PASTE: A Novel Prime-editing Technique
A technical & theoretical overview of how Cancer works and how we can cure it using PASTE
Cancer, one of the most common diseases throughout human history, claimed over 10 million lives in 2021. This is no statistical anomaly, as scientists predict it will result in nearly 20 million deaths in 2040. With massive improvements in modern medicine, from tumour-detection AI algorithms to radio-chemotherapy, one might expect these numbers to decrease, but this is simply not the case. However, this has now changed overnight with the introduction to a novel CRISPR technology known as PASTE.
To first understand how scientists can utilize PASTE to eradicate cancer, it’s integral to understand the fundamentals behind how cancer arises and spreads.
Simply put, cancer is a disease in which some of the body’s cells grow uncontrollably and spread to other parts of the body. Cancer can start almost anywhere in the human body, and usually originates following genetic errors. This is critical as DNA provides a set of instructions telling the cell what functions to perform, as well as how to grow and divide. With incorrect instructions, the cell cannot function correctly, and the accumulation of more of these ‘faulty’ cells results in cancer.
What is Cancer & How does it spread?
Simply put, cancer is a disease in which some of the body’s cells grow uncontrollably and spread to other parts of the body. Cancer can start almost anywhere in the human body, and usually originates following genetic errors. This is critical as DNA provides a set of instructions telling the cell what functions to perform, as well as how to grow and divide. With incorrect instructions, the cell cannot function correctly, and the accumulation of more of these ‘faulty’ cells results in cancer.
- Proto-oncogenes are involved in cell growth and division. However, when these genes are altered in certain ways or are more active than normal, they may become cancer-causing genes (or oncogenes), allowing cells to grow, while avoiding apoptosis. Apoptosis is the in-built mechanism encoded in cells to self-destruct if the DNA is changed, but mutations in oncogenes allow the cell to bypass this process, allowing the cell to grow indefinitely
- Tumour suppressor genes inhibit cell growth and division. Cells with certain alterations in tumour suppressor genes may divide in an uncontrolled manner.
- DNA repair genes are involved in fixing damaged DNA. Cells with mutations in these genes tend to develop additional mutations in other genes and changes in their chromosomes, such as duplications and deletions of chromosome parts. Together, these mutations may cause the cells to become cancerous.
It is also important to understand the concept of growth factors. A growth factor is a substance, usually a protein (recall that proteins enable all bodily functions) capable of stimulating cell proliferation. Usually, cells need growth factors to grow. However, cancer cells may make their own growth factors, or have growth factor pathways that are stuck in the “on” position. This constantly tells the cell to multiply. By manufacturing growth factors, the cancerous cell can even induce neighbouring cells to multiply uncontrollably. Mutations can even change the promoter of growth factors. Usually, growth factors are released in specific circumstances (i.e. too little vitamin A). However, if the gene can be translocated to another promoter, the growth factors may be manufactured in a different circumstance (i.e. too much vitamin A). Lastly, there can be duplication in the number of promoters of a growth factor. There can be 3 genes instead of 1 that encode for the production of the growth factor, resulting in over-production and over-stimulation
Another hallmark of cancer cells is their “replicative immortality,” a fancy term for the fact that they can divide many more times than a normal cell of the body. In general, human cells can go through only about 40–60 rounds of division before they lose the capacity to divide, “grow old,” and eventually die (known as the Hayflick limit). Cancer cells can divide many more times than this, largely because they express an enzyme called telomerase, which reverses the wearing down of chromosome ends (telomeres) that normally happens during each cell division. The Hayflick limit is reached when the chromosome ends get fully worn off, but since cancer cells can regrow the telomeres, there is no limit dictating how much they multiply.
Let’s look at two specific genes that when mutated, result in cancer:
- The ras proto-oncogene (named for Rat sarcoma — a connective tissue cancer) codes for the Ras protein. Its main role is as a cell-signalling protein to relay the signal from a Growth Factor into the cell which ultimately stimulates the cell cycle/cell division. But certain mutations in the ras gene can lead to the production of a hyperactive. Ras protein triggers the cell cycle even in the absence of growth factor, resulting in increased cell division.
- The p53 gene normally safeguards against excessive cell division. The gene codes for a transcription factor/protein that inhibits the cell cycle. But if mutated, would not be able to safeguard the normal cell cycle and cells would uncontrollably divide → cancer. The p53 gene can also be activated by damaged DNA to activate other ‘suicide genes’ that initiate apoptosis.
Mutations in Ras account for 30% of cancers and mutations in p53 account for 50% of cancers, making them an excellent point of research in the field of gene editing.
Now that we better understand what cancer is and how it’s caused, let’s learn about how PASTE works and how it can be used to stop cancer.
