The CRISPR Revolution: Tracing the History of Genome Editing

Imagine being able to edit the genetic code of any living organism with precision and ease. This is the revolutionary promise of CRISPR technology—a tool that has transformed genetics and biotechnology in a remarkably short time.

Let’s delve into the captivating history of CRISPR, exploring the key discoveries and brilliant minds that paved the way for this groundbreaking advancement.

1987: The First Glimpse of Mysterious Repeats

The story begins in 1987, when a team of Japanese scientists led by Yoshizumi Ishino at Osaka University stumbled upon unusual repeating sequences in the DNA of Escherichia coli while studying a gene called iap. These repeats were separated by unique sequences, but their purpose remained a mystery for years.

1993-2005: Unveiling the CRISPR Locus

Francisco Mojica at the University of Alicante in Spain was intrigued by these enigmatic repeats. Throughout the 1990s, he studied similar sequences in various microorganisms and noticed they shared common features. In 2000, he proposed that these repeats formed a distinctive genetic locus.

By 2002, the term CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) was coined by Ruud Jansen and colleagues, standardizing the nomenclature and drawing more attention to these sequences.

In 2005, Mojica and other independent groups, including Gilles Vergnaud and Alexander Bolotin, discovered that the unique sequences between the repeats matched fragments of viral DNA. This led to the hypothesis that CRISPR serves as an adaptive immune system in bacteria, allowing them to “remember” and defend against viral invaders by incorporating snippets of viral DNA into their own genomes.

2005: Discovery of Cas Genes and the PAM Sequence

Alexander Bolotin and his team at the French National Institute for Agricultural Research (INRA) were studying Streptococcus thermophilus when they identified a novel protein associated with the CRISPR system, later named Cas9 (CRISPR-associated protein 9). They also discovered the Protospacer Adjacent Motif (PAM), a short DNA sequence essential for the CRISPR system to recognize and target viral DNA accurately.

2006-2007: Theoretical Framework and Experimental Proof

In 2006, Eugene Koonin at the National Institutes of Health proposed a theoretical model suggesting that CRISPR is part of an adaptive immune system in prokaryotes. He outlined how bacteria could acquire resistance to viruses by integrating fragments of viral DNA into their genomes.

Building on this theory, in 2007, Philippe Horvath and colleagues at Danisco (now part of DuPont) provided the first experimental evidence. They demonstrated that S. thermophilus could acquire resistance to bacteriophages by incorporating phage DNA into its CRISPR loci, confirming the adaptive immune function of CRISPR systems.

2008-2011: Deciphering the Molecular Mechanism

Researchers began to unravel how the CRISPR-Cas system operates at the molecular level:

? 2008: John van der Oost and his team at Wageningen University in the Netherlands showed that bacteria transcribe the viral DNA snippets in CRISPR loci into small RNA molecules called CRISPR RNAs (crRNAs). These crRNAs guide Cas proteins to target and cleave invading viral DNA.

? 2008: Luciano Marraffini and Erik Sontheimer at Northwestern University made a pivotal discovery that CRISPR interference targets DNA rather than RNA. This finding differentiated CRISPR from other gene-silencing mechanisms like RNA interference (RNAi).

? 2010: Sylvain Moineau and colleagues at Laval University in Canada demonstrated that Cas9, guided by crRNAs, creates precise double-stranded breaks in viral DNA, effectively neutralizing the threat.

? 2011: Emmanuelle Charpentier at Ume? University in Sweden discovered a second RNA molecule essential for the CRISPR-Cas9 system, called the trans-activating CRISPR RNA (tracrRNA). She showed that tracrRNA pairs with crRNA to form a complex that guides Cas9 to its DNA targets, which is crucial for its DNA-cutting activity.

2011-2012: Engineering CRISPR for Gene Editing

The realization that the CRISPR-Cas9 system could be harnessed for targeted DNA cleavage led to groundbreaking advancements:

? 2011: Virginijus ?ik?nys and his team at Vilnius University in Lithuania cloned the CRISPR-Cas9 system from S. thermophilus and demonstrated that it could function in a different organism, E. coli. They purified the Cas9 protein and showed that it could be programmed to cut DNA at specific sites by altering the crRNA sequence.

? 2012: Emmanuelle Charpentier collaborated with Jennifer Doudna at the University of California, Berkeley. Together, they simplified the system by fusing the crRNA and tracrRNA into a single synthetic guide RNA (sgRNA). This innovation streamlined the process of directing Cas9 to specific DNA sequences, making it a more practical and versatile tool for gene editing. They demonstrated that this engineered CRISPR-Cas9 system could be programmed to cut any DNA sequence by designing a corresponding sgRNA.

2013: CRISPR-Cas9 Applied to Mammalian Genome Editing

The leap from prokaryotic systems to eukaryotic genome editing occurred in 2013:

? Feng Zhang at the Broad Institute of MIT and Harvard successfully adapted the CRISPR-Cas9 system for use in human and mouse cells. His team engineered Cas9 and demonstrated targeted DNA cleavage in mammalian cells, effectively editing genes within these organisms. They also showed that the system could target multiple genes simultaneously and facilitate homology-directed repair.

? Simultaneously, George Church’s lab at Harvard Medical School independently achieved similar results, showcasing the potential of CRISPR-Cas9 in human genome engineering.

These pioneering studies marked a significant turning point, opening the door for researchers worldwide to utilize CRISPR-Cas9 for gene editing across a wide array of organisms. The technology’s simplicity and efficiency revolutionized genetics, biotechnology, and medicine.

Conclusion: A New Era in Genetic Engineering

The history of CRISPR is a testament to human curiosity, collaboration, and ingenuity. From the initial discovery of mysterious DNA repeats to the development of a versatile genome-editing tool, CRISPR-Cas9 has dramatically expanded our ability to manipulate genetic material.

This journey highlights the importance of fundamental research and international cooperation, involving scientists from Japan, Spain, France, the Netherlands, the United States, Sweden, Lithuania, and many other countries.

As we reflect on these milestones, we recognize that CRISPR technology has not only advanced our understanding of genetics but also holds immense potential to address some of humanity’s most pressing challenges, from treating genetic diseases to enhancing agricultural productivity.

For a more detailed narrative on the history of CRISPR research, consider reading “The Heroes of CRISPR” by Eric S. Lander in the January 14, 2016 edition of Cell.

Thank you for joining this exploration of CRISPR’s remarkable history—a story that continues to unfold, shaping the future of science and medicine.