Rewriting the history books and genetic mutations alike through “Prime Editing”

Take a moment. Close your eyes. Imagine each and every cell within your body as an infected, sick and broken organism from the moment you were birthed to the moment it leads to your miserable demise. This describes the unfortunate reality for approximately 20% of the human population born with genetic disorders. The question “Why me?” consumes the mind of the individual knowing they’re completely helpless in regards to the life-draining mutation they’ve inherited. There’s simply no way out. Right…?

Insert genome editing…

Dating back to the dawn of humanity, the human race has been obsessed with altering the natural course of life; whether it was developing medical treatments to fight diseases to a higher level of success, plastic surgery to modify our appearances or sex change surgeries. This obsession has evolved to a molecular level concerning the human genome.

Genome editing is defined as a method of genetic engineering that allows humans to modify or change the genetic information of a living cell or organism through changing their unique DNA sequences. There are a multitude of various technologies that allow for genetic information to be added, removed or adjusted in the human genome with the intent of preventing hereditary mutations in individuals. The prominent players in the genome editing league include TALENs, ZFNs and more importantly, CRISPR.

What is CRISPR?

Clustered Regularly Interspaced Short Palindromic Repeats (a mouthful, I know!) more commonly referred to as CRISPR-Cas9 is a technology developed from a natural process in prokaryotic cells in order to edit strands of DNA in the human genome. In bacteria, this process is used as a defense mechanism to fight viral DNA; the bacteria captures and remembers specific segments of the virus’ DNA and stores it for future reference. Once the bacteria perceives a threat from the virus again, it uses a guide RNA in order to target the virus’ DNA which is then cut and disabled by the Cas9 endonuclease.

Scientists working in the field of molecular biology observed that the sheer effectiveness of this editing system could be utilized in regards to human DNA. The CRISPR-Cas9 genome editing technology was born. In this technology, scientists formulate a complex by the name of the Crisper-Cas9 complex which allows for gene editing to occur. It consists of a guide RNA (gRNA) which has the ability to target a specific loci and allow the cleavage of the targeted sequence of DNA. Once the identified DNA sequence is cut, we rely on DNA repair pathways in order for the mutation to be disabled; we can also insert very basic DNA sequences to an extremely limited extent. The video shown below explains the inner-workings of the CRISPR-Cas9 technology:

Brief explanation of CRISPR-Cas9

Wait, so essentially this disruptive technology allows for genetic mutations to be prevented and treated in every individual? That sounds too good to be true. Bingo!

It is too good to be true…

CRISPR-Cas9 has an abundance of issues and uncertainties that question it’s potential and future in genome editing. This technology relies on two DNA repair pathways once the double strand break is created; Non-Homologous End Joining (NHEJ) and Homology-directed repair (HDR). NHEJ is extremely susceptible to failure with the result being unintended insertions or deletions categorized as “off-target” edits with the repercussions being detrimental. HDR is the recessive pathway in DNA repair, the infrequency of this pathway causes it to be extremely inefficient and ineffective to rely on when editing genes. The precariousness of CRISPR-Cas9 is why the practicality and application of this is heavily debated.

The two DNA repair pathways relied upon when using CRISPR-Cas9

The solution for these inadequacies is a recently developed technology called “Prime Editing.” Prime Editing is a CRISPR-based system that focuses on searching for and editing the targeted DNA sequence related to the genetic mutation rather than simply disabling it as CRISPR-Cas9 suggests. It’s been concluded that, in theory, prime editing can be the solution to approximately 89% of all genetic mutations/variations that cause disease in the human genome; which is astronomical in comparison to CRISPR-Cas9.

As we established previously, the inconsistencies when using CRISPR-Cas9 originate from the double strand break in the DNA helix which triggers the two DNA repair pathways. Prime editing proves to be beneficial in that aspect due to it’s utilization of the CRISPR-Cas9 fundamentals in order to create a single strand break in the DNA as opposed to a double strand break. How does it do that? Let’s begin at the two main components that are necessary for this process to occur.

