A fresh study leveraging a new prime editing approach brings to the table a bunch of hands-on and computer-based tools to dive deep into the genetic changes happening in cancer cells.
Talking about genetic variants linked to diseases opens up a vast and intricate field. Here, changes in loads of different genes caused by a disease can lead to various kinds of mutations in each gene involved. «Some mutations might just switch one DNA piece for another, while others might add or remove big chunks»[source: “Mutation, Repair and Recombination” – National Library of Medicine].
In a piece out on Nature Biotechnology on March 12, 2024, “High-throughput evaluation of genetic variants with prime editing sensor libraries” from MIT’s Biology Department, the team sheds light on how cancer genomes are a «mix of single nucleotide changes and big shifts in how many copies of genes there are. This mix can mess with many genes in different ways, depending on the type of change, what the gene does, and the biological setting».
They point out that knowing a tumour’s genetic makeup is crucial for understanding how the disease starts, grows, and responds to treatment. Yet, we’re still figuring out what the heck thousands of mutations in cancer cells mean for how genes and proteins act. Getting a handle on this could hugely help tailor cancer treatments down the line, particularly for patients with specific genetic quirks in their tumours.
TAKEAWAYS
Prime editing: what it’s all about and why it’s different from CRISPR Cas9
The team at the Massachusetts Institute of Technology has pinpointed prime editing as the go-to method for whipping up and shaping – before taking a closer look and evaluating – the genetic variants of cancer genomes, right down to the tiniest detail, within their natural genomic setting. The goal? To get a grip on how these variations influence cancer development and, more critically, how they react to treatment, all within their actual biological environment. But, before we dive in, let’s clarify a few things.
Prime editing is a way of tweaking genomes that builds on the CRISPR tech that labs all over the globe have been using since 2012. It lets scientists tweak DNA sequences in plants, animals, and humans with a lot more accuracy and control than before, sidestepping the unwanted edits that can sometimes happen with other methods.
The first to chat about this were researchers from the Merkin Institute of Transformative Technologies in Healthcare at the Broad Institute of Harvard, including David R. Liu from MIT. In a piece for Nature on 21 October 2019, “Search-and-replace genome editing without double-strand breaks or donor DNA” the crew laid out how prime editing works. Unlike CRISPR Cas9, it doesn’t snip through both strands of DNA; instead, it uses a guide RNA to hit the exact spot that needs changing.
In a more recent article, dropped in the August 2023 edition of Trends in Biotechnology – “Prime editing: advances and therapeutic applications” – prime editing is described as «a neat “search and replace” method. It can swap any base pair, tack on a bit here, trim a bit there, all without breaking the DNA apart».
Creating genetic variants where they naturally occur
David R. Liu is now pointing out how prime editing steps up to meet one of the huge challenges that comes with CRISPR-based genome editing, which is to let scientists «create any kind of genetic variant right in its natural setting».
But when we’re talking about cancer cell genomes, reaching this goal meant they had to come up with a special technique that could make the editing process even sharper and more reliable.
The guide RNAs (called “pegRNA”) that prime editing uses to get CRISPR enzymes to the exact DNA spot for cutting are known for their varying levels of success. This leads to a bit of “variability” (or in simpler terms, “noise”) in the results from pegRNAs that don’t make the change correctly, as the researchers put it:
«One thing that makes using prime editing to check out genetic variants a bit tricky is how the editing success can vary a lot between different pegRNAs. Previously, a bunch of computer-based tools were made for designing pegRNAs, including some smart algorithms that can whip up guide RNA sets meant to make really efficient changes. But even with these high-tech pegRNAs, you still need to do a bunch of tests to make sure they work as expected, and there’s no promise they’ll work the same in every type of cell»
So, to cut down on this natural unpredictability, the team came up with a way that leans on “synthetic target sites” to help figure out how well each guide RNA does its job.
Putting it simply, by checking how often each pegRNA used by the system gets the modification right at these synthetic sites, this fresh approach has given the researchers a way to obtain an empirical measurement of the efficiency of each pegRNA.
