Prime Editing: The Next Gen

We are in the midst of a Genome Editing revolution that can transform mankind. Arguably, its transformational impact can be more profound than that of information technology, as it can change life as we know it, and not just our lifestyles!

While humans have been engineering life in plants and livestock for thousands of years through basic techniques like selective breeding, our understanding of the code of life, DNA (Deoxyribonucleic Acid), and its double helix structure (see figure under Crispr/Cas9 Technique subheading) only developed in the second half of the 20th century. Since then, we have been fascinated with the DNA, having developed many commercial applications from genetic studies, and have also sequenced the entire 3.2 billion base pairs of the human genome in 2003. The importance placed on genetic research is evidenced by the consistent center stage it has had in Nobel Prize ceremonies for several years.

While early gene therapy focused only on minimizing the consequence of a genetic mistake, we have now advanced to gene editing with the advent of the technology to create site-specific Double Strand Breaks (DSB) in the DNA (discussed later). Although this technology is still in clinical stage, the discovery of the CRISPR/Cas9, derived from the adaptive immune system of a bacteria, in 2012, has enabled widespread research in the field, due to its easy access and low cost. CRISPR has become a disruptive technology that is creating a lot of excitement in microbiology.

Before CRISPR/Cas9, zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) were used for gene editing. Similar to Cas9, these are also fusion proteins, and use DSB for edits, but they lack application to a broad range of target DNA sequences. Also, CRISPR is significantly cheaper (approximately 150 times), faster and more precise.

Prime Editing is considered the next version of CRISPR/Cas9. That said, unlike the obsolescence typical of software versions, both Prime Editing and CRISPR are currently expected to co-exist. The well established CRISPR editing tool will remain a preferred method for edits to larger gene sequences until researchers further enhance Prime Editing and resolve its delivery issue discussed in later sections.

PRIME EDITING

Prime Editing was introduced in 2019 by the researchers from Broad Institute of MIT and Harvard, who include the pioneers of Base Editing as well that was introduced a few years before.

As mentioned earlier, Prime Editing is a refinement of CRISPR/Cas9, and therefore, for a comprehensive understanding of this technique, we have adopted the logical sequence of explaining CRISPR/Cas9, followed by Base Editing and then Prime Editing. There are commonalities and improvements as we navigate from one to the other and this article tries to bring that out for the reader.

Before we start, it will be useful to mention the analogy used for these three recent techniques — CRISPR/Cas9 is like a pair of scissors, Base Editing is like a pencil and Prime Editing is like a word processor that can precisely search and replace.

Interesting! Let’s now elaborate on it.

CRISPR/Cas9 Technique

Let’s get the acronym out of the way first!

CRISPR/Cas9 stands for Clustered Regularly Interspaced Short Palindromic Repeats / CRISPR-Associated Proteins 9.

Simply put, the CRISPR/Cas complex consists of CRISPR, a spacer DNA sequence, and Cas9, a fusion protein. They are found in certain bacteria and archaea. A common source is the antiviral immune system of the bacteria, Streptococcus Pyogenes. In 2012, Jennifer Doudna and Emmanuelle Charpentier introduced the use of CRISPR/Cas9 for programmed gene editing which was the start of the current revolution being witnessed in microbiology.

The Cas9 protein has two endonucleases (i.e. two pairs of scissors), that can cut both strands of a DNA. The DNA is a spiral ladder-like structure that has sugar-phosphate as its backbone with the middle lines representing the nucleotides or base pairs. There are four bases, adenine (A), thymine (T), guanine (G) and cytosine (C ); and interestingly A pairs only with T and G pairs only with C. The order of these bases determines the DNA’s instructions or genetic code.

If you are wondering what a gene is, it is simply a subset of the DNA, possibly consisting of several hundreds of base pairs, that is capable of producing a particular protein. Erroneous substitution, insertion or deletions in these base pairs is referred to as gene mutation and can be the cause of several diseases. The mutation can be caused due to hereditary or external factors.

Process Chart

The above diagram depicts the DNA and the CRISPR/Cas9 editing process. The DNA is in the nucleus of our cells. Once CRISPR/Cas9 enters the cell nucleus, the gRNA (single strand, shown in red) is able to open up the DNA strand and search for the 20 bases that are complementary to its own 20 base sequence, and it binds to this target site to form base pairs. This step serves as a guide for the Cas9 protein (shown as J-shaped blue bubble) to the target site.

Thereafter, the two scissors of Cas9 nick both the DNA strands 3–4 base pairs ahead of a marker called PAM sequence that immediately follows the target site. PAM stands for Protospacer Adjacent Motif, but again, simply put, it is a short 2–6 base pair sequence located downstream from the target site (shown in labels).

After this double strand break, the cell is expected to self-repair the DNA and it has two pathways. If the objective of the editing technique is to disable the gene as it is causing a disease, the error-prone Non-Homologous End Joining (NHEJ) pathway is allowed to ligate (i.e. attach) the DNA strands via random deleting or inserting bases and thus completely disrupting the gene sequence.

