Just after breakfast, sitting in his laboratory at the University of Alicante, Francisco Mojica stared at his computer screen in dismay. It was the 1990s, and he had just created a database of DNA sequences of extreme bacteria\u2014organisms that lived in conditions that would kill almost any other life. These bacteria inhabited salt-saturated lakes\u2014organisms adapted to be “salt lovers,” as their scientific name, halobalilia, implied.labiotech+1<\/a>\u200b<\/p>\n\n\n\n But instead of the usual, orderly DNA sequences he expected, Mojica stumbled upon something strange: regularly repeated DNA fragments, separated by spaces with ever-changing sequences<\/strong> . They were like repeated words in a strange verse\u2014”word-space-word-space-word.” Interesting. Almost like an archive. But an archive of what?wikipedia<\/a>\u200b<\/p>\n\n\n\n Little did he know that he had just discovered one of the most groundbreaking technologies that would fundamentally revolutionize medicine, agriculture, and biology over the next two decades.<\/p>\n\n\n\n The history of CRISPR (acronym for Clustered Regularly Interspaced Short Palindromic Repeats) begins earlier, in Japan.bitesizebio<\/a>\u200b<\/p>\n\n\n\n In 1987, while working on the gene encoding alkaline phosphatase in E. coli<\/em> , Japanese scientist Yoshizumi Ishino<\/strong> and his team had an unexpected surprise. While cloning DNA for an experiment, they stumbled upon fragments of DNA that were repeated\u2014a highly unusual finding. These sequences were organized into clusters and were regularly distributed along the bacterial genome.biocompare+1<\/a>\u200b<\/p>\n\n\n\n Ishino and his team published their observations, but their significance was never fully explored. This discovery languished in the scientific literature, like a hidden treasure waiting for adventurers.biocompare<\/a>\u200b<\/p>\n\n\n\n Flash forward to the year 2000. Francisco Mojica, now a researcher at the University of Alicante, was working on a different question: how do bacteria from extreme environments adapt to changes in salt concentration? But his curiosity quickly veered elsewhere.labiotech<\/a>\u200b<\/p>\n\n\n\n Using access to growing genetic databases, he began comparing these strange, repetitive sequences Ishino had previously discovered. Imagine his surprise when he discovered that the same repeats appeared in the genomes of bacteria studied around the world\u2014in microorganisms from the ocean, soil, and caves<\/strong> .biocompare<\/a>\u200b<\/p>\n\n\n\n In 2000, he and his colleagues published work showing that this cluster was highly evolutionarily conserved\u2014and therefore must have meant something important. Its very preservation over millions of years of evolution indicated that nature does nothing without reason.biocompare<\/a>\u200b<\/p>\n\n\n\n But that was just the beginning.<\/p>\n\n\n\n A few years later, while comparing databases, Mojica noticed something extraordinary: DNA fragments nested between repeats in the bacterial genome were identical to fragments of the genomes of viruses (bacteriophages) that attack bacteria<\/strong> .labiotech<\/a>\u200b<\/p>\n\n\n\n Not just any fragments \u2013 but fragments of actual viruses infectious to those same bacteria!<\/p>\n\n\n\n It was an immediately logical presumption: if a bacterium stores fragments of a virus’s DNA in its cell, it must have acquired this genetic material somehow<\/strong> . And if it holds them, it must need them for something.labiotech<\/a>\u200b<\/p>\n\n\n\n Mojica hypothesized: CRISPR is a bacteria’s adaptive immune system<\/strong> . Once a virus attacks a bacterium and it becomes infected, part of the virus’s genome is squeezed into the CRISPR archive. The next time the same virus tries to attack that bacterium (or its descendants), the immune system will “remember” it and attack it.wikipedia+1<\/a>\u200b<\/p>\n\n\n\n Sounds almost like memory? Because that’s exactly what it is\u2014 biological, genetic memory<\/strong> .<\/p>\n\n\n\n In 2003, Mojica wrote a paper proposing this theory. He submitted it to Nature<\/strong> , one of the world’s most prestigious scientific journals. It was rejected. He tried the Proceedings of the National Academy of Sciences<\/strong> . It was rejected.wikipedia<\/a>\u200b<\/p>\n\n\n\n Then Molecular Microbiology<\/strong> . Refusal.wikipedia<\/a>\u200b<\/p>\n\n\n\n Nucleic Acids Research<\/strong> . Once again \u2013 refusal.wikipedia<\/a>\u200b<\/p>\n\n\n\n He was frustrated, but he didn’t give up. The paper finally made it to the Journal of Molecular Evolution<\/strong> in February 2005. It wasn’t Nature, but it was a publication. Importantly, that same year, independently of Mojica’s work, another laboratory published similar findings.flagshippioneering+1<\/a>\u200b<\/p>\n\n\n\n But something changed. Scientists began to listen.