Throughout the annals of history, individuals have accomplished remarkable feats, navigating epochs of adversity, toil, and misfortune with a tenacious spirit that propelled them to achieve unprecedented breakthroughs. One groundbreaking achievement stands amidst fictional narratives, elevating what was once deemed improbable to the realm of possibility — Clustered Regularly Interspaced Short Palindromic Repeats, commonly known as CRISPR. Etching itself into the forefront of scientific innovation, CRISPR is a technology that research scientists use to selectively modify the deoxyribonucleic acid (DNA) of living organisms. While CRISPR might seem like a concept from the realm of science fiction, its roots extend into our cultural imagination through iterations in books and movies. In cinematic storytelling, the idea of genetic sequencing and manipulation found expression in movies like "Gattaca," where a future society relies on advanced genetic engineering for societal stratification. This film anticipates themes related to precision gene editing and its societal implications, echoing some of the principles underlying CRISPR technology. In the literary world, novels such as "Oryx and Crake" by Margaret Atwood delve into speculative futures where genetic engineering plays a central role. Through these cultural representations, the concept of manipulating genes, now a reality with CRISPR, has long captured our collective imagination. In the following exploration, we delve into the intricacies of this cutting-edge technology, unraveling the complex narrative that connects CRISPR's origins to its profound implications for the future.
Origin of CRISPR
The emergence of CRISPR gained public attention in the early '90s when Spanish scientist Francisco Mojica identified the first CRISPR locus, officially reported in 1993. Throughout the decade, he discerned common features in seemingly disparate repeat sequences, coining the term "CRISPR" in 2002. By 2005, he associated these sequences with bacteriophage genomes, proposing the hypothesis that CRISPR functions as an adaptive immune system.
Spanish microbiologist Francisco Mojica explains his CRISPR discovery in Sydney. Image by ABC
Cas9 and PAM Discovery
Image by Integrated DNA Technologies
In May 2005, Alexander Bolotin from the French National Institute for Agricultural Research (INRA) delved into the genome of Streptococcus thermophilus, a type of bacteria commonly found in yogurt that plays a crucial role in the fermentation process, turning milk into yogurt. Known for its ability to thrive in warm temperatures, this bacterium is well-suited for the yogurt-making environment. Notably, the CRISPR array discovered showcased novel CRISPR-associated genes (Cas genes), a set of genes often found in proximity to CRISPR sequences in the genomes of bacteria and archaea. In its explanation, the CRISPR-Cas system works by storing small pieces of viral DNA, known as spacers, within the organism's own genome.
Random mutagenesis in vegetatively propagated crops: opportunities, challenges and genome editing prospects - Scientific Figure on ResearchGate. Available from: https://www.researchgate.net/figure/CRISPR-Cas9-system-is-a-monomeric-protein-consisting-of-a-single-Cas9-enzyme-that-makes-a_fig5_354101491 [accessed 22 Dec, 2023]
Today, Cas genes, including one encoding a significant protein with nuclease activity, are now recognized as Cas9. Bolotin's team, in this genetic odyssey, identified a recurring sequence at the spacers' edge — the protospacer adjacent motif (PAM), a vital cue for target recognition. This revelation was a eureka moment, unleashing the transformative power of Cas9 as a genetic maestro capable of precise gene editing. In this narrative of scientific intrigue, Bolotin's exploration marked a turning point, paving the way for revolutionary advancements in genetic manipulation.
Hypothetical scheme of adaptive immunity
Eugene Koonin, Ph.D. NIH Distinguished Investigator
Evolutionary Genomics Research Group, NLM/NCBI. Image by NIH
A year later, in March 2006, the intriguing scientific saga unfolded as Eugene Koonin, a visionary researcher from the US National Center for Biotechnology Information, embarked on a computational odyssey. His quest involved deciphering clusters of orthologous groups of proteins, and in this digital expanse, Koonin unfurled a hypothetical scheme for CRISPR cascades, envisioning them as the unwavering sentinels of bacterial immunity.
