file name, not only on a sticker. A power cut that warms a freezer of editor mRNA is a reason to quarantine the tube until a control edit works, not a reason to interpret a blank locus as biology.

Synthesis quality matters more as the RNA lengthens. Ask for the sequence you designed, including the scaffold, and check the paperwork on arrival. A truncated product will nick or will do nothing, and the cells will not tell you which half was missing.

What the enquiry needs

State that the experiment is prime editing, name the editor fusion class if you have chosen one, and include the spacer, the scaffold, the reverse-transcription template and the primer-binding site as separate fields. Say whether a second nick is part of the plan, what the cell type is, and whether delivery is plasmid, RNA or protein. Verification of the templated interval can be discussed via the CRISPR validation sequencing enquiry reference. Use that page as an enquiry reference for the method. It is an independent reference for the discussion, not a finished genotyping plan. The molecular biology pathway is the broader context. Ask whether a quotation is possible. A request that says only knock-in will be answered with the wrong reagent class.