However, if the objective is to precisely edit the gene, a donor DNA is introduced as a template, with the desired gene edits, for the cell to perform the repair work through recombination. This pathway is called Homology Directed Repair (HDR).

The gRNA refers to the guide RNA, which is a combination of engineered single strand of 20 complementary nucleotides or bases known as crisprRNA (shown in bold red) that is used to locate the target site, and the tracrRNA (shown in light red) that is used as a scaffolding to bind the gRNA and Cas9 protein to form a complex.

CRISPR/Cas9 can be delivered to the cell using physical delivery methods (microinjection; electroporation), viral delivery methods (adeno-associated virus (AAV); full-sized adenovirus and lentivirus), and non-viral delivery methods (liposomes; polyplexes; gold particles), and each have their relative merits based on the purpose of the experiment.

It is interesting to note that since CRISPR broke into the gene editing scene, plenty of vendors have mushroomed that provide a complete CRISPR editing tool kit at affordable prices, enabling widespread research in gene editing. The human DNA has 3.2 billion base pairs, and then there are countless living organisms, thus there is no dearth of research opportunities for young microbiologists! That said, the safety and ethical risk associated with this mass availability of a potent technology is a cause of concern and is touched upon in a later section.

Base Editing Technique

Researchers have been concerned with the risk of double strand break shattering the integrity of the DNA in the CRISPR/Cas9 technique. More specifically, it can cause translocation of DNA, activation of p53 that can induce cell death or cellular senescence. In other words, Crispr/Cas9 can be mutagenic with a complex mix of undesirable (off-target) results. The technique’s use of double strand break has been tagged as “DNA vandalism” by some.

Base Editing evolved as an alternative that does not require this double DNA cleavage and instead uses a deaminase enzyme to perform a more precise gene editing. While gRNA and Cas9 are used in this technique as well, the Cas9 scissors are deactivated through point mutation, and therefore it is referred to as dead Cas9 or dCas9. The said deaminase enzyme is infused in the dCas9.

Process Chart

As is standard, the process starts by the gRNA opening up the DNA and binding to the complementary target site (as shown above), thus guiding the enzyme infused dCas9 to the target site. Then the deaminase enzyme is then able to chemically convert a single base in the DNA sequence without causing any breaks in the strand that may alter the surrounding sequence. This enzyme is capable of converting C to T, G to A, A to G and T to C.

As implied from the process, Base Editing is limited to single base edit and therefore is useful in point mutations only. That said, a large portion of the known human pathogenic genetic variants are point mutations, and therefore, this is a safe and powerful tool. On the flip side, similar to CRISPR/Cas9, Base Editing is also prone to off-target edits.

Prime Editing Technique

The hype around Prime Editing is well deserved. The technique attempts to overcome the inefficiencies of CRISPR/Cas9, and the single base limitation plus inefficiencies of Base Editing.

Prime Editing is a search and replace gene editing technique that does not use double strand breaks and does not need donor DNA. This technique makes the following three notable modifications to the conventional CRISPR/Cas9:

1. Modification of Cas9 — the Cas9 is modified by mutating one of its two endonucleases thus allowing only a single strand break. The modified Cas9 is called Cas9 H840A nickase.
2. Enzyme infusion of Cas9 — The Cas9 H840A nickase is fused with a mutant version of an enzyme Reverse Transcriptase, that is widely used in molecular biology and is considered as a very stable performer. This infused Cas9 nickase is called the Prime Editor.
3. Modification of gRNA — The Prime Editing Guide RNA or pegRNA is a modified gRNA that is substantially larger (>100bases vs. standard 20 bases) as it has a primer binding sequence (PBS) and a template containing the desired RNA sequence added at its 3’ end.

Process Chart

As is standard, the Cas9 nickase is guided to the DNA target site by the pegRNA that opens up the DNA strands and binds to the strand. Thereafter, the Cas9 nicks only a single strand generating a flap. Thereafter, the PBS, located on the pegRNA, binds to the DNA flap and the edited RNA sequence is reverse transcribed using the reverse transcriptase. The edited strand is then incorporated into the DNA at the end of the nicked flap, and the target DNA is repaired with the new reverse transcribed DNA.

The original DNA segment is removed by a cellular endonuclease. This leaves one strand edited, and one strand unedited.

In the newest PE system, the unedited strand can be corrected to match the newly edited strand by using an additional standard gRNA. In this case, the unedited strand is nicked by a Cas9 nickase and the cell uses the newly edited strand as a template to repair the nick, thus completing the edit.

Prime Editing is considered a game-changer in genome editing as it preserves the benefits of the other CRISPR techniques and solves their inherent disadvantages. Arguably, it has the potential to correct up to 89% of disease-causing mutations and is engineered to make more precise edits than other gene editing techniques.

The main challenge facing Prime Editing is its delivery system. The molecular components of this technique are too big to deliver to the cell using current methods. Researchers have to focus on solving the delivery issue, if they hope to commercialize the use of Prime Editing for in vivo applications like medicinal drugs.

