By John Rennie, Science Editor & Journalist

Modern biotechnology dawned in the early 1970s when researchers first began to cut and splice the DNA of living things to endow them with desirable new genetic traits. Since then, biotechnologists have pushed ahead to many victories, but that success did not come easily. Compared to the ambitions that scientists and entrepreneurs have had for it, genetic engineering has often been costly and slow to execute, uncertain in its results, and impractical for use on some organisms.

Meanwhile, nature’s simplest organisms quietly guarded a superior way to edit genomes, which they first evolved as a defense hundreds of millions of years ago. The recent discovery of that secret, and its development into a dazzlingly superior method of genome engineering generally known as CRISPR, has electrified the life sciences in less than a decade—and challenged the scientific community to use it wisely. And although CRISPR is not without controversies, none can dispute the singular importance of biologists Emmanuelle Charpentier and Jennifer Doudna in bringing it to light. For that exceptional work, they were named as the winners of the Dr. Paul Janssen Award for Biomedical Research in 2014.

Born outside Paris in 1968, Emmanuelle Charpentier was always encouraged in diverse academic and artistic pursuits by her parents. She shared her mother’s interests in psychology, but she also favored philosophy, mathematics, and especially medicine. She vowed early in life to make a contribution to people’s medical well being.

Charpentier pursued undergraduate study of microbiology, genetics, and biochemistry in Paris at University Pierre and Marie Curie (UPMC) between 1986 and 1992. The life of a researcher suited her, she learned: the science nourished her curiosity about nature’s secrets and she enjoyed working as part of a collaborative team. She continued with graduate work at Institut Pasteur (1992-1995) and at UPMC (1993-1995), during which she studied the molecular mechanisms underlying antibiotic resistance. It also led her to the related study of how bacteria interact with their surroundings during the process of infecting hosts.

“Along my career, I have always developed tools to improve the precision of genetics studies, whether in bacteria or mice” Charpentier explains.

Stints as a post-doctoral fellow took her to Institut Pasteur (1995-1996), Rockefeller University (1996-1997), New York University’s Langone Medical Center (1997-1999), St. Jude Children’s Research Hospital (1999) and the Skirball Institute of Biomolecular Medicine (1999-2002). She then returned to Europe as the head of a laboratory at Vienna University, in its Institute of Microbiology and Genetics. Charpentier had been focusing her work on how bacteria sometimes use small RNA molecules to regulate the expression of their genes. In Vienna, that interest led her to think about new discoveries that were just being made about a peculiar feature in the genomes of many bacteria (and other ancient cells named archaea): odd clusters of repeating nucleotide sequences punctuated by short spacers of DNA seemingly stolen from viruses. Bacterial geneticists called them clustered regularly interspaced short palindromic repeats—or more conversationally, CRISPR.

Microbiologists’ hypothesis in the early 2000s was that CRISPR was a piece of something like an immune system in bacteria that defended them against viruses to which they (or their ancestors) had been exposed. For the bacteria, CRISPR was like a wall of genetic wanted posters for known viral threats. But details of how CRISPR enables this defense were a mystery when Charpentier first began to think about it. With her colleagues, Charpentier quietly began to dig into the problem. They identified specific small RNA molecules associated with CRISPR that interacted with a DNA-slicing enzyme called Cas9. Charpentier suspected that these RNAs, which included bits of the viral gene sequences, guided Cas9 to targets in viral DNA and allowed it to destroy viruses infecting the cell. The concept was unorthodox because, hitherto, only complexes of proteins guided by RNA were known to perform that kind of DNA-targeting function in cells.

Further experiments, completed after Charpentier moved to Umeå University in 2009, established important proof for her bold idea. She showed that when CRISPR RNA containing viral sequences is transcribed, it matures by forming a duplex with another bit of RNA (called tracrRNA) and then binding to Cas9. The resulting complex seemed to have all the pieces needed to thwart a viral attack. Charpentier first presented her discovery at a conference in 2010, and then more formally in a paper in Nature in 2011.

