There is no shortage of optimism about the scientific potential of CRISPR–Cas9, a technique that can precisely alter the genomes of everything from wheat to elephants. But there is a great deal of confusion over who will benefit financially from its use. On 10 March, the US Patent and Trademark Office (USPTO) will begin an investigation into who deserves the patent on using CRISPR–Cas9 to edit genes. This ‘patent interference’ could determine who profits from CRISPR in coming years. Already, companies have sprung up to take advantage of the technique in agriculture, industrial biotechnology and the treatment of human diseases. One firm, Editas Medicine in Cambridge, Massachusetts, raised US$94 million when it went public on 2 February, even though it does not expect to enter clinical trials until 2017. Nature takes a look at what the interference proceeding entails and what it could mean for the fate of CRISPR–Cas9. One patent claim comes from a team led by molecular biologist Jennifer Doudna at the University of California, Berkeley, and microbiologist Emmanuelle Charpentier, now at Umeå University in Sweden and the Max Planck Institute for Infection Biology in Berlin. They published a 2012 paper demonstrating that the Cas9 enzyme can be directed to cut specific sites in isolated DNA (M. Jinek et al. Science 337, 816–821; 2012), and initiated their patent application on 25 May 2012. Another team, led by Feng Zhang at the Broad Institute of MIT and Harvard in Cambridge, Massachusetts, published a 2013 paper demonstrating the application of CRISPR–Cas9 in mammalian cells (L. Cong et al. Science 339, 819–823; 2013). Zhang’s team began a patent application on 12 December 2012. Although the Berkeley team filed first, the Broad team submitted its application to an expedited review programme, and was awarded the patent in April 2014. The Berkeley team then requested a patent interference against the initial Broad patent plus 11 related Broad patents. On 11 January, the USPTO granted Berkeley’s request. A relic from the past. Until a few years ago, the United States awarded patents to those who could show that they were the first to invent, rather than simply the first to file the patent. Under that system, when competing inventors claimed to have created the same invention first, the USPTO declared an interference proceeding to determine which deserved the patent. The United States switched to a first-to-file system in March 2013. But several key CRISPR–Cas9 patents were filed before the change. A panel of three USPTO patent judges will hear evidence from both sides to establish which team invented the application of CRISPR–Cas9 for gene editing. Much of the action will be carried out over the telephone or through written documents. But there will probably be some oral arguments, and these could include testimony from the academic inventors. Patent interferences can be highly technical, says John Conley, a legal scholar at the University of North Carolina in Chapel Hill. “It’s hard for me to cite anything more convoluted in the law than this,” he says. “It’s mind-boggling.” The USPTO panel will probably try to determine not only which team was the first to use CRISPR–Cas9 for gene editing, but which conceived of the invention first. The process could be messy. During the era of ‘first-to-invent’ patents, some companies kept ‘inventor’s notebooks’: when someone at the firm thought of a new invention, they were to write it down in the notebook and have the entry notarized in case it came into play during future patent disputes. Few academic labs go to such lengths. The law that did away with the United States’ first-to-file policy also introduced changes intended to expedite interferences. But a verdict on the CRISPR patents could still be months, or even years, away. And given the high financial stakes, many expect the losing party to appeal against the USPTO interference decision, further dragging out the process. Not necessarily. In its filings to the Securities and Exchange Commission, Editas Medicine highlighted a potential interference claim by a Seoul company called ToolGen. Having multiple interferences over the same patent is rare, says Conley, but possible. No. The Broad and MIT team also fast-tracked several of its applications at the European Patent Office (EPO), and has been awarded several patents so far. Doudna’s single application is pending. Although the EPO does not have an interference process, outside parties can formally object to a patent. By 11 November 2015, the deadline for objections to the Broad’s first European CRISPR–Cas9 patent, nine parties had come forward — launching an opposition procedure that can take years to resolve. Once that process is finished, participants can appeal. This adds another four or five years to the clock, says Michael Roberts, a partner at the intellectual-property law firm Reddie & Grose in Cambridge, UK. For this reason, Roberts believes that it will be several years before there is clarity on the earliest CRISPR–Cas9 patents in Europe.
