Prime editing is a 'search-and-replace' genome editing technology in molecular biology by which the genome of living organisms may be modified. The technology directly writes new genetic information into a targeted DNA site. The most basic prime editor uses a fusion protein, consisting of a catalytically-impaired programmable endonuclease linked to an engineered reverse transcriptase, and a prime editing guide RNA (pegRNA), capable of identifying the target site and providing the new genetic information to replace the target DNA nucleotides. It mediates targeted insertions, deletions, and base-to-base conversions without the need for double strand breaks (DSBs) or donor DNA templates.
The technology has received mainstream press attention due to its potential uses in medical genetics. It utilizes methodologies similar to precursor genome editing technologies, including CRISPR/Cas9 and Base editing.
Prime editing has been used on some animal models of genetic disease, in various plants, and in bacteria. The first human trials for happened in 2024, with both patients enlisted being effectively cured of chronic granulomatous disease.
Components
Cas9 prime editors
Major components
Prime editing involves three major components:
Prime editing guide RNA (pegRNA): capable of (i) identifying the target nucleotide sequence to be edited, and (ii) encoding new genetic information that replaces the targeted sequence. The pegRNA consists of an extended single guide RNA (sgRNA) containing a primer binding site (PBS) and a reverse transcriptase (RT) template sequence. During genome editing, the primer binding site allows the 3' end of the nicked DNA strand to hybridize to the pegRNA, while the RT template serves as a template for the synthesis of edited genetic information. Cas9 nickase: With the exclusion of certain variants, all Cas9 prime editors use a nickase. Normally, Cas9 is a nuclease, slicing DNA in half to make blunt-ended double-stranded breaks. The protein uses two domains to do this. The HNH domain cleaves the target strand, the strand of DNA bound to guide RNA, and the RuvC domain cleaves the non-target strand, the target strand's compliment. By swapping the protein's 840th amino acid from a histidine to an alanine, the HNH domain is prevented from cutting. This generates a H840A nickase which can only make a single-stranded break (a nick) in the non-target strand. Most prime editors use a H840A nickase, but inverse prime editors take an opposite approach, instead using a D10A nickase. Here, the 10th amino acid is changed from aspartic acid to alanine. This prevents RuvC from functioning but preserves the HNH domain. Reverse transcriptase: an enzyme that synthesizes DNA from a single-stranded RNA template.
Accessory components Certain editors have various accessory components:
sgRNA: Small RNA protector protein (La): circRNA: MCP: Rep-X: Cys4: T2A:
Cas12 prime editors
Main components Cas12 prime editing involves five major components:
crRNA: Cas12a nickase or nuclease: Reverse transcriptase: circRNA: MCP:
Accessory components
additional crRNA:
OMEGA prime editors
Editing mechanism
Original (used by PE1 and 2) Genomic editing takes place by transfecting cells with the pegRNA and the fusion protein. Transfection is often accomplished by introducing vectors into a cell. Once internalized, the fusion protein nicks the target DNA sequence, exposing a 3'-hydroxyl group that can be used to initiate (prime) the reverse transcription of the RT template portion of the pegRNA. This results in a branched intermediate that contains two DNA flaps: a 3' flap that contains the newly synthesized (edited) sequence, and a 5' flap that contains the dispensable, unedited DNA sequence. The 5' flap is then cleaved by structure-specific endonucleases or 5' exonucleases. This process allows 3' flap ligation, and creates a heteroduplex DNA composed of one edited strand and one unedited strand. The reannealed double stranded DNA contains nucleotide mismatches at the location where editing took place. In order to correct the mismatches, the cells exploit the intrinsic mismatch repair (MMR) mechanism, with two possible outcomes: (i) the information in the edited strand is copied into the complementary strand, permanently installing the edit; (ii) the original nucleotides are re-incorporated into the edited strand, excluding the edit.
PE3 strategy Despite its increased efficacy, the edit installed by PE2 might still be removed by mismatch repair of the edited strand. To counter this, the Prime Editor 3 strategy comes with an extra component borrowed from base editing. In addition to the editor itself, PE3 uses a standard Cas9 nickase to exploit DNA repair systems by targeting the opposite non-edited strand. Following the nick on the non-edited strand, DNA repair will use the edited strand as a template, consequentially improving the odds of success.
However, there are drawbacks to this system as the unaltered strand can be nicked prematurely and lead to additional undesired indels.
Paired prime editing HOPE strategy: use two complimentary guide RNA pairs to increase chances of edit inclusion (just like Rin and Len guys!)
PE4 and 5 strategies Prime editor 4 utilizes the same machinery as PE2, but also includes a plasmid that encodes for dominant negative MMR protein MLH1. Dominant negative MLH1 is able to essentially knock out endogenous MLH1 by inhibition, thereby reducing cellular MMR response and increasing prime editing efficiency.
Prime editor 5 utilizes the same machinery as PE3, but also includes a plasmid that encodes for dominant negative MLH1. Like PE4, this allows for a knockdown of endogenous MMR response, increasing the efficiency of prime editing.