Already it was clear to her that if the CRISPR-Cas9 system could locate DNA targets in cells so quickly and efficiently, it held immense potential as a tool for genetic engineering. Yet to realize that possibility, she needed more detailed, structural understanding of how the system worked.

The needed expertise presented itself a few months later, when Charpentier attended a conference in Puerto Rico and met Jennifer Doudna, an eminent structural biologist and Howard Hughes Medical Institute investigator at the University of California in Berkeley. Doudna, too, had been studying RNAs involved in the CRISPR system for several years. Unlike Charpentier, Doudna had not started off in life imagining a scientific career for herself. Yet, growing up on the Big Island of Hawaii, she had been fascinated by the extraordinary variety and adaptations in the natural world around her. At Pomona College, she studied biochemistry. In 1985, she moved on to graduate work at Harvard University with Jack Szostak, who had pioneered the study of how early RNA molecules with the right chemical properties might have kicked off life’s evolution. In his biochemistry laboratory, Doudna could concentrate on developing novel ribozymes—RNA molecules that have catalytic chemical properties like enzyme proteins.

In 1991 she headed to the University of Colorado at Boulder and the laboratory of Thomas Cech, who had recently shared a Nobel prize for the discovery of ribozymes. There, she began working on crystallizing and learning the three-dimensional structure of a particular ribozyme (at that time, scientists knew the folded structure of only one other RNA molecule). She continued that project after joining the faculty of Yale University in 1994, and eventually succeeded in 1996. The experience only cemented Doudna’s conviction that knowledge of RNA structures was essential to understanding their function. When the opportunity to have her own laboratory at the University of California, Berkeley, arose in 2000, Doudna took it in part because she would have handier access to the synchrotron x-ray source at Lawrence Berkeley National Laboratory, which would help with the crystallization studies.

At Berkeley, Doudna had turned her attention to CRISPR-Cas systems, intrigued by many of the same questions that had drawn in Charpentier. When she met Charpentier at the conference in Puerto Rico in 2011, they hit it off personally and also recognized that they brought complementary strengths to the study of CRISPR-Cas. Notwithstanding the huge gulf between their laboratories—Doudna’s on the West Coast, Charpentier’s in Vienna—they vowed to collaborate.

The fruit of their joint effort was their epic paper in Science in August 2012, which showed that the stored viral sequence in a mature CRISPR RNA duplex does indeed direct its associated Cas9 enzyme to slice up the corresponding viral DNA whenever and wherever it manifests in a cell.

Yet the paper did more than explain how bacteria use CRISPR to defend themselves against viruses. It also showed that researchers could use tailored RNA to program CRISPR-Cas9 complexes to slice a DNA double helix wherever they wanted. In conjunction with a cell’s DNA-repair mechanisms, they could delete a gene or insert a new one. Moreover, CRISPR’s usefulness for genome regulation may be at least as important as for genome editing. With CRISPR, scientists can introduce programmable transcription factors to cells’ DNA. With those, they can reversibly silence or deactivate specific genes without altering them. As Doudna noted during a TED talk in 2015, “We realized that we could harness its function as a genetic engineering technology—a way for scientists to delete or insert specific bits of DNA into cells with incredible precision—that would offer opportunities to do things that really haven't been possible in the past.”

Genome researchers and biotechnologists have rushed to use the new CRISPR technique. With every passing year, the number of published papers citing the Charpentier-Doudna paper has increased exponentially.

Charpentier and Doudna have continued to improve on the technique, as have other researchers including Feng Zhang of the Broad Institute at the Massachusetts Institute of Technology and George M. Church of Harvard (who were each independently working on developing CRISPR for genome modification at the same time as Doudna and Charpentier).

