CRISPR-Cas9 is a technology that enables precise modifications to an organism’s genetic code. This tool allows researchers to add, remove, or alter specific DNA sequences, offering unprecedented control over gene expression. Its development has transformed molecular life sciences, opening new avenues in various fields. CRISPR-Cas9 holds significant potential to address a range of biological and medical challenges.
Early Bacterial Observations
The story of CRISPR-Cas9 begins with observations of unusual DNA sequences in bacteria and archaea. In the 1980s, Japanese researchers, including Yoshizumi Ishino, first reported repetitive DNA sequences in Escherichia coli. However, the significance of these repeats remained unclear at the time.
A decade later, in the early 1990s, Spanish molecular biologist Francisco Mojica extensively studied similar repetitive sequences in the archaeal organisms Haloferax and Haloarcula species. He noted their peculiar, regularly interspaced nature, comprising short palindromic repeats. Mojica’s work in 2000 was instrumental in recognizing that these sequences shared common features, leading him to coin the acronym CRISPR, standing for Clustered Regularly Interspaced Short Palindromic Repeats, in 2002.
Mojica dedicated over a decade to understanding these repeating structures. This foundational work laid the groundwork for future discoveries by characterizing the CRISPR locus.
Unraveling the Immune System
The initial identification of CRISPR sequences posed a mystery regarding their biological role. The breakthrough in understanding their function came from scientists investigating how bacteria defend themselves against viral infections. In the early 2000s, molecular biologists Philippe Horvath and Rodolphe Barrangou, working at Danisco, observed that bacteria developed resistance to bacteriophages, viruses that infect bacteria.
Their research, particularly in Streptococcus thermophilus, revealed a correlation between acquired viral resistance and the incorporation of viral DNA fragments into the CRISPR loci. They discovered that bacteria integrated small segments of viral genetic material, known as “spacers,” into their own CRISPR regions. These spacers served as a genetic memory, allowing the bacteria to recognize and defend against subsequent infections by the same viruses.
Horvath and Barrangou demonstrated that these CRISPR sequences, along with associated CRISPR-associated (Cas) proteins, form an adaptive immune system in bacteria. This system enabled bacteria to acquire specific immunity against invading viruses and plasmids by targeting their nucleic acids in a sequence-specific manner. Their work in 2005 established that CRISPR-Cas systems provide acquired resistance against viruses in prokaryotes.
Repurposing for Precision Gene Editing
The understanding of CRISPR’s natural immune function paved the way for its adaptation as a gene-editing tool. In 2011, Emmanuelle Charpentier identified tracrRNA, an RNA molecule that was a component of the bacterial CRISPR-Cas system. She showed it played a part in disarming viruses by cleaving their DNA.
Charpentier then collaborated with Jennifer Doudna, an expert in RNA. Their combined efforts led to a paper published in Science in 2012, demonstrating how the bacterial CRISPR-Cas9 system could be repurposed to make precise cuts in any DNA molecule. They proved that the Cas9 enzyme, guided by a single guide RNA (a fusion of tracrRNA and crRNA), could be programmed to target and cut specific DNA sequences.
Following this, several research groups demonstrated the system’s utility in eukaryotic cells, including those from mice and humans. Feng Zhang’s group adapted the CRISPR-Cas9 system for use in mammalian cells, publishing their findings in 2013. The ability to precisely cut DNA with a programmable guide RNA allowed scientists to introduce changes, such as adding, removing, or altering genetic material, transforming the field of gene editing.