Thus, as mentioned earlier, the different CRISPR based genome-editing techniques are currently expected to co-exist as they will be needed for different types of edits. Prime Editing, although the preferred technique in most instances, it may not be able to make large DNA insertions or deletions that CRISPR/Cas9 is capable of as the pegRNA strand is already long and stretching it any longer will likely make it vulnerable to various enzymes in the cell. It feels like further enhancement of these techniques is likely in the near future!

Why Prime Editing & other CRISPR based techniques are a big deal?

As noted earlier, the CRISPR based genome editing techniques are viewed as revolutionary in biotechnology. For several decades, we have been unable to precisely target parts of the staggeringly long genome (for example, 3.2 billions base pairs in humans), that dance around in the cell. CRISPR has made this possible in an easy and low cost manner, and this has opened up research in a plethora of opportunities covering agriculture, livestock, human health and also the controversial heritable genome editing.

In human health, there are over 75,000 pathogenic genetic variants that have been identified. Existing genome editing methods using nuclei (TALENs and ZFNs) and base editors only have the potential of correcting a portion of those variants. CRISPR/Cas9 is a significantly faster, cheaper and more accurate gene editing tool than previous techniques, and Prime Editing further enhances CRISPR’s precision and flexibility. This is critical for human use, given the low tolerance for error.

In agriculture, gene editing has the capability of making plants that not only produce higher yields, like Lippman’s tomatoes, but also ones that are more nutritious and more resistant to drought and pests. These are features that may help crops endure more extreme weather patterns predicted in the coming years. Presently, research and development labs are testing the potential of gene editing tools to solve a variety of food-related concerns for both consumers and growers. Such experiments include reduced-gluten wheat, a mushroom that doesn’t brown when bruised or cut, soybeans lower in unhealthy fats, and even protecting global chocolate supply like the candy maker Mars, who has been making an effort to strengthen cacao’s ability to fight off a virus that’s devastating the crop in West Africa.

By 2050, the global demand for animal-based food products is estimated to increase by 70%. Genome editing of livestock is an important tool which enables breeders to improve animal welfare, performance and efficiency, and is creating a path towards a more sustainable future for livestock agriculture. Currently, genome editing of livestock is limited to specialized laboratories because of the complexity of techniques available for the delivery of genome editing on zygotes and reproductive cells. Three cutting-edge reproductive technologies including zygote electroporation, zygote transduction of recombinant adeno-associated virus (rAAV), and surrogate sire technology will provide livestock breeders with a new toolkit of delivery strategies.

Genome editing is one of the most interesting prevention and treatment methods to human diseases. Although the majority of research on genome editing is done to understand diseases using cells and animal models, scientists are still determining whether there is a safer and more effective use in people. This research is being conducted on a wide variety of diseases including single-gene disorders such as cystic fibrosis, hemophilia, and sickle cell disease. As well, this technology could also have the capability of treating and preventing more complex diseases including cancer, heart disease, mental illness, and human immunodeficiency virus (HIV) infection.

Heritable Genome Editing

Ethical, safety and social concerns arise when genome editing technologies are applied to germline or reproductive cells. So far the majority of the editing is limited to somatic cells, that only affect certain tissues, and are not hereditary. However, genome edits to germline cells or an embryo can be undertaken with similar ease as for somatic cells.

Currently there is broad consensus to maintain a moratorium for germline editing. It is prohibited by law in 40 countries and by a binding treaty in Europe. Despite these measures, in November 2018, Chinese researcher He Jiankui claimed to have successfully created the first human genetically edited babies that caused authorities to quickly respond with a regulation that anyone manipulating the human genome by gene-editing techniques, like CRISPR, would be held responsible for any related adverse consequences.

Germline editing has tremendous benefits of eradicating incurable diseases such as HIV/AIDS, sickle-cell anemia and multiple forms of cancer and preventing known and future diseases from becoming an epidemic. However, on the flip side, they can be used to create “designer babies”, i.e. humans with perfect traits, which is a far more controversial application and is currently heavily scrutinized.

There are ethical, safety and social concerns associated with such applications. Also, such alterations are untested and therefore the irreversible unintended consequences of heritable genome editing are also not known.

Safety is a major concern as well given this technology is susceptible to use in manipulation of viruses, the transfer of genes in order to use them as a weapon, or corporate America exploiting crops and animals in order to manufacture traits to meet economic needs, without ethical consideration.

Social concerns arise if the benefits of gene editing are not equally available in society due to patents, affordability or other restrictions.

Opinion

As is evident, this is one of the most fascinating topics that has captivated today’s world. We are at a cusp of making important decisions that will determine the future of mankind and our environment by harnessing the potential of this technology.

Eradicating incurable diseases, increasing food productivity, extending human lifespan, reversing aging, ability to sustain in currently inhospitable environments (like Mars etc) are very promising applications.