From the corrective snipping of defective sections of human DNA to the emergence of new and hazardous genetic imperfections, the line is thin, particularly in cases involving diseases with an irregular and complex genomic background.
Since its discovery in 2012, CRISPR Cas9 – the most advanced tool in genomic editing – has been under continuous investigation by the global scientific community for its potential diagnostic and therapeutic uses.
Its applications range from correcting genetic defects underpinning certain rare diseases to tackling conditions such as muscular dystrophy, cystic fibrosis, neurological disorders like Alzheimer’s and Parkinson’s, and specific types of cancer.
The distinctive feature of CRISPR Cas9, compared to previous gene editing techniques, lies in the streamlined and accelerated genome modification process it introduced. With CRISPR Cas9, the procedure involves identifying and cutting the faulty DNA sequence, followed by the integration of the corrected one.
TAKEAWAYS
The risks of CRISPR Cas9: the irreversible genetic cut-and-paste
CRISPR Cas9, a genetic cut-and-paste tool, has shown potential risks from the outset: the Cas9 protein can make off-target cuts that are irreversible and permanent. This concern has driven ongoing efforts over the years to refine the technique, aiming particularly at making genetic modifications reversible.
To step back briefly and revisit what the universal acronym CRISPR stands for (Clustered Regularly Interspaced Short Palindromic Repeats), it refers to segments of DNA found in bacteria. These segments help microorganisms detect and dismantle the genomes of viruses similar to those that created the palindromic repeats. Essentially, CRISPR acts as a natural defence mechanism for bacteria against external attacks.
The observation and study of this defence mechanism have progressively led to advanced genetic engineering techniques for manipulating DNA in plants, animals, and humans. Initial research into what would later be known as “CRISPR” began in 1987 at Osaka University, Japan. The actual acronym was introduced in 2001 to unify the terminology for various bacterial DNA sequences previously referred to differently in scientific literature.
Further breakthroughs included the identification of a CRISPR system within “Streptococcus pyogenes” bacteria, utilising the Cas9 protein. This protein functions as a “molecular scissor,” defending against pathogens.
In 2012, scientists Emmanuelle Charpentier and Jennifer A. Doudna formalised this system as a groundbreaking tool capable of cutting targeted DNA sequences in plant, animal, and human cells, removing and replacing them with others. Yet, despite CRISPR Cas9’s ability to target specific defective genome sections, it remains true that «in certain conditions, repair can lead to new genetic defects, as seen in chronic granulomatous disease».
This warning comes from a team of scientists in the Division of Gene and Cell Therapy at the University of Zurich’s Institute for Regenerative Medicine. Their findings, published in “Gene editing of NCF1 loci is associated with homologous recombination and chromosomal rearrangements” (Communications Biology, October 2024), shed light on these complications. Let’s explore this further.
The impact of molecular scissors on Chronic Granulomatous Disease
Chronic Granulomatous Disease (CGD) is a rare inherited genetic condition affecting approximately one in 120,000 individuals. The immune system of those with CGD is compromised, unable to effectively combat and clear certain bacteria and fungi from the body. As a result, patients often suffer from recurrent and potentially life-threatening infections [source: National Institute of Allergy and Infectious Diseases].
From a DNA sequence perspective, the study team explains, «it is caused by the absence of two nucleotides in the NCF1 gene. This genetic defect results in the inability to produce an enzyme complex crucial for immune defence against bacteria and fungi».
The focus of the research was to use CRISPR Cas9 to cut the faulty DNA sequence and insert the missing nucleotides.
The experiment was conducted on cultured immune cells exhibiting the same genetic defect as those found in individuals with CGD. During these lab tests, new defects were observed in some of the edited cells. Specifically, the authors point out, «entire sections of the chromosome were missing precisely at the site of the cut-and-paste».
The cause of this issue lies in the unique «genetic constellation» of the NCF1 gene, «which appears three times on the same chromosome: once as an active gene and twice as pseudogenes. These pseudogenes share the same sequence as the defective NCF1 but do not typically contribute to the formation of the enzyme complex due to their lack of cellular expression». So, what exactly happened?
The risks of CRISPR Cas9 when failing to differentiate between multiple gene variants
Among the risks associated with CRISPR Cas9’s molecular scissors, researchers at the University of Zurich highlight the possibility of accidental DNA strand cuts at multiple points on the chromosome. This occurs because the system cannot reliably distinguish between different versions of a specific gene, such as NCF1: «When the cut sections are subsequently rejoined, entire segments of DNA may become misaligned or missing. The consequences are unpredictable and, in the worst-case scenario, may contribute to diseases such as certain types of leukaemia».
