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Prime editing is derived from CRISPR, but more accurately and no byproduct (don't need to break DNA helix, no additional mutation).

David Liu developed a prime editing machine called base editor (inspired by CRISPR cas9). It has higher precision compared to CRISPR but a smaller range of DNA is targeted. Prime editing changes the targeted DNA chemically, rearranging it from the atom level.

Prime editing is still in a test level for human but it has huge potential to treat genetic diseases. Most genetic diseases are caused by point mutation and prime editing made to correct these point mutations.

Prime editing uses a base editor to target one specific DNA sequence, however, DNA sequence comes in pairs. If one DNA sequence is changed they would be unmatched. The cell could change either DNA sequence to correct it. In order for the cell to perform the wanted result base editor need to be used to change the paired DNA sequence slightly.

Components
Components of prime editing Prime editing involves three major components:


 * A 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.
 * A fusion protein consisting of a Cas9 H840A nickase fused to a Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase.
 * Cas9 H840A nickase: the Cas9 enzyme contains two nuclease domains that can cleave DNA sequences, a RuvC domain that cleaves the non-target strand and a HNH domain that cleaves the target strand. The introduction of a H840A substitution in Cas9, through which the 840º amino acid histidine is replaced by an alanine, inactivates the HNH domain. With only the RuvC functioning domain, the catalytically impaired Cas9 introduces a single strand nick, hence the name nickase.
 * M-MLV reverse transcriptase: an enzyme that synthesizes DNA from a single-stranded RNA template.
 * A single guide RNA (sgRNA) that directs the Cas9 H840A nickase portion of the fusion protein to nick the non-edited DNA strand.

Mechanism
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 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.

Development process
During the development of this technology, several modifications were done to the components, in order to increase its effectiveness.

Prime editor 1
In the first system, a wild-type Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase was fused to the Cas9 H840A nickase C-terminus. Detectable editing efficiencies were observed.

Prime editor 2
In order to enhance DNA-RNA affinity, enzyme processivity, and thermostability, five amino acid substitutions were incorporated into the M-MLV reverse transcriptase. The mutant M-MLV RT was then incorporated into PE1 to give rise to (Cas9 (H840A)-M-MLV RT(D200N/L603W/T330P/T306K/W313F)). Efficiency improvement was observed over PE1. PE3 system for prime editing

Prime editor 3
Despite its increased efficacy, the edit inserted by PE2 might still be removed due to DNA mismatch repair of the edited strand. To avoid this problem during DNA heteroduplex resolution, an additional single guide RNA (sgRNA) is introduced. This sgRNA is designed to match the edited sequence introduced by the pegRNA, but not the original allele. It directs the Cas9 nickase portion of the fusion protein to nick the unedited strand at a nearby site, opposite to the original nick. Nicking the non-edited strand causes the cell’s natural repair system to copy the information in the edited strand to the complementary strand, permanently installing the edit.

Implications
Although additional research is required to improve the efficiency of prime editing, the technology offers promising scientific improvements over other gene editing tools. The prime editing technology has the potential to correct the vast majority of pathogenic alleles that cause genetic diseases, as it can repair insertions, deletions, and nucleotide substitutions.

Advantages
The prime editing tool offers advantages over traditional gene editing technologies. CRISPR/Cas9 edits rely on non-homologous end joining (NHEJ) or homology-directed repair (HDR) to fix DNA breaks, while the prime editing system employs DNA mismatch repair. This is an important feature of this technology given that DNA repair mechanisms such as NHEJ and HDR, generate unwanted, random insertions or deletions (INDELs) byproducts which complicate the retrieval of cells carrying the correct edit.

The prime system introduces single-stranded DNA breaks instead of the double-stranded DNA breaks observed in other editing tools, such as base editors. Collectively, base editing and prime editing offer complementary strengths and weaknesses for making targeted transition mutations. Base editors offer higher editing efficiency and fewer INDEL byproducts if the desired edit is a transition point mutation and a PAM sequence exists roughly 15 bases from the target site. However, because the prime editing technology does require a precisely positioned PAM sequence to target a nucleotide sequence, it offers more flexibility and editing precision. Remarkably, prime editors allow all types of substitutions, transitions and transversions to be inserted into the target sequence.

Because the prime system involves three separate DNA binding events (between (i) the guide sequence and the target DNA, (ii) the primer binding site and the target DNA, and (iii) the 3’ end of the nicked DNA strand and the pegRNA), it has been suggested to have fewer undesirable off-target effects than CRISPR/Cas9.

Limitations
There is considerable interest in applying gene editing methods to the treatment of diseases with a genetic component. However, there are multiple challenges associated with this approach. An effective treatment would require editing of a large number of target cells, which in turn would require an effective method of delivery and a great level of tissue specificity.

As of 2019, prime editing looks promising for relatively small genetic alterations, but more research needs to be conducted to evaluate whether the technology is efficient in making larger alterations, such as targeted insertions and deletions. Larger genetic alterations would require a longer RT template, which could hinder the efficient delivery of pegRNA to target cells. Furthermore, a pegRNA containing a long RT template could become vulnerable to damage caused by cellular enzymes.

Overall, much research will be needed before prime editing could be used to correct pathogenic alleles in human diseases.

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Delivery method
Base editors used for prime editing is a lab evolved protein, it does not exist in the nature. Which makes it very challenging when delivering it into human body. One of the successful way of inject base editor into animal and plants are assimilate the base editor with a natural virus. Which delivers the base editor by infecting the host. It has shown positive results in plants and animal, but remains questionable in human. Moreover, there are many other ways of delivering methods are being tested that is inspired by CRISPR-Cas9 systems. Other non viral methods are nano-particles, hydrodynamic injection, electroporation, etc. Nevertheless, the are many obstacles on how to deliver the base editors into human body safely and ethically.