1980 - The early years
CRISPR systems exist naturally in a variety of microbes (prokaryotes and archaea). Although the CRISPR genome editing system is in wide use today, it is only thanks to basic research into bacterial immune systems that we even stumbled across the CRISPR system in nature. Many microbes have employed CRISPR systems to protect themselves against pathogens (viruses and foreign DNA elements) for millions of years.
In the 1980s, biologists began to notice that many bacterial genomes contain arrays of small repeated segments of DNA interspersed with segments of DNA with no clear function.
The combined arsenal of snipped segments of viral DNA and Cas enzymes helps the bacterium to recognize identical invading viral DNA, and target it for destruction. As the bacterium encounters more viruses (or foreign DNA elements from other microbes), the CRISPR array grows, providing the bacteria and its offspring with an expanded library of biological antivirus support.
2005 - The discoveries begin
The CRISPR DNA sequences are like genetic fingerprints filed away from past viral invaders. If the invaders show up inside the bacteria again, a CRISPR-associated (Cas) protein recognizes and destroys them.
Jennifer Doudna, a professor at the University of California, Berkeley, and a Howard Hughes Medical Institute investigator, got started on CRISPRs over coffee. She shared a café table and her interest in RNA with her colleague Jill Banfield, a Berkeley professor and geobiologist, who was studying the sequences of natural bacterial communities. Banfield said her data were revealing many CRISPR sequences that often matched virus sequences. Banfield asked Doudna to meet for coffee to talk about the data. “There was no evidence for it at the time [in 2006], but it seemed intriguing that bugs were acquiring sequences from viruses that could potentially be turned into RNA,” Doudna recalled.
While Doudna was investigating this possibility, another research group confirmed her suspicions about CRISPRs as RNAs in 2008. But it was still another four years before CRISPR became a practical genome editing tool used in non-bacterial cells.
Genome editing isn’t a new technology. It allows scientists to add, subtract or modify sequences in the genome as they would text in a word processor. Genome editing uses molecular tools that modify the genome in specific cells and, before CRISPR, the most advanced editing tools were zinc finger nucleases and a similar technology called TALENs (Transcription Activator-Like Effector Nucleases). Zinc finger-genome editing recently made news when they were used in HIV treatment. The Zinc Fingers were engineered to disable the CCR5 gene in immune cells, allowing them to become resistant to HIV infection. However, both Zinc Fingers and TALENs are very difficult to engineer. CRISPR proponents say their method is much easier and much cheaper.
2011 - Think about Gene Editing
“I didn’t really start thinking about using CRISPRs for genome editing until late in 2011 when we were collaborating with Emmanuelle Charpentier’s lab,” Doudna said. The Charpentier lab at the Helmholtz Centre for Infection Research had data showing that one of the genes in the CRISPR system had the ability to cut DNA, what was exactly what you need for genome editing.
Across the country, bioengineer Feng Zhang was starting his first faculty position in 2011 at the Broad Institute at MIT. Zheng recalls that exploring the MIT campus one afternoon, he was mistaken for a freshman undergrad. He was just 29, but had already proven to be a brilliant scientist.
Zhang was born in China but moved to Des Moines, Iowa as a child. “People were really genuine and very nice, it was a great place to grow up,” Zhang said. As a high school sophomore in Des Moines, he started volunteering four to five hours a day at the Human Gene Therapy Research Institute. “That’s when I really started to appreciate biology as an engineering discipline. I like to tinker with things,” he said.
As a senior in high school, he won third place in the Intel Science Talent Search and went onto Harvard University for a degree in chemistry and physics. Zhang finished his PhD in bioengineering in 2009 at Stanford where he worked with mentor Karl Deisseroth in developing optogenetics, an innovation that was named 2010’s “Method of the Year” by Nature Methods. He joined George Church and Paolo Arlotta, both professors at Harvard, for a short postdoc in genome editing. Last year, Zhang was named one of the “brilliant ten of 2013” by Popular Science.
Zhang first learned about CRISPR at a scientific advisory board meeting at the Broad Institute soon after he started there. “One of the researchers who works on infectious disease mentioned CRISPR during his talk… I went online to see how people are using it and what is it. Then I realized that CRISPR is actually a nuclease that cuts DNA. That instantly made me start thinking about harnessing the CRISPR system to do genome editing,” he said. Zhang had been working on the TALEN genome editing technology but was growing increasingly frustrated with the difficulty of making new TALENs. Suddenly this other pathway opened before him.