<\/p>\n\n\n\n Although Mojica proposed the hypothesis, experimental evidence came from a completely different direction \u2013 from laboratories that were studying\u2026 ferments for yogurt production.pmc.ncbi.nlm.nih<\/a>\u200b<\/p>\n\n\n\n In 2005, Philippe Horvath’s<\/strong> team at Danisco (yes, the dairy giant!) investigated how Streptococcus thermophilus<\/em> bacteria \u2013 used to produce yogurt and cheese \u2013 could be resistant to infectious viruses (bacteriophages).pmc.ncbi.nlm.nih<\/a>\u200b<\/p>\n\n\n\n Horvath and his colleagues demonstrated experimentally what Mojica had proposed theoretically: when S. thermophilus<\/em> was infected with a new bacteriophage, the bacterium integrated new sequences derived from the phage’s genome directly into its CRISPR region of DNA<\/strong> . Even better, the next time the same phage tried to infect descendants of that bacterium, they were resistant.pmc.ncbi.nlm.nih<\/a>\u200b<\/p>\n\n\n\n This was not just a theory \u2013 it was experimental proof of a working biological immune system.pmc.ncbi.nlm.nih<\/a>\u200b<\/p>\n\n\n\n Separately, that same year, Vytautas Siksnys<\/strong> from Lithuania published a paper showing that the CRISPR system from one bacterium ( S. thermophilus<\/em> ) could be transferred to a completely different species\u2014 E. coli<\/em> \u2014and it would work there. This was important because it demonstrated the universality of the system.flagshippioneering<\/a>\u200b<\/p>\n\n\n\n In the following years, scientists around the world began to study CRISPR in more detail. Fiona Barrangou<\/strong> and others demonstrated exactly how CRISPR works\u2014how bacteria “learn” to recognize viruses and use this knowledge to protect themselves.nature<\/a>\u200b<\/p>\n\n\n\n Several variants of CRISPR systems have been discovered \u2013 CRISPR-Cas9<\/strong> , CRISPR-Cas12a<\/strong> , and others. Each system had its own Cas proteins \u2013 enzymes that perform the actual “cutting” of DNA.pmc.ncbi.nlm.nih<\/a>\u200b<\/p>\n\n\n\n It turned out that Cas9<\/strong> , from Streptococcus pyogenes<\/em> (the bacterium that causes angina), was particularly remarkable. When prompted by guide RNA, it would precisely cut DNA exactly where instructed.pmc.ncbi.nlm.nih<\/a>\u200b<\/p>\n\n\n\n By 2011, scientists knew almost everything they needed to know about CRISPR in nature. But no one had yet considered: what if, instead of letting bacteria do what they do naturally, we scientists taught Cas9 to do what we wanted?<\/strong><\/p>\n\n\n\n In 2011, at a scientific conference in San Juan, Puerto Rico, two scientists from different sides of the Atlantic met by chance.pmc.ncbi.nlm.nih<\/a>\u200b<\/p>\n\n\n\n Jennifer Doudna<\/strong> , a protein structuralist at the University of California, Berkeley, specialized in studying biological mechanisms at the molecular level. Emmanuelle Charpentier<\/strong> , a microbiologist at Ume\u00e5 University in Sweden, also studied bacterial immune systems.innovativegenomics+1<\/a>\u200b<\/p>\n\n\n\n They talked about CRISPR. Doudna was fascinated; Charpentier was an expert. They decided to collaborate.pmc.ncbi.nlm.nih<\/a>\u200b<\/p>\n\n\n\n Before Doudna and Charpentier deepened their collaboration, Charpentier had made a significant discovery in her Ume\u00e5 lab. She was studying CRISPR with Streptococcus pyogenes<\/em> and discovered something that would change everything.mpg<\/a>\u200b<\/p>\n\n\n\n It turned out that in addition to krRNA (CRISPR RNA) and Cas9, there was a third, critically important component: tracrRNA (trans-activating crRNA)<\/strong> . This was a small but crucial RNA molecule.pmc.ncbi.nlm.nih+1<\/a>\u200b<\/p>\n\n\n\n This was a groundbreaking observation because the tracrRNA turned out to be a “bridge”\u2014it connected Cas9 to the krRNA in such a way that Cas9 knew where to look and where to cut.pmc.ncbi.nlm.nih<\/a>\u200b<\/p>\n\n\n\n Now Doudna and Charpentier had all the pieces of the puzzle. In their UC Berkeley\/Ume\u00e5 lab, they worked