An Introduction to Prime Editing

Introduction

What’s so great about prime editing?

The CRISPR Cas9 system (colloquially referred to as CRISPR) is an interesting method of genetic editing. When first introduced, CRISPR Cas9 was revolutionary. Its effectiveness, speed, and ease of use contributed towards its popularity. However, despite its merits, CRISPR isn’t perfect. Despite being able to effectively modify gene sequence, using CRISPR often produces unintentional byproducts. These erroneous differences may be small but when it comes to genetic editing, precision is key.

Take a look at this partial DNA sequence:

CCT GAG GAG

This is a section of the normal DNA of the Beta Globin gene. This sequence of DNA is responsible for conveying instructions for the production of Beta Globin, a protein that makes up hemoglobin. Compare this sequence with the following:

CCT GTG GAG

Now this is a mutated DNA sequence of the Beta Globin gene. The difference between the two sequences is the fifth letter.

Although seemingly inconsequential, the slight change is responsible for sickle cell anemia, a serious, damaging genetic disorder. I say this to demonstrate that the slight inconsistencies accompanying CRISPR can have seriously detrimental effects. This lingering potential for error prohibits and limits CRISPR, particularly in relations to its therapeutic applications. So what’s the solution?

Let me tell you a little about prime editing

First introduced in 2019 by researchers at MIT and Harvard, this relatively new technique is an extension of the CRISPR system. It exists to push the threshold of genetic editing and to avoid the difficulties and problems of CRISPR. Specifically, prime editing addresses issues resulting from cutting double stranded breaks (DSB) by which both DNA strands are snipped. Doing so destabilizes the structure of the DNA and leads to a multitude of problems such as the random editing of DNA by the cell reparation functions. Consequently, CRISPR Cas9 which uses DSB, is unreliable and unpredictable. Prime editing, on the other hand, utilizes a single stranded break. Therefore, it is able to avoid excessive and unpredictable modification that faces CRISPR Cas9 making it a useful and effective tool towards geneticists. In the article, we will explore the different aspects of prime editing like how prime editing works and why it is important.

How does CRISPR Cas9 (CRISPR) work:

Don’t worry, we’ll get into more of prime editing soon but to understand some key concepts, let me first get you more acquainted with the CRISPR Cas9 system. Here we go!

CRISPR, as stated above, is genetic editing technology. Although it is slowly becoming obsolete, understanding how CRISPR works is useful in understanding prime editing. So how does CRISPR function?

Well, the system is based on the inherent defence mechanisms of microorganisms which utilize the Cas (CRISPR associated) 9 protein and RNA to destroy invaders. These organisms capture the DNA of invaders, produce countering gRNA (guide RNA) to identify the DNA of threats, (viruses, etc.), and utilizes Cas9 proteins to cut the DNA. These mechanisms were appropriated in prime editing. For CRISPR Cas9, the editing processes are as follows:

1. A gRNA attaches itself onto the Cas9 protein.
2. A gRNA navigates itself and the Cas9 to the target and binds itself to the targeted sequence of DNA.
3. Cas9 is then utilized to cleave two strands of the double helix identified by the gRNA
4. Cell attempts to find loose genetic material to repair the area. Scientists can take advantage of this and provide edited material, therefore, modifying the area.

This is an image which summarizes the workings of CRISPR quite nicely.

Prime editing (components) :

Prime editing is a complicated process which involves various components working together to accomplish genetic editing. Each of them have a specific role and distinct purpose.

As in CRISPR Cas9, prime editing involves the Cas9 protein however prime editing utilizes a modified variant called the Cas9 nickase which is programmed to nick a singular strand DNA. In both of these methods, the Cas9 protein functions as the “molecular scissors,” slicing DNA sequences.

Prime editing also utilizes a pegRNA. (prime editing guide RNA) This is similar to the gRNA utilized for CRISPR Cas9. Both types of RNA strands primarily function to identify targeted areas for their respective methods. However, pegRNA is also responsible for stabilizing DNA strands and contains the template RNA required to construct the substitute for the target sequence.

Another component of prime editing is the reverse transcriptase enzyme (RT). It is responsible for reverse transcription. This means the reverse transcriptase can interpret a provided RNA template and create a substitute DNA strand.

To recap, the primary components of prime editing are:

Cas9 nickase

• responsible for cutting DNA
• Is a modified Cas9 protein
• Combines with reverse transcriptase enzyme to form the prime editor

pegRNA

• responsible for providing instructions to RT
• responsible for guiding prime editing components towards targeted DNA sequence
• stabilizes single stranded break

Reverse transcriptase enzymes:

• Interprets RNA
• Responsible for creating substitute DNA strand
• Combines with Cas9 nickase to form the prime editor

Image showing three major components of prime editing: Cas9 nickase, pegRNA, and reverse transcriptase

Prime Editing (how does it work?):

Prime editing begins when the reverse transcriptase and the modified Cas9 protein (known as the Cas9 nickase) forms a complex called the prime editor. The prime editor and the pegRNA both attach onto the target sequence with the pegRNA binding to both strands of DNA to retain structural stability. The DNA strands near the target sequences are then opened up. This breaks the established bonds between the two strands. It prepares the area for further editing.