The prime editor complex is a combination of two enzymes essential in the process of gene editing. The first component of the editor is the Cas9 enzyme; in prime editing, this enzyme is slightly modified in comparison to it’s older brother used in CRISPR-Cas9. Cas9 H840A is variant of the original Cas9 enzyme which has been engineered on a molecular level in order to create a nick in a single strand of DNA for the revising of genetic information to begin. The second piece of the puzzle is an enzyme referred to as the reverse transcriptase; the purpose for this enzyme is to convert the RNA encoded template for the genetic change provided by the pegRNA into a DNA sequence used to complete the editing process.

The prime editing guide RNA, more commonly referred to as pegRNA, is a modified guide RNA that has the capability to target a specific, pre-programmed loci in the DNA helix and direct the prime editor complex there to a high degree of accuracy. In addition to this, the pegRNA carries an encoded RNA version of the desired edit for the reverse transcriptase to convert into a functioning DNA sequence to be edited in.

Components involved in Prime Editing

So… How do these components result in an edited DNA sequence?

The tools mentioned above fuse together to become one complete prime editing unit ready to eliminate genetic mutations once and for all. To begin, scientists identify a specific loci in the human genome where the process will take place. The pegRNA is programmed to target this mutation and point the prime editor complex to the correct part of the DNA helix where it is then bound via the targeting sequence. The modified Cas9 nickase then creates a single strand break in the helix which allows for the primer binding site in the pegRNA to bind to the nicked DNA strand while the reverse transcriptase undergoes it’s process. During this, the reverse transcriptase is taking the encoded RNA information and converting it into a unique DNA sequence that undergoes a letter by letter transfer into the nicked spot of the DNA strand and solidifying the edit in the single nicked strand. A natural endonuclease in the genome then removes the unedited old DNA strand and we now have an edited strand of DNA. The process is done now, right? Right…? Wrong. In order for the edit to take effect, the other unaffected strand of DNA needs to harness the edit but it doesn’t have the means to do so. Another guide RNA called the single guide RNA (sgRNA) then takes the complex to the unedited strand of DNA to create a nick there as well. The unedited strand then utilizes the edited strand as a template in order to repair the break in the strand and as a result, both strands of DNA are edited and the mutation is then eliminated.

The entire Prime Editing process outlined

Is Prime Editing better than CRISPR-Cas9?

Through extensive research and lab experiments, it’s been concluded that prime editing proves to be more efficient than CRISPR based on two main factors. Prime editing had the ability to edit a higher quantity of cells with success in comparison to CRISPR-Cas9; on average, 20–50% of cells were successfully improved through prime editing while researchers struggled to reach double digits when using CRISPR-Cas9. Moreover, prime editing is the originator of far less off-target effects such as unintended insertions and deletions that CRISPR-Cas9 is exceptionally prone to due to it’s double strand break. The effects of these unintended changes in the human genome include tumorigenesis which is the formation of a tumor in the individual, something prime editing solves.

What is the significance of the Prime Editing technology?

Prime editing proves to be a major player in the genome editing industry due to it’s one-of-a-kind ability to successfully complete all twelve possibilities regarding base-to-base changes which as a result solves around 89% of the 75,000 genetic mutations. Previous technologies such as base editing could only complete four base-to-base edits (C — T, G — A, A — G and T — C); and CRISPR-Cas9 lacks the ability to complete base-to-base edits at all, it just disables the gene and relies on the repair mechanism to fix the gene on it’s own. This technology is revolutionary due to it’s extreme flexibility and precision that nothing else offers at this point in time; and I think I speak for everyone when I express how excited I am to see how things unfold for prime editing and what the future holds…

Well… What does the future hold?

Although prime editing seems like an immense advancement in the world of genome editing (and it is!), this technology was recently introduced in 2019 and we do not have enough information at this point to determine the heights that prime editing has the potential to reach. There are a multitude of distinct obstacles that this technology needs to overcome before being widely accepted by society and before application of prime editing can occur in humans. Aspects such as delivery methods, the immense size of the prime editor on a molecular level and the long-term effects on human cells all need to be studied and investigated thoroughly before any assumptions can be made about the future of prime editing. However, prime editing will likely act as an additional tool to the original CRISPR-Cas9 and base editing systems as each have their own pros and cons. It’s appropriate that these systems will have a relationship of coexistence in society. It is certainly an exhilarating time to be involved in the world of genome editing and there’s no doubt that this progress will prove to be beneficial for humanity. Now all we can do is wait and see…

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