together (communicating across the ocean) to assemble CRISPR-Cas9 into something that could be a controllable tool.embryo.asu<\/a>\u200b<\/p>\n\n\n\n Their key contribution was elegant: instead of using two separate RNA molecules (krRNA and tracrRNA), they combined them into a single molecule<\/strong> , which they called single guide RNA (sgRNA)<\/strong> .embryo.asu<\/a>\u200b<\/p>\n\n\n\n Why was this important? Because it simplified the technology. Instead of programming two different RNAs, scientists now had to program just one. It was like going from using a computer with two buttons to one with a single large button labeled with what you wanted to do.mpg+1<\/a>\u200b<\/p>\n\n\n\n In their experiment, Doudna, Charpentier and their team (including Martin Jinek and Michael Hauer from Berkeley, and Krzysztof Chylinski and Ines Fonfara from Ume\u00e5) set up a laboratory scene:embryo.asu<\/a>\u200b<\/p>\n\n\n\n And they waited.<\/p>\n\n\n\n What happened: Cas9 precisely cut the DNA exactly where the guide RNA told it to<\/strong> . Not just anywhere\u2014right there.embryo.asu<\/a>\u200b<\/p>\n\n\n\n But that wasn’t the goal. Doudna and Charpentier were pursuing something much bigger: demonstrating that the CRISPR-Cas9 system can be programmed like a hyper-precise tool that scientists can target to ANY DNA sequence<\/strong> .embryo.asu<\/a>\u200b<\/p>\n\n\n\n When Doudna and Charpentier showed they could program five different guide RNAs, each targeting a different site in the DNA, the idea was clear: It could work for any sequence a scientist wanted to edit<\/strong> .embryo.asu<\/a>\u200b<\/p>\n\n\n\n Their manuscript reached the editorial office of Science<\/em> in 2012.pmc.ncbi.nlm.nih<\/a>\u200b<\/p>\n\n\n\n In the June 2013<\/strong> issue of Science<\/em> , an article was published: “RNA-guided genetic engineering of human pluripotent stem cells.” The title didn’t sound revolutionary, but its content was incredible.pmc.ncbi.nlm.nih<\/a>\u200b<\/p>\n\n\n\n The article included a detailed description of the three CRISPR-Cas9 components:pmc.ncbi.nlm.nih<\/a>\u200b<\/p>\n\n\n\n And importantly , they showed how all three could work together as a programmed, precise DNA editing tool<\/strong> .pmc.ncbi.nlm.nih<\/a>\u200b<\/p>\n\n\n\n But that was only part of it. Doudna and Charpentier proposed something radical: What if scientists could use this system not only in bacteria, but also in eukaryotic cells\u2014like human cells?<\/strong>pmc.ncbi.nlm.nih<\/a>\u200b<\/p>\n\n\n\n The scientific world reacted with madness.<\/p>\n\n\n\n In 2013, just a few months after Doudna-Charpentier’s publication, Feng Zhang<\/strong> of the MIT Broad Institute published his own paper in Science<\/em> .embryo.asu<\/a>\u200b<\/p>\n\n\n\n Zhang took the CRISPR-Cas9 described by Doudna and Charpentier and demonstrated that it could be delivered into living mouse and human cells and edit their genome<\/strong> .embryo.asu<\/a>\u200b<\/p>\n\n\n\n It was a massively important demonstration. Theoretically, it worked in a tube. But would it work in living cells? Yes, and Zhang is proof.embryo.asu<\/a>\u200b<\/p>\n\n\n\n Now scientists had not only a conceptual tool, but a practical tool.<\/p>\n\n\n\n Six months after Doudna-Charpentier’s publication, dozens of labs around the world had already begun experimenting with CRISPR-Cas9.news.berkeley<\/a>\u200b<\/p>\n\n\n\n Here’s why CRISPR was so transformative compared to previous technologies (such as ZFNs \u2013 Zinc Finger Nucleases, and TALENs \u2013 Transcription Activator-Like Effector Nucleases):ijisrt<\/a>\u200b<\/p>\n\n\n\nFirst Spark: The Strange Sequence from 1987<\/h2>\n\n\n\n
The Key to the Puzzle: Francisco Mojica Discovers the Function<\/h2>\n\n\n\n
Eureka Moment: Virus in Bacterial DNA<\/h2>\n\n\n\n
A path through the groves of scientific journals<\/h2>\n\n\n\n
Experimental Proof: Horvath and Siksnys Show It Works<\/h2>\n\n\n\n
2006-2011: Developing the Foundation<\/h2>\n\n\n\n
The San Juan Meeting and the History That Was Written<\/h2>\n\n\n\n
2011: Charpentier Discovers the Third Missing Piece<\/h2>\n\n\n\n
2012: The Turning Point When Nature Became a Tool<\/h2>\n\n\n\n
Experiment: Testing in Dish<\/h2>\n\n\n\n
\n
Science Publication: The Moment When Everything Changed<\/h2>\n\n\n\n
\n
The Year After: Feng Zhang and the First Editions in Mammalian Cells<\/h2>\n\n\n\n
Revolution: Six Months, Thousands of Articles<\/h2>\n\n\n\n