Soon after, the target area is nicked. Similarly in CRISPR Cas9, prime editing utilises the Cas9 protein as its “scissors.” However, contrastingly, prime editing involves the nicking of only one strand which creates a single stranded break. By doing so, prime editing avoids the issues CRISPR Cas9 faces when making a double stranded break.

After the single stranded break is created, the reverse transcriptase then interprets the RNA template found in the pegRNA and utilizes it to create reversed transcribed DNA. Cellular functions proceed to cut loose the old DNA and substitute it with the constructed strand with the edit.

One strand of DNA now has the intended edit. However, prime editing is not finished. Although one strand is edited, it cannot bond with the corresponding sequence on the second strand until both strands compliment each other. To resolve this issue, the Cas9 nickase cuts the unedited section and the inherent functions of the cell rely on the edited sequence as a blueprint to fix the missing area. Therefore, both strands of DNA are bonded and the edit is complete. Prime editing has finished its job!!!

Why is prime editing important?

Genetic disorders, defined simply, are diseases resultant of mutations in one’s DNA. These disorders, which cause pain and serious injury, include terrible diseases like sickle cell anemia or Tay-Sachs disease. Over 20% of the global population has some form of a genetic disorder. Those afflicted have none, if not few, available solution. As well, because of the hereditary nature of contracting genetic disorders, the number of the affected will continue to exponentially increase. In the face of such a dire future, a potential solution lies in the field of genetic editing: prime editing. This method can rewrite many deficient DNA sequences, more safely, and efficiently than other editing methods. There are approximately 75,000 types of mutations which can cause genetic diseases. Dr. Liu, a researcher who co-wrote a paper on prime editing, estimates that prime editing can correct 89% of all DNA errors which cause genetic diseases. Its safety, versatility, and preciseness, in essence, is why prime editing is so important. It represents a potential safe solution to certain disorders. In all, prime editing is a definite step towards a future where those severely affected by genetic diseases can be cured.

Comparison between prime editing and CRISPR Cas9:

Processes:

As prime editing expands on CRISPR Cas9, many similarities can be found between the two methods. Like CRISPR, prime editing begins when a guide RNA brings the necessary components to a target sequence. For CRISPR, this guide is the gRNA which binds to a singular strand of DNA. However, prime editing utilizes a variant called the pegRNA. Instead of functioning simply as a guide, the pegRNA also provides information for the reverse transcriptase. The pegRNA also differs as it binds to two DNA strands rather than binding to a single one like the gRNA does. Significant differences can also be found during the cutting processes. Whereas CRISPR Cas9 utilises a Cas9 protein to cleave two DNA strands at the target sequence, prime editing uses a variation called the Cas9 nickase which snips off DNA on one strand causing a single stranded break. Due to the differences in their respective processes, the remainder of each method is quite different. As CRISPR caused a double stranded, it relies on cell functions to re heal the wound. In instances of genetic editing, this would be where scientists would give the cell edited genetic material to substitute the target DNA with the intended edit. For prime editing, the reverse transcriptase and the pegRNA work together to form substitute DNA with the intended edit. It is then where the cell naturally cuts off the old DNA and closes the single stranded break with the constructed DNA. When this is completed, the cell notices that the other unedited strand is not complementary to the edited strand. Therefore, it uses the edited strand as a blueprint and completes the edit on the other strand.

Application:

Differences in application between CRISPR and prime editing are related to how each of their respective processes function. A particular difference is how each method cuts DNA. While CRISPR Cas9 causes a double stranded break which has more off target effects, prime editing introduces a more stable single stranded break. Prime editing, therefore, is much more safe and predictable. As such, it can be used in more situations and is consequently the preferred method for use on humans. In early trials, prime editing was used to rewrite DNA mutations which cause sickle cell anemia and Tay-Sachs disease. Prior to this, genetic editing methods were not advanced enough to efficiently accomplish this. Because of its versatility and relative safeness, prime editing can surpass CRISPR.

The future of prime editing:

Prime editing is new and as such, the potential of prime editing has not been fully discovered. In the future, prime editing can elicit developments related to designer babies, embryos who have been genetically edited. Although controversial and ethically ambiguous, prime editing can serve useful regarding developments and research on designer babies. As the most advanced method of genetic editing, prime editing also serves an instrumental role in revolutionizing the way we treat genetic disorders. Although not all hereditary illnesses can be fixed through prime editing, prime editing is a definite stepping stone to future technologies. In all, prime editing paves the way to a future where genetic disorders are a past issue and genomic modification is as safe and effective as ever. I for one, am excited to see this happen.

I hope this article allows you (the viewer) to gain comprehensive knowledge on prime editing. Thanks for reading. xD