Development and discovery
The first version of CRISPR could cut DNA; the second had base editing capabilities; and the third had Prime editing, which allowed for any tiny modifications, such as base edits, insertions, and deletions. However, because CRISPR was only useful for tiny deletions and insertions, scientists have been unable to edit genes on the scale of kilobase. With the creation of PASTE, we can “drag and drop” kilobases’ worth of DNA.
But how does one go from the first generation of CRISPR to this 4th-gen “drag and drop”? The AbuGoot lab realized that they could likely do it using Prime editing, alongside additional modifications. Let’s look at what prime editing is and the additions scientist made it allow them to scale up the number of bases PASTE can edit.
Components of Prime Editing
Similar to CRISPR, prime editing requires the presence of a Cas endonuclease and a single guide (sg) RNA. However, prime editing differs from 1st-gen CRISPR as it edits sequences without generating a double-stranded break, which resulted in a higher likelihood of mutations seen in CRISPR. Moreover, instead of traditional Cas9, prime editing utilizes Cas9 nickase, which, true to its name, ‘nicks’ the DNA. Cas9 nickase is then fused to a reverse transcriptase. The fusion is referred to as the prime editor (PE).
In prime editing, instead of sgRAN, it utilizes prime editing guide RNA (pegRNA), which is substantially larger than standard sgRNAs. The pegRNA is a sgRNA with a primer binding sequence (PBS) and the template containing the desired RNA sequence is added at the 3’ end. The resultant molecule is the PE:pegRNA complex, which is used to facilitate genomic editing within the cell.
Process of Prime Editing
The PE:pegRNA complex binds to the target DNA, and Cas9 nicks only one strand, generating a flap. The PBS, located on the pegRNA, binds to the DNA flap and the edited RNA sequence is reverse transcribed using reverse transcriptase. The edited strand is 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. The newly edited strand is used as a template to repair the nick, thus completing the edit.
How PASTE works
Building on the prime-editing system, researchers at AbuGoot lab believed they could first insert a small recombination site (delivered by attB-site containing ‘atgRNA’) in the prime edited position, which can then be recognized by a serine integrase. Serine integrases are members of the serine recombinase superfamily that catalyze site-specific integration — in other words, insert a large chunk of DNA to the attachment site.
The research coined this new approach PASTE — Programmable Addition via Site-specific Targeting Elements.
Phillip Markolin shares a pretty good analogy to better understand this:
“Imagine DNA as a power cable. If you want to introduce an ‘insert’ at a specific position, usually you would have to cut the cable and re-attach the respective ends, which is pretty messy, error-prone and difficult. Now prime editing allows you to basically put another cable on top of the power cable, while nicking the bottom, getting a small patch of extra ‘string’ to become part of the whole cable. With the PASTE system, you don’t just use a small patch of cable but attach instead what amounts to a plug-and-socket module (attB site) at the specific position. You still nick the bottom cable, making the plug-socket module an intrinsic part of the cable. The plug and socket can then be detached easily and an arbitrary-length extension cable (with their own plug&socket named AttP) can be put in. This is what the serine integrase of PASTE does”.
With that in mind, you can check out figure 1 below. The top left depicted a large fusion protein consisting of SpCas9, M-MLV reverse transcriptase, and the Bxb1 integrase. This protein complex performs all the work required for genomic edits. In the middle, the attB integration site is the ‘plug-socket’ addition and the to-be-inserted gene(top right) is the ‘extension cable’. Finally, integration leaves a ‘plug/socket’ pair both left and right of the gene (red triangles).
New Possible Cure for Cancer?
Now that we understand the mechanisms that drive the PASTE system, we can apply them to new cancer treatments. In nature, we have observed that larger animals have similar rates of cancer as smaller animals. This intrigued scientists, as intuitively, having more cells increases your cases of contracting cancer. It was then discovered that the larger the animal, the more copies of tumour-suppressor genes it had. For example, elephants had 20 copies of the p53 gene, compared to our one copy. Therefore, if a mutation were to occur in this one copy of the gene, there are 19 more to carry out the role of suppressing tumours.
What if we were to insert another copy of the p53 gene into the human genome? Thanks to the work of genome sequencing efforts, we have fully mapped out the p53 gene. Furthermore, the p53 gene is 20 kb in size, making the PASTE system perfect to leverage, which can currently insert sequences up to 36 kb. This would be done by adding the protein complex that edits the genome to a lipase, which can cut the cellular membrane that envelops all cells in our body. By applying this solution to the origin of the tumour, we can effectively stop the spread of the tumour. From there, medical professionals can perfect surgery to remove the tumour. Even if they leave a small portion of the tumour, there is no longer the threat of cancer recurrence as it would not grow to a critical amount.