To mitigate these negative impacts, several alternative approaches to correcting the genetic defect underlying Chronic Granulomatous Disease have been developed. These include modified versions of the CRISPR system components and the integration of protective elements designed to minimise the likelihood of cutting the chromosome at multiple sites. However, none of these solutions has proven entirely effective in preventing the side effects of genetic cut-and-paste techniques.
More specifically, the research team explains, to ensure precise identification of the genetic mutations to target, they «have established a method based on ddPCR, a rapid and reliable technique for quantifying the NCF1 gene and its surrounding sequences».
ddPCR (short for Droplet Digital PCR) is a molecular biology technique that selectively replicates DNA segments by partitioning the sample into thousands of droplets: «This allows for the amplification of target molecules within each individual droplet» [source: Droplet-based digital PCR (ddPCR) and its applications – TrAC Trends in Analytical Chemistry, 2023].
Nevertheless, even with this solution, the researchers report that although «a modest reduction in chromosomal alterations was observed, the concurrent decline in editing efficiency could likely hinder its future clinical applicability».
Glimpses of Futures
The study outlined reveals a critical flaw in gene therapies based on CRISPR Cas9 and highlights efforts to address it, offering valuable insights for developing genetic modification interventions tailored to Chronic Granulomatous Disease. Undoubtedly, years of research will be required, but the direction is clear.
To anticipate potential future scenarios, let us now use the STEPS framework to analyse the impacts of advancements in the study of CRISPR Cas9 risks across Social, Technological, Economic, Political and Sustainability dimensions.
S – SOCIAL: the application of the CRISPR Cas9 system as a therapy for diseases with a complex genetic framework – characterised by pseudogenes that misdirect the action of the molecular cut-and-paste, as in Chronic Granulomatous Disease – is called into question by chromosomal rearrangements caused by the presence of multiple targets. This presents us with a crossroads, requiring, on the one hand, the abandonment of genomic editing in such cases, given the additional risks to patients’ health, and, on the other hand, the pursuit of a tailored genetic scissor for diseases like the one examined by the Swiss scientists. The results obtained from the study point in the latter direction, suggesting, for the future, the necessity of a careful evaluation of the specific genomic context (excluding the presence of homologous regions) and opening the door to the correction of genetic defects underlying a wider range of rare diseases.
T – TECHNOLOGICAL: in recent years, the discovery of new CRISPR systems (a study from November 2023, conducted by the National Center for Biotechnology Information of the US National Institutes of Health in collaboration with the McGovern Institute for Brain Research and the Broad Institute, both affiliated with the Massachusetts Institute of Technology, identified over a hundred) signals the emergence of increasingly precise genetic cut-and-paste technologies with even more refined mechanisms of action. Combined with progress in research on the risks of CRISPR Cas9, this points to a future – within the next two decades – where its application could expand to a broader range of genetic diseases.
E – ECONOMIC: ìin a hypothetical future scenario where the type of studies described leads to CRISPR Cas9 overcoming all its limitations and becoming a viable therapy for many genetic diseases, it will become urgent for governments worldwide to revise the pricing frameworks for specialised outpatient services. These would need to include advanced technological interventions such as those related to genetic engineering, which are still, in many cases, excluded and entirely borne by families. According to data from a study on the economic impact of rare diseases in countries like Germany, France, and Italy (Rare disease burden of care and the economic impact on citizens in Germany, France and Italy, published in late October 2023), the average annual cost per patient currently stands at €107,000.
P – POLITICAL: from a political perspective, in the future, the greatest impact of an increasingly accurate and efficient genomic editing system. – potentially becoming a therapeutic tool for genetic disorders – will always be linked to the commitment of governments and institutions to ensuring safety. This will involve overseeing the effects of CRISPR Cas9 on the overall health of patients and continuously monitoring its potential long-term risks. Safeguarding human health must remain at the core of the objectives for gene therapies developed using molecular scissors.
S – SUSTAINABILITY: as genomic editing technologies evolve for therapeutic applications, their impact on sustainability lies in the imperative to ensure universal access to gene therapies. This aligns with the principles of healthcare equity and the right to meaningful social support for individuals living with rare genetic disorders.
