Earlier today, the The Royal Swedish Academy of Sciences, Stockholm, Sweden, awarded the 2020 Nobel Prize in Chemistry to Emmanuelle Charpentier and Jennifer A. Doudna “for the development of a method for genome editing”. CRISPR-Cas9, the technique that brought the duo the coveted prize, has raised excitement and controversy in almost equal measures during its decade of existence. Here, we take a look at the brief yet tumultuous history of this pathbreaking technique, and examine how these ‘genetic scissors’ stand to revolutionize biological research.
“The power to control our species’ genetic future is awesome and terrifying. Deciding how to handle it may be the biggest challenge we have ever faced.”
Jennifer Doudna and Samuel H. Sternberg,
Pioneers of CRISPR-based gene-editing technology,
Excerpt from their book A Crack in Creation: Gene Editing and the Unthinkable Power to Control Evolution, 2017
In November 2018, Antonio Regalado, senior editor for biomedicine at MIT Technology Review, came across a curious set of medical documents posted in a Chinese registry for clinical trials. In a study apparently aimed at making babies resistant to HIV, smallpox, and cholera, a team of Chinese researchers had genetically modified human embryos before transplanting them into human uteruses. And all the while, the broader scientific world remained blissfully unaware.
Regalado broke his story on 25 November 2018. The same day, He Jiankui, the lead scientist behind the project, uploaded a series of videos on YouTube, announcing the discovery. Three days later, He presented his results at an international conference in Hong Kong, leaving scientists across the world reeling in shock. In an attempt to reduce the risk of HIV transmission, He and his team had altered the genomes of a set of human embryos created using in-vitro fertilization, then transplanted them into the uteruses of healthy women. This had led to the birth of twin babies – Lulu and Nana – the world’s first gene-edited humans. The researchers had done this by using a technology known as CRISPR to inactivate a gene called CCR5, variants of which have been shown to provide resistance against HIV.
The fallout from the announcement was immediate and far-reaching. The study was widely considered to be premature, unnecessary, and unethical. The very next day, Jiankui was suspended from the university he worked at, and an investigation was launched by Chinese authorities. Several notable researchers, including Nobel laureate David Baltimore, publicly made statements condemning the research and its negligence of ethical considerations. He’s research had been carried out in secret and not published in any peer-reviewed journal. There were irregularities in the informed consent provided by the parents of the babies, and authorities alleged that certain key ethical approval documents had been forged.
Moreover, evidence connecting the engineered form of CCR5 to HIV-resistance was nowhere near as strong as was claimed, nor had the two baby girls ever been exposed to HIV (though their father was stated to be HIV-positive). It was very possible that He had inactivated a healthy gene for no reason – in fact, one which has been suggested to play a role in memory function and recovery from strokes.
While he would be forever marked with the distinction of creating the world’s first gene-edited babies, history may also remember He as someone who set the field of genetic editing back by years by muddying the ethical waters and setting off a knee-jerk reaction against an immensely powerful technology that was still taking its baby steps at the time of his announcement.
Tinkering around in genomes
While tools to cut, join, and read DNA sequences have been around since the 1970s, the field had stalled until recently when it came to making precise genetic changes in complex organisms like mice or humans. Techniques existed, but these were fiddly, expensive, and time-consuming.
All of this changed in the early 2010s, however. CRISPR, the technique that He and his colleagues used to introduce the gene mutation, is less than a decade old but has the potential to revolutionize our ability to understand as well as manipulate the biological world.
Genes, in their most basic forms, are functional units of DNA – the hereditary material contained in our cells and passed on to our offspring when we reproduce. Like an instruction manual, our DNA contains a written record of almost all the information needed to assemble and maintain a fully-functioning human. This book is written in an alphabet containing only four letters – A, T, G, and C, each letter a small chemical structure known as a ‘base’. Three billion such bases are arranged in a precise sequence, folded, packed, and stuffed into a volume four hundred million times smaller than a droplet of water.