Several biotech start-ups have jumped in to commercialize applications of CRISPR technology, including CRISPR Therapeutics (co-founded by Charpentier), Caribou Biosciences and Intellia Therapeutics (both co-founded by Doudna), and Editas Medicine (co-founded by Doudna, Zhang and others. The intellectual property rights surrounding CRISPR are currently in dispute: a U.S. patent on the technique was initially granted to Feng Zhang and the Broad Institute, but Doudna and Charpentier objected that they had filed for a patent first; an investigation into who should hold the rights is now pending with the U.S. Patent and Trademark Office. Nevertheless, the technique remains freely available for use by academic researchers around the world, and they are making the most of it.

Already, agricultural researchers are applying CRISPR to engineer pest- and disease-resistant wheat, rice, oranges, soybeans and other crops. Animal researchers have been making disease-resistant pigs, and ones with “humanized” organs that might be safe donors for transplants to human beings. Biomedical investigators used it to treat mice with a condition resembling Duchenne muscular dystrophy. Entomologists are exploring the possibility of altering the genetics of wild mosquito populations to reduce their ability to spread malaria and Zika virus.

CRISPR is also of intense interest to medical researchers as a means of treating heritable illnesses such as cystic fibrosis and sickle cell disease, as well as DNA-infiltrating ones like HIV. It is widely believed that within a decade a CRISPR-based therapy may be tested against a human illness—possibly a blood disease in adults, since blood cell lines are easier to manipulate than most tissues are, and non-heritable genetic alterations to adults are less ethically fraught.

Acclaim and prestigious honors for Charpentier and Doudna have been abundant because of CRISPR. In addition to the Janssen Award, they shared the 2015 Breakthrough Prize in Life Sciences, the 2015 Gruber Prize in Genetics, and the 2016 Canada Gairdner International Award (along with Feng Zhang), among many others. Charpentier received the Leibniz Prize of the German Research Foundation in 2016 and was elected as a member of the Royal Swedish Academy of Sciences. (Doudna is also a member of the National Academy of Sciences, but she was elected to that body in 2002 on the strength of her ribozyme structural work.) Charpentier and Doudna are widely regarded as likely recipients of a Nobel Prize in the near future.

Today, in her Berlin laboratory as a director at the Max Planck Institute for Infection Biology, Charpentier continues to study bacterial CRISPR systems and to work on further refinements of the gene editing technology that sprang from it. But she has also returned to work on some of the other broad mysteries in biology that have always interested her, such as the interactions between bacteria and their hosts and the molecules that govern that relationship at the genetic and biochemical level.

Doudna, meanwhile, has become extremely involved in the ongoing international discussion about how to apply CRISPR wisely. As she and other scientists have warned, enthusiasm for the technique should not blind anyone to the risks of unintended consequences. Treating genetic conditions in ailing body tissues like muscle sounds sensible; the almost irresistible temptation to alter human embryos or germline tissues for disease prevention in future generations should not be indulged lightly. Ethicists worry about CRISPR being used too casually to “improve” humans by eliminating conditions that are not strictly pathological, and that altered DNA passing on to future generations—a situation that could be a boon or a eugenic nightmare. Ethicists worry about the eugenic nightmares that could come from “improvements” made to the DNA of future generations. Eugenicists worry that benign efforts to eliminate genetic illnesses from the human population permanently could too easily slide into the eugenic nightmare of “improving” humans by deleting traits that are not pathological, merely unpopular.

For that reason, Doudna has helped to spearhead a movement among scientists to discuss and govern CRISPR technology. In November 2015, an international assembly of them met and over three days worked out a set of accords that declared a temporary, voluntary moratorium on most potential CRISPR experiments on humans. Under the terms of that agreement, experiments on human ova would proceed only if the potential benefits of an experiment greatly exceeded the potential harm, and only on human embryos that had no possibility of being brought to term.

“I am excited about the potential for genome engineering to have a positive impact on human life, and on our basic understanding of biological systems,” Doudna wrote in a commentary for Nature in December 2015, adding, “But I also think that today's scientists could be better prepared to think about and shape the societal, ethical and ecological consequences of their work.”

READINGS

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