Looking into cancer cells’ genetic variants: spotlight on the TP53 gene
The test run of the new prime editing tech took a deep dive into one gene in particular, TP53. This gene’s famous for having genetic variants in over half the folks dealing with cancer. Yet, these variations are mostly uncharted territory, as the research crew points out.
Digging deeper, they looked at human lung adenocarcinoma cells that had over a thousand different tweaks to this specific gene. The mutations put in place by the study’s authors, thanks to the new method, led to checking how well these sick cells could hang on, seeing how each tweak affected the cell’s form.
What jumped out from this first round of experiments was how different the MIT team’s findings were from earlier studies. «Previous work involved editing genes to stick artificial copies of the mutated TP53 gene into the cancer cell». Interestingly, the mutations introduced with this fresh take on prime editing, even though they stopped the cancer-fighting p53 protein from teaming up into a quartet, still let the cancer cells keep on living. Meanwhile, earlier research had reached the complete opposite end, marking those same mutations as “harmless”.
This really highlights, according to the researchers, a scenario where the genetic makeup of cancer cells, when sparked in the lab, only really comes to light if those changes are crafted and shaped in their natural genomic setting, not by sticking to artificial methods.
Glimpses of Futures
While the experimentation is in its early stages, the authors are looking toward the future application of their prime editing technique for the study of genetic variants observed in other genes linked to oncological diseases. The primary goal is twofold: to develop patient-specific treatments tailored to cure the specific mutations of that particular gene and to predict how an individual’s tumour might respond to a certain drug treatment.
Using the STEPS framework, let’s now attempt to foresee possible future scenarios by analysing the impacts that the evolution of the new prime editing technique for the generation, modelling, and analysis of genetic variants of tumour genomes in their native context might have from a social, technological, economic, political, and sustainability perspective.
S – SOCIAL: the approach taken by the Massachusetts Institute of Technology is aimed at broadening our understanding of the complex world of pathogenic genetic variants, proposing – in a future scenario – to develop increasingly personalised and never standard therapies. More specifically, in oncology, the ability to generate precise genetic variants of a given tumour genome within its biological environment – thanks to genomic editing – to see if these kill the diseased cells or let them reproduce, opens up broad prospects where the patient, with their unique characteristics and highly personal genetic profile, is at the absolute centre of the therapeutic scene.
T – TECHNOLOGICAL: in the future – the research team anticipates – the technique of “synthetic target sites”, used to measure the efficiency level of the guide RNAs utilised by the prime editing system and thus reduce its inherent variability, might, for example, be employed to study the impact of endogenous genetic variants on drug resistance or other cellular phenotypes relevant to cancer. Furthermore, additional methods could be developed to address the inherent variability in editing efficiency and make it an even more reliable tool in investigating genetic variants.
E – ECONOMIC: from an economic standpoint, the most critical aspect of a future possible adoption of the new prime editing technique to induce (and study) genetic variants of tumour cells in their genomic context, is related to costs, still high for all operations related to genetics and not supported by our National Health System. In this regard, as of January 2024, the new outpatient specialist fee schedule has come into effect, which, however, has not yet included many medical genetics services, including those for the early diagnosis of rare diseases. This remains a significant issue to be resolved if we are to keep pace with scientific research and its discoveries for the benefit of human well-being and health.
P – POLITICAL: from a political perspective, the impact of a future evolution of the new prime editing technique for generating and analysing the genetic variants of tumour genomes in their natural environment will necessarily have to go through a policy that supports the exemption regime for medical services of a genetic nature, which, as mentioned, are still excluded from the outpatient specialist fee schedule in our country.
S – SUSTAINABILITY: in a possible future scenario still marked by the absence of financial support from the National Health System for oncological expenses that involve the use of genomic editing systems for the search for personalised therapies, the community will still have to deal with the moral imperative, inherent in every democratic country, which mandates supporting the economically and socially most vulnerable people, especially in cases of physical and/or psychological hardship and distress.