Breaking away from past conjectures that the Cas proteins might form an avant-garde DNA repair ensemble, Koonin's revelation danced on the idea of inserts homologous to phage DNA nestled within the natural spacer array. This conceptual shift marked a seismic departure, redefining the CRISPR narrative. Published in the chronicles of scientific exploration by Dr. Kira Makarova and her team in 2006, Koonin's visionary proposal laid the groundwork for understanding CRISPR cascades as the vigilant gatekeepers of bacterial defense, weaving a tapestry of adaptive immunity in the intricate dance of genetic guardianship.
Dr Kira Markova (Image by NLM)
CRISPR's Experimental Triumph
Philippe Horvath (Image by National Inventors Hall of Fame)
In a groundbreaking moment in 2007, Philippe Horvath and the scientific pioneers at Danisco France SAS unveiled a transformative chapter in the CRISPR saga. Working with the widely-used bacterium Streptococcus thermophilus in the dairy industry, they embarked on a quest to understand how it combats phage attacks — a recurring challenge in industrial yogurt production. Through meticulous experimentation, Horvath and his colleagues definitively demonstrated the adaptive nature of CRISPR systems. These microscopic guardians showcased their prowess by integrating new phage DNA into the CRISPR array, arming themselves for the impending battle against invading phages.
Image by MDPI
The revelation that Cas9 might be the sole protein orchestrating this defensive symphony marked a scientific triumph, unraveling the intricate details of CRISPR's interference mechanism. This experimental feat not only illuminated the adaptive immunity encoded in CRISPR but also paved the way for a revolution in genetic defense strategies.
CRISPR and DNA targets
Luciano Marraffini (Image by The Rockefeller University)
Erik Sontheimer (Image by UMASS Chan Medical School)
In December 2008, Luciano Marraffini and Erik Sontheimer of Northwestern University upended assumptions about CRISPR. Contrary to expectations and preconceived notions that CRISPR mimics eukaryotic ribonucleic acid (RNA), a molecule that serves as a crucial intermediary in the transfer of genetic information from DNA to protein synthesis in cells, they revealed that it plays a DNA-centric symphony. This unexpected revelation challenged the scientific community, suggesting CRISPR's potential beyond bacteria. Marraffini and Sontheimer then hinted at transformative applications beyond its microbial origins.
Image by BYJU'S
In 2011, the foundational work on CRISPR-Cas9 took place, but the revolutionary discovery regarding its use as a "cut-and-paste" tool for genome editing was officially reported in 2012. Jennifer Doudna and Emmanuelle Charpentier from the University of California, Berkeley, published their landmark paper in 2012 detailing the application of CRISPR-Cas9 for precise genome editing. Feng Zhang, George Church, and others also contributed to the rapid advancement of CRISPR technology around the same time. Therefore, while early work on CRISPR started in 2011, the key breakthroughs and the transformative realization of its potential occurred in 2012.
CRISPR-Cas9 Revolution: From Genome Editing Discovery to Applications in Disease Treatment and Beyond
CRISPR pioneers (from left to right): George Church, Jennifer Doudna, Feng Zhang, and Emmanuelle Charpentier. Image by SCIENCE
In 2012, Jennifer Doudna and Emmanuelle Charpentier, along with Feng Zhang from the Massachusetts Institute of Technology (MIT) and George Church from Harvard University, made a groundbreaking discovery. By designing guide RNA to target a specific region in the genome, they found that the CRISPR-Cas9 system could be employed as a "cut-and-paste" tool to modify genomes. This DNA-editing tool enables CRISPR-Cas9 to remove or introduce new genes and also able to silence or activate genes.
CRISPR-Cas9 harnessed for genome editing
Eukaryotic Cell Diagram by Sciencefacts.net
In a groundbreaking achievement in January 2013, Feng Zhang reached a pivotal milestone by successfully applying CRISPR-Cas9 for genome editing in eukaryotic cells. Eukaryotic cells, known for their complexity and structural organization, serve as the fundamental building blocks of organisms within the domain Eukaryota. Zhang's remarkable feat included the precise editing of genes in mammalian cells, including those of humans. This adaptation of CRISPR-Cas9 represented a significant leap forward in the field of genetic engineering, expanding the potential applications of this revolutionary technology beyond its initial use in bacterial systems. The precision afforded by CRISPR-Cas9 in editing the genome of eukaryotic cells paved the way for novel frontiers in genetic research and therapeutic possibilities. This groundbreaking achievement heralded a transformative era in molecular biology, offering unprecedented opportunities for understanding, manipulating, and potentially treating genetic conditions in complex organisms.
