Prime Editing: Snipping DNA Like Scissors

Millions of people in the world have genetic linked diseases. Some of these diseases have no cure and patients pass away earl​y. However, prime editing can vastly decrease these numbers with high edge gene therapy technology.

What is Prime editing?

Prime editing can:

1. Replace any “letters” (the letters that make up DNA are A,T,G,C) and can have 12 possible combinations of letters.
2. Remove a precise number of nucleotides.
3. Insert missing nucleotides or replace a string of disease causing ones.
4. Does not require double-stranded DNA cuts or separate DNA templates.

The components of prime editing:

Prime editing is done by the prime editor (PE) and pegRNA complex. The two components of the prime editor structure are cas9 nickase and reverse transcriptase. The pegRNA complex has three components: the gRNA target sequence, the primer binding sequence (PBS), and the template containing the desired RNA sequence added to the 3’ end. Unlike CRISPR, prime editing edits sequences without a double-stranded break of the DNA, so both the cas9 protein and RNA are modified. We have three versions of prime editors: PE1, PE2, PE3/PE3b. PE1 does insertions, deletions, and base transversions. PE2 improved the efficiency of PE1. PE3 and PE3b repair the mismatched sequences.

How does prime editing work:

Prime editing is a tool that modifies an individual’s genome. The PE:pegRNA complex initiates the process by binding to the target DNA. The cas9 nickase nicks only one strand of the DNA. The PBS on the pegRNA structure binds the nicked flap and the RNA sequence is reverse transcribed with the reverse transcriptase. The new DNA strand is attached to the end of the nicked DNA segment and the target DNA with the new reverse transcribed DNA. The cellular endonuclease removes the original DNA segment, which leaves one strand edited and one strand unedited. In PE3 and PE3b, the unedited strand is again nicked by cas9 nickase and the newly added strand is used to repair the nick. Both the strands are edited.

Why is prime editing important?

Prime editing can cure about 89% of genetic diseases! Imagine being diagnosed with a genetic disease, something that until recently, patients are told to go home and make the most of their time. Prime editing can help because it can edit the unhealthy or mutations in the DNA. Not only is this technology a huge advancement, but it is also a new way of viewing genetic diseases.

A few genetic diseases that prime editing can be applicable to:

• Cystic fibrosis
• Tay-Sachs
• Sickle cell anemia
• Etc.

The billions of DNA sequences are made of adenine, thymine, cytosine, and guanine (or A,T,C,G). RNA is read in pairs of three’s, and is translated into an amino acid chain. An amino acid chain folds into a functioning protein. However, if there was a mutation in the DNA (a change in one of the A,T,C,G or nucleotide), then it can negatively affect a resulting protein. This leads to some of the most common and fatal genetic diseases; Prime editing is important because it can correct these mistakes and errors from the DNA.

How is Prime editing different than CRISPR?

What is CRISPR?

CRISPR stands for clustered regularly interspaced short palindromic repeats. CRISPRs are made of nucleotides and spacers. Nucleotides are made of nitrogenous bases, a phosphate group, and a 5 carbon sugar. Spacers, as a defence mechanism, have a copy of the DNA from the virus’ that have previously attacked the cell. It also has the cas9 protein, which is made of crRNA and tracrRNA. crRNA is the copy of the target RNA and the tracrRNA holds the crRNA in place in cas9. When a new virus enters and if the cas9 matches the crRNA to the RNA of the virus, it kills the virus. When a new virus enters the cell and the cas9 does not have a copy of the RNA, the cas9 will make a copy of the RNA and store it in the spacers and still will kill the virus. Some studies concluded that the crRNA and tracrRNA can be simplified to a single guide RNA. And it is this RNA that guides the cas9 protein to the desired location where the protein will make a double-stranded break of the target DNA. Also, short DNA sequences known as PAMs serve as an extra guidance to mark the target site for the cas9 to cut. Once the DNA is cut, the cell undergoes natural self-repair mechanisms to fix itself.

Differences of CRISPR and prime editing:

Prime editing does not make double-stranded breaks in the DNA; instead, it utilizes the cas9 nickase to make cuts in one strand of the double helix. While CRISPR makes a cut in both the strands of the double helix. CRISPR does self repair and is more prone to errors while prime editing gives better control of the gene to edit.

Also, prime editing reduces the number of unintentional changes to the genome by inserting changes that scientists want to make to the DNA itself. Unlike CRISPR, which relies on the cell’s natural repair system.

In CRISPR cas9 is the protein that cuts the DNA and in prime editing, it is the cas9 nickase. In CRISPR the RNA is single guide RNA (sgRNA) while the RNA in prime editing is pegRNA.

Lastly, prime editing can make more changes in the DNA base pairs. A wider spectrum of combinations are possible. CRSIPR can make four possible combinations (C-T, G-A, T-C, A-G) while prime editing can make twelve.

Prime eding cannot make the big DNA insertions that CRISPR cas9 is capable of, so it cannot replace CRISPR.

What might the future hold for prime editing? And are there any hurdles?

Prime editing can revolutionize gene therapy and could be a starting point for a potentially larger technology. As the technique is new, more research is going on.

“Prime editing is the beginning, rather than the end of a long-standing aspiration in the molecular life-sciences to be able to make any DNA change in any position of a living cell or organism, including potentially human patients with genetic diseases,” — David Liu, founder of prime editing.

There are ethical concerns and questions not about prime editing, but the umbrella subject of gene editing itself.

This technology can be applied not only to genetic diseases, but also to agriculture and world hunger. All in all, prime editing is a profound change that can have serious impacts.