The codes contained within individual genes can be used to build proteins, which can take a dizzying variety of forms and carry out most of the functions that keep us alive. Being able to alter the sequences of genes can provide researchers with two main advantages. First, though the human genome contains over 30,000 genes, the precise role played by most of them is still a mystery. By disabling or ‘knocking out’ specific genes in model systems, we can observe which functions are disrupted and thus guess at what that gene might be doing under normal conditions. The second, more well-known application of gene-editing is therapeutic. Many diseases that are either caused by malfunctioning genes or believed to have genetic components can perhaps be treated by directly editing our genetic makeup.
However, while researchers have been able to edit the genes of bacteria, viruses and other simpler organisms for many years, doing so in mammals has been both expensive and technologically challenging. But the field took a leap forward in 2012, when two labs independently hit upon an idea based on a little-known, almost forgotten peculiarity of bacteria and their immune systems.
“We have discovered the secret of life!”
Francis H. Crick,
Co-discoverer of the structure of DNA,
Cambridge, England, 1953
A molecular mug-shot gallery
In 1987, a team of Japanese scientists studying the genome of E. coli, microbiologists’ favourite gut bacteria, stumbled upon certain strange DNA sequences that appeared to be repeated at regular intervals but served no known function. They mentioned this oddity in their report but could offer no explanation as to their significance.
Pretty soon, scientists were observing similar sequences in other species of bacteria. In fact, the sequences, which had been named Clustered Regularly Interspaced Short Palindromic Repeats, or CRISPR for short (pronounced ‘crisper’), seemed to be near-ubiquitous in bacteria and were even found in archae, another ancient branch of one-celled microorganisms. It would be another two decades before the mystery over their function would be resolved, primarily through the efforts of Spanish scientist Francisco Mojica, as well as other teams of scientists throughout the world.
The most crucial clue lay not in the CRISPR sequences themselves, but in what resided between them. The CRISPR regions were separated by short sequences called spacers, and researchers realised that these spacers bore a strong resemblance to the genomes of a completely different class of beings – viruses. It turned out that the CRISPR sequences, their spacer regions, and a set of genes located close to the CRISPR sites, called CRISPR-ASsociated proteins (or Cas proteins) form an elaborate and ingenious adaptive immune system that bacteria use to fight off attacks from viruses.
When our bodies encounter a foreign particle, like a bacterium or virus, our immune cells recognize it and create antibodies to mark it for destruction. This response is highly specific – we desire to kill off the disease-causing microorganisms (also known as “pathogens”) but would prefer to leave our own cells and our body’s many harmless inhabitants in peace. Once the germs are destroyed, the memory of this attack remains in our body, so that if we encounter the same bacteria or virus again, we can quickly mount an armed defence.
Just like us, bacteria are also plagued by pathogens, primarily viruses of various kinds. Viruses that attack bacteria are known as phages, and they are remarkably good at latching on to the surface of bacterial cells and injecting their genetic material – DNA or RNA (a close cousin of DNA with minor chemical differences) – into the bacteria’s body. There, they hijack the bacteria’s cellular machinery and use it to make numerous copies of themselves which eventually burst out from their unfortunate host to attack other bacteria in the vicinity. Bacteria and viruses have, therefore, been evolving in a constant arms race against each other for millions of years – the viruses keep coming up with new strategies to infect the bacteria, while the bacteria devise methods to thwart these efforts, sending the viruses back to the drawing board – and the cycle continues.
One of the ways by which bacteria fight viral attacks is using special enzymes called nucleases, whose sole aim in life is to find stretches of DNA (or RNA) and destroy them. Here, a key problem arises. The bacteria’s own genetic material is made up of DNA, and unless there is a way to recognise viral DNA specifically, the nucleases may end up chewing up the bacteria’s own genome, effectively pressing a self-destruct button. This is where the beauty and ingenuity of the CRISPR system come into play.
Every time a virus infects a bacterial cell and is successfully fought off, the bacteria takes a portion of the virus’s snipped DNA and inserts it into its own genome. These stretches are flanked by the CRISPR sequences to mark them as ‘foreign’ and over time, this forms the bacteria’s personal little library of virus attackers. Noted science journalist Carl Zimmer calls this a “molecular most-wanted gallery”. The bacteria then copy these sequences into short “guide RNA” molecules and hand them off to a protein called Cas9, which happens to be a nuclease. But unlike garden-variety nucleases that, like patrolling police officers, are forever on the lookout for suspicious characters, Cas9 is more of a special agent, armed with a photo and a description of the suspect.
