CRISPR, an acronym for Clustered Regularly Interspaced Short Palindromic Repeats, is a gene-editing system. It acts like precise molecular scissors, capable of cutting and modifying DNA at specific locations within an organism’s genome. The simplicity, cost-effectiveness, and precision of CRISPR make it a tool for rewriting genetic code in nearly any organism.
Early Microbial Discoveries
Initial observations of CRISPR began with genetic sequences in bacteria. In 1987, Japanese researchers identified unusual repetitive DNA segments in Escherichia coli. Later, in 1991, similar patterns were noted in Mycobacterium bovis. These findings were genetic curiosities with no clear biological purpose.
The investigation into these repetitive sequences continued in the early 1990s, notably by Francisco Mojica, a graduate student at the University of Alicante, Spain. While studying the archaeon Haloferax mediterranei and its adaptation to high salinity, Mojica observed repeated DNA sequences. He noticed that these sequences, approximately 30 bases long, appeared repeatedly, separated by unique stretches of DNA.
Mojica recognized that these repeat sequences shared common features. Through correspondence with Ruud Jansen, Mojica coined the term “CRISPR” in 2001, first used in print in 2002. These clustered regularly interspaced short palindromic repeats were initially a genetic signature whose function remained unknown.
Unveiling the Bacterial Immune System
CRISPR’s biological role as an adaptive immune system in bacteria and archaea was revealed in the early 2000s. In 2005, Francisco Mojica’s group realized that the “spacer” DNA sequences found between the CRISPR repeats were identical to snippets of viral DNA. This observation suggested that bacteria might be incorporating fragments of invading viruses into their own genomes.
Further research by Philippe Horvath and Rodolphe Barrangou provided experimental evidence that solidified CRISPR’s role in bacterial defense against viruses, known as bacteriophages. When a bacterium encounters a virus, it can integrate a piece of the viral genome into its CRISPR array, effectively creating a genetic memory of the invader.
This memory allows the bacterium to recognize and defend against future infections by the same virus. The CRISPR system works with CRISPR-associated (Cas) genes, which encode proteins. These Cas proteins, guided by RNA transcribed from the CRISPR array, precisely target and cleave the DNA of invading viruses or plasmids, neutralizing the threat.
Transforming CRISPR into a Gene Editor
The ability of CRISPR-Cas systems to precisely target and cut DNA led to their use as gene-editing tools. In 2012, research teams independently demonstrated how the bacterial CRISPR-Cas9 system could be simplified and reprogrammed to edit DNA in non-bacterial cells, including human cells.
Jennifer Doudna and Emmanuelle Charpentier showed that the Cas9 enzyme, guided by a synthetic single-guide RNA (sgRNA), could cut specific DNA sequences. The guide RNA is a customizable molecule, typically around 20 nucleotides long, that matches the target DNA sequence. This mechanism allows for precise modifications to the genome.
Around the same time, Feng Zhang and George Church also published work demonstrating CRISPR-Cas9’s use for gene editing in eukaryotic cells. These simultaneous discoveries advanced molecular biology, showcasing CRISPR-Cas9’s potential for targeted gene disruption, deletion, correction, or insertion.