Zhang immediately got to work using CRISPRs for genome editing. He loved the simplicity of using an RNA-based system compared with the previous protein-based systems like TALENs. “An RNA pairs with one of four bases: A, U, G, or C. So it’s much easier [and cheaper] to design a new RNA to recognize a new DNA sequence than it is to develop a new protein,” Zhang said.
2012 - Spreading the word
Doudna and Charpentier published their observation that CRISPR is an RNA-guided DNA-cutter that could be used for genome editing in June 2012. Zhang published a more precise and efficient CRISPR editing system in January 2013. In the year since then, the use of CRISPRs has exploded. “I think [Addgene] is one of the reasons we’re seeing such a rapid uptake,” Doudna said.
...and Spreading the Plasmids
Addgene is a non-profit library for small DNA plasmids, like CRIPSR. It grew out of a common frustration felt by researchers. Until recently, if a researcher wanted something novel—say Feng Zhang’s CRISPR tools— she would have to contact Zhang, fill out a material transfer agreement, and wait while Zhang’s lab made the tools, and mailed them. In fact, these requests for his new CRISPR tools were overwhelming the Zhang lab within weeks of the publication of his method. Luckily, Addgene was in place to take over.
Addgene was the brainchild of Melina Fan, a graduate student at Harvard University who was having a hard time getting plasmids from other labs. After finishing her PhD in 2004, Fan teamed up with her husband, Benjie Chen, a computer scientist, and her brother Kenneth Fan, a businessman, to start a company that would help share plasmids. It’s free for researchers to deposit their plasmids and it costs $65 to request a plasmid. The fee covers operating costs of the repository and conducting quality control. Zhang’s CRISPR tools have now been sent out over 12,000 times. They’re the most requested on AddGene.
Zhang can’t imagine CRISPR’s success without Addgene. “When I first started to work in the genome editing field, I started by studying zinc finger nucleases… but it was very difficult to get the reagents from the zinc finger researchers,” Zhang said. He could have bought the technology from a company, but he couldn’t afford it. “Because of that I always felt that if we are building a research tool, we needed to make it open,” Zhang said. He kept his promise. “We filed a patent application, but we put the [CRISPR] reagent on AddGene,” Zhang said.
CRISPRs are already available to edit the genomes of Arabadopsis, tobacco, sorghum, rice, fruit flies, worms, mice, rats, pigs, and even monkeys. The technology can be applied to edit genomes in cells and animals to mimic human diseases. It can be used to edit the genomes in plants, making them more resistant to pests without using non-plant DNA as with GMOs. As a quick, cheap, and increasingly precise method to edit human DNA, CRISPR is already being explored as a way to change the genomes of humans with cystic fibrosis, cancer, Parkinson’s, and other diseases.
2013 - Startups entering the space
In the short term, Doudna believes Editas Medicine will focus on targets similar to those of the new HIV therapeutics, where cells are edited outside the body and then put back into the patient. “Longer term goals are to be to be able to do editing in tissues, but there you have to think about delivery and that’s a bigger challenge,” Doudna said.
Editas was founded in 2013 with $43 million in Series A venture capital financing led by leading health care venture capital firms Flagship Ventures, Polaris Partners and Third Rock Ventures with participation from Partners Innovation Fund.
2016 - What comes next?
Though they’re now working on Editas Medicine, Doudna and Zhang haven’t given up studying CRISPRs in their own labs. Says Zhang, “We know that [CRISPRs] can lead to off-target mutations. We are trying to use protein engineering and structural biology to better understand how to improve the specificity. There are many CRISPR systems out there and many of them could be harnessed for biotechnology application, so we’re also exploring those,” he added.
Doudna shares the excitement, “The past year has been the craziest of my career, really fun and exciting, but I haven’t had time to do much else,” she says. “There are so many interesting things to pursue and so many other people pursuing them as well that we feel intensely involved in the research at the moment.” In addition to her work with Editas Medicine, she co-founded Caribou Biosciences in 2011 to develop CRISPRs for research applications and diagnostics. Plus Doudna says that her lab is “actively working to understand molecular mechanisms to figure out if we can improve the technology for particular applications.”
Just 18 months ago CRISPRs and bacterial immunity seemed like an esoteric area of science, she recalls. “Now it’s obvious that this is an interesting area to pursue that is related to human health,” Doudna said.