Twin prime editing The "twin prime editing" (twinPE) mechanism reported in 2021 allows editing large sequences of DNA – sequences as large as genes – which addresses the method's key drawback. It uses a prime editor protein and two prime editing guide RNAs.
PASTE Drag and drop using an Integrase
PASSIGE Performs way better than PASTE
Nucease prime editing
PEDAR
PETI
pegRNA improvements and modifications
Engineered pegRNA (epegRNA) Prime editing efficiency can be increased with the use of engineered pegRNAs (epegRNAs). One common issue with traditional pegRNAs is degradation of the 3' end by exonucleases. While the 5' end is protected by protein, the 3' end is fully exposed. If targeted by an exonuclease, the RT template and primer binding sequence could partially or fully vanish. The nickase (or nuclease) will make the right cut, but reverse transcription either won't happen or will create an incomplete product. Degraded prime editors compete with functional ones when targeting the same location, thus leading to decreased PE efficiency. epegRNAs have a structured RNA motif added to their 3' end to prevent degradation.
Two RNA pseudoknots were tested. One was a modified prequeosine1-1 riboswitch aptamer (evopreQ1), while the other was from the Moloney murine leukemia virus (referred by the paper as mpknot). mpknot was chosen because at the time, prime editors exclusively used a MMLV reverse transcriptase and this motif may have helped with recruiting it. evopreQ1 chosen because it was smaller than the first (only 42 nucleotides) and would minimize unintentional RNA secondary structures from interfering with editor performance. Choosing this would be more cost effective, as shorter RNA strands are easier to synthesize.
Xrn1 resistant pegRNA (xr-pegRNA) Xr-pegRNA is similar to epegRNA in that it adds an additional RNA motif after the primer binding sequence. In this case, these are Xrn1-resistant RNAs (xrRNAs), a group of conserved knot-like structures found in flaviviruses. They impede viral RNA degradation and are highly resistant to unfolding. When testing xrRNAs from five different viruses (the Murray Valley encephalitis virus, West Nile virus, Dengue virus, Yellow fever virus, and Zika virus), researchers found that they improved the efficiency of both PE2 and PE3 strategies while producing around the same or less indels as traditional pegRNA. This finding was consistent across different genes and cell types. Though using a xrRNA-PE3 strategy generally improved efficiency, there were a few sites where xrPE2 barely differed from xrPE3. In terms of which xrRNA motif worked best, the exact answer varied by target. Granted that, the Zika motif generally functioned well for both PE2 and PE3 strategies.
Compared to epegRNA, xr-pegRNA has a similar edit-to-indel ratio and improvement in efficiency. However, unlike the RNA pseudoknot from the Moloney murine leukemia virus (mpknot), xr-pegRNA doesn't need an RNA linker to ensure structural stability. This is similar to the evopreQ1 motif, though even that sometimes requires one. Because the linker isn't necessary, using xr-pegRNA would offer better predictability and could prove more convenient.
Addition of an RNA G-quadruplex Much like epegRNA and xr-pegRNA, this modification involves adding a 3' RNA motif to prevent the RT template and primer binding site from degrading. Though, in contrast with the first two (which add a RNA pseudoknot), this attaches a G-quadruplex, a completely different structure. These are composed of one or multiple four-guanine rings stabilized by a monovalent cation (an ion with a single positive charge like sodium or potassium).
To keep the pegRNA as short as possible, researchers tested small G-quadruplexes from three different sources: MT3, TERRA, and NRas. Overall, the quadruplex from NRAS provided the greatest benefit, while the one from TERRA had an inconsistent performance. Regardless of which quadruplex was used, the RT template and primer binding site lengths had no effect on the increased efficiency. Furthermore, at the sites tested, there was no significant difference in off-target effects between traditional and G-quadruplex pegRNA. The modification induced a higher amount of indels than regular pegRNA, but this was generally proportional to the increased editing level. Comparing the G-quadruplexes to the evopreQ1 motif from epegRNA, each performed around the same. This is notable, as all three G-quadruplexes were shorter than the motifs used in both xr-pegRNA and epegRNA (mpknot and evopreQ1).
Split pegRNA with a MS2 stem-loop aptamer
Engineered non-canonical pegRNA (npegRNA)
Protein modifications in animals Prime editing was developed in the lab of David R. Liu at the Broad Institute and disclosed in Anzalone et al. (2019). Since then prime editing and the research that produced it have received widespread scientific acclaim, being called "revolutionary" and an important part of the future of editing. During the development of this technology, several modifications were done to the components, in order to increase its effectiveness.
Prime editor 1 (PE1) Prime editor 1 was a prototype designed to have the minimum components necessary. It consisted of a wild-type Moloney murine leukemia virus (MMLV) reverse transcriptase attached to Cas9 H840A nickase by a flexible protein linker. Originally, the linker was too short and the reverse transcriptase was unable to do its job. However, extending it to 8-15 bases led to detectable editing. During development, the linker was attached to either the C- or N-terminal of the Cas9 protein. Linkage the C terminal proved more efficient, so it was chosen as the final design. Detectable editing efficiencies were observed.
Prime editor 2 (PE2)