When a virus next attacks the cell, Cas9 is there, guide RNA in hand, and if its sequence matches the virus’s DNA, Cas9 makes a precise cut to dispose of the invader neatly. Since the original library of spacer sequences remains intact, the bacteria can transfer this immune memory to their offspring. In this regard, the bacteria manage to steal a march on us humans who, despite our Shakespeare and the stock market, are still unable to pass on our acquired immunity to our children.
Researchers piecing together this system were elated by its elegance, and one of its first applications was in the dairy industry, where Danish food-processing company Danisco harnessed the system to create phage-resistant bacteria for yoghurt and cheese production. It took a few more years, before two teams of researchers simultaneously and independently hit upon the idea of tricking this system and turning it to our own uses.
It is easy to synthesize RNA strands in the laboratory. By the simple expedient of providing the Cas9 nuclease with a new guide RNA, it can be used to precisely target any gene sequence we want. Not only can this be used to inactivate rogue genes, but completely new sequences can also be inserted in the genome by strategically including new pieces of DNA in the mix. And all this can be done several times faster and at a fraction of the cost of any other gene-editing technique.
In 2012, French microbiologist Emmanuel Charpentier and American biochemist Jennifer Doudna published a landmark paper demonstrating that the CRISPR-Cas9 system can be reprogrammed to target an entirely new sequence of DNA in bacteria (a team led by Virginijus Siksnys from Lithuania also published very similar results at the same time). A year later, Feng Zhang’s team from the Broad Institute of MIT and Harvard was the first to demonstrate the use of this system in human and mouse cells – CRISPR was no longer a weapon of bacteria alone.
Pathbreaking innovation or playing God?
While a protracted legal battle over patenting this technology followed these announcements, researchers across the world were quick to grasp the potential applications of this technology. CRISPR has been used to improve the quality of crops, cure genetic ailments in mice, screen drugs to treat cancer, and has even been incorporated into proposed strategies for controlling vector-borne diseases like malaria. Recently, during the COVID-19 pandemic, a team of Stanford bioengineers came up with a CRISPR-based technique called PAC-MAN that might be able to neutralize the novel coronavirus’s genome.
However, the short history of this technology has also been fraught with controversies. As He Jiankui’s experiment has shown, in the absence of international guidelines and consensus on the use of CRISPR, and in the face of its immense projected profitability, we risk prematurely unleashing genetic changes upon the world using a technique whose safety has not yet been adequately tested. In particular, researchers warn against the fact that many genes play complex roles in our bodies, and disabling or altering them without a complete understanding of their function can lead to more harm than good.
The technique of editing germline cells (eggs and sperms) or embryos also give rise to tricky ethical questions – to what extent do we have the right to play with the biology of future generations? How do we mitigate unforeseen consequences that may arise decades down the line? And finally, in a world still bearing the scars of 20th-century eugenics, to what extent do we risk exacerbating existing societal inequalities and prejudices when parents get their hands on technology that might allow them to create ‘designer babies’?
In 2019, a Chinese court sentenced He Jiankui to three years in prison and a fine of 3 million Chinese Yuan (about Rs 3.2 crores) for having “forged ethical review documents and misled doctors into unknowingly implanting gene-edited embryos into two women.” A few months earlier, the World Health Organization (WHO) had put out a statement urging regulatory authorities throughout the world to disallow any further work that might result in the birth of gene-edited babies “until its implications have been properly considered.”
In the early 1970s, when the recombinant DNA technology revolution was just beginning, the international scientific community took an unprecedented step and placed a self-imposed moratorium on research until proper biosafety guidelines could be agreed upon and drawn up. Whether such drastic measures are necessary or even enforceable in today’s world remains to be seen. However, one thing is for certain. “Just because we are not ready for scientific progress does not mean it won’t happen,” writes Doudna in her book A Crack in Creation: Gene Editing and the Unthinkable Power to Control Evolution. Neither science nor society is a dispassionate observer of our reality. Whatever the 21st century has in store for us, it would be the conjunction of the two that will decide the future of our species.
Author’s note: A version of this article was first published on “Bandhan”.