Image by LABIOTECH
Controversial Case and Ethical Debate
In 2018, the scientific community was shocked when Chinese biophysicist He Jiankui publicly announced, through a series of YouTube videos, a groundbreaking event — the genetic alteration of twin sisters' genes named Lulu and Nana. Utilizing CRISPR-Cas9, he modified the DNA in the embryos of the twin girls, rendering them resistant to human immunodeficiency virus (HIV) inherited from their parents. This historic moment triggered extensive ethical debates and raised concerns about the unexplored terrain of manipulating the human germline.
He Jiankui speaking about the success of genetic alteration (Image by MIT Technology Review)
At the heart of the discourse was the apprehension regarding potential unforeseen consequences and the long-term effects of gene editing, prompting questions about safety and the possibility of unintended mutations. Compounding the issue, the scientific community criticized the lack of transparency and insufficient ethical review. This episodic period propelled itself to the forefront, leading to a call for robust ethical guidelines and international collaboration in the rapidly advancing field of gene editing.
The subsequent year saw Jiankui listed as one of Time's 100 most influential people of 2019, categorized under "Pioneers." However, some media outlets labeled him a "rogue scientist," "China's Dr. Frankenstein," or a "mad genius." In a legal turn, a court in Shenzhen found Jiankui guilty of "illegal medical practices," sentencing him to three years in prison, accompanied by a 3 million Yuan fine, as reported by the Chinese news agency Xinhua.
Genetic Scissors awarded Nobel in 2020
Charpentier and Doudna received Nobel Prize in 2020. Image by Niklas Elmehed, Nobel committee
In 2020, after eight years of publishing their research together, Charpentier and Doudna were awarded the Nobel Prize in Chemistry — making them only the sixth and seventh women in history to win a chemistry prize. It had been just eight years since Doudna and Charpentier co-authored their first paper, demonstrating the immense possibilities of CRISPR-Cas9. That year, Claes Gustafsson, chair of the Nobel Committee for Chemistry, commented, “There is enormous power in this genetic tool, which affects us all. It has not only revolutionized basic science but also resulted in innovative crops and will lead to groundbreaking new medical treatments.” Since then, the technology has exploded, with doctors beginning to experiment — seeking cures for genetic disorders such as sickle cell disease and hereditary blindness. In other fields, the use of technology includes creating new crops, and some researchers are even trying to use CRISPR to bring species back from extinction.
Future of CRISPR
The journey of CRISPR weaves through time, tracing its humble origins in the '90s to groundbreaking achievements that have reshaped the landscape of genetic research and the world of medicine. It stands as a testament to human ingenuity and the relentless pursuit of knowledge. The capability to precisely edit the complex genetic code of living organisms, once a figment of science fiction, is now a powerful reality, offering unprecedented opportunities and posing profound ethical questions.
The bestowal of the Nobel Prize in Chemistry upon Jennifer Doudna and Emmanuelle Charpentier in 2020 not only celebrated their remarkable contributions but also underscored the transformative impact of CRISPR on our understanding of life itself. Peering into the unknowable future, CRISPR holds the potential to revolutionize medical treatments, providing tailored solutions for genetic disorders, infectious diseases, and even cancers. However, this quest for expanding life expectancy has raised the world’s ethical concerns.
Image by Google DeepMind
The power to manipulate the very fabric of life, this god-like ability, demands a judicious balance between scientific exploration and ethical responsibility. The lessons from the controversial case of He Jiankui serve as a stark reminder of the imperative to tread cautiously, ensuring that the promise of CRISPR is realized without compromising our ethical compass.
In this era of unprecedented possibilities, the story of CRISPR unfolds as a narrative of human curiosity and its potential to reshape the course of biology. The chapters yet to be written will undoubtedly reveal new dimensions of discovery, challenge our preconceptions, and invite us to navigate the ethical complexities of playing genetic maestros — and certainly, the future will be defined by the ethical choices we make.
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