Researchers from the Max Planck Institute for Infection Biology in Berlin, the Umeå University in Sweden and the Helmholtz Centre for Infection Research in Braunschweig have now discovered a feature of the CRISPR-associated protein Cpf1 that has previously not been observed in this family of enzymes: Cpf1 exhibits dual, RNA and DNA, cleavage activity. In contrast to CRISPR-Cas9, Cpf1 is able to process the pre-crRNA on its own, and then using the processed RNA to specifically target and cut DNA. Not requiring a host derived RNase and the tracrRNA makes this the most minimalistic CRISPR immune system known to date. The mechanism of combining two separate catalytic moieties in one allows for possible new avenues for sequence specific genome engineering, most importantly facilitation of targeting multiple sites at once, the so-called multiplexing. CRISPR-Cas is part of the immune system of bacteria and is used to fight viruses. In the CRISPR-Cas9 system, the enzyme Cas9 cuts the virus DNA at a location specified by an RNA molecule – known as CRISPR RNA (crRNA) in complex with another RNA, the so-called tracrRNA. This puts the pathogens out of action. In 2011, Emmanuelle Charpentier and her co-workers described that the system consists of two RNAs forming a duplex (tracrRNA and pre-crRNA), with tracrRNA maturing pre-crRNA to crRNA, in the presence of the protein Cas9 (formerly named Csn1). A year later Emmanuelle Charpentier and colleagues demonstrated that tracrRNA and crRNA together, be it in form of the duplex of two guide RNAs or a fused single guide RNA, are required to specifically guide the Cas9 enzyme to the matching target DNA sequence. Since then, CRISPR-Cas9 has taken laboratories by storm. Both scientists and clinicians have great hopes for it: the latter aims to use the enzyme scissors to cure severe genetic diseases. "Although the workings of CRISPR-Cas9 sound simple, the details of the mechanisms involved are rather subtle," says Charpentier, Director at the Max Planck Institute for Infection Biology. Before the crRNA molecule can show the Cas9 protein the cutting point, it must be transformed into its final form itself: RNA-cleaving proteins are needed so that a functioning crRNA arises. One of these is RNase III. In 2011, Charpentier discovered that this enzyme is involved in the crRNA maturation process along with tracrRNA. The researchers have now discovered that the immune defence mechanism of some bacteria is simpler in structure than CRISPR-Cas9. In addition to Cas9, these bacteria use the enzyme Cpf1 for cleaving foreign DNA. The results now show that Cpf1 can cut both RNA and DNA. Cpf1 first removes sections of the crRNA and thereby assists the maturation. Additional maturation enzymes like RNase III are not required. The mature RNA-molecule then guides Cpf1 to its target section on the DNA. Cpf1 thus has a dual function: it enables the functioning of crRNA and then cleaves the DNA in a sequence specific manner. In addition, unlike Cas9, Cpf1 is not depending on the help of a tracrRNA molecule to reach its destination. Consequently, it is even simpler in structure than CRISPR-Cas9. "CRISPR-Cpf1 is a plug-and-play system with no additional component needed. In contrast, CRISPR-Cas9 needs in its natural setting an assistant to activate the system," explains Charpentier. "If the CRISPR-Cpf1 system provides any tangible added value over the CRISPR-Cas9 system when it comes to eukaryotic gene editing remains to be elucidated. However, it is stunning to see how evolution has succeeded to yield a dramatically minimalistic but effective immune system to fight invading viruses", says Charpentier. "There may be more such systems to be found in nature in the future, the search for them is already in full swing." Explore further: Revolutionizing genome engineering: Review on history and future of the CRISPR-Cas9 system published More information: Ines Fonfara et al. The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA, Nature (2016). DOI: 10.1038/nature17945
In a new approach to genome editing called “base editing,” researchers have engineered the CRISPR-Cas9 enzyme to modify individual DNA bases more efficiently and more accurately than with existing gene-editing methods. Many genetic diseases arise from single-base mutations, or point mutations. David R. Liu, postdoc Alexis C. Komor, and coworkers at Harvard University developed the new base-editing process and showed that it can correct point mutations in living cells, suggesting the method could lead to therapies that correct genetic errors that cause human diseases (Nature 2016, DOI: 10.1038/nature17946). Researchers currently use zinc finger nucleases, transcription-activator-like effector nucleases, and CRISPR-Cas9 for genome editing. All three tools initiate genome editing by cutting both strands of DNA. Cellular machinery reduces the efficiency of these editors’ ability to correct point mutations by trying to repair such double-strand breaks with so-called indels, random DNA insertions or deletions. By avoiding double-strand breaks, the new approach causes far fewer indels and corrects point mutations more efficiently. Liu and coworkers began by using a Cas9 mutant (dCas9) that doesn’t cut DNA but retains native Cas9’s ability to bind to target DNA sequences. In this case, the team wanted to target C:G base pairs and change them to T:A. They made their first base editor by fusing the mutant to cytidine deaminase, which converts cytosine (C) to the RNA base uracil (U), thus changing targeted C:G base pairs to U:G. Cellular DNA repair and replication machinery can correct such U:G mismatches to U:A or T:A. But cells’ DNA repair machinery can also change U:G back to C:G, so the researchers made a second base editor by appending to their first base editor a small protein called uracil glycosylase inhibitor, which prevents that switchback. In human cells, the second base editor increased the efficiency of base editing several-fold, compared with the first. Liu and coworkers then restored one of the DNA-cutting active sites in dCas9, creating a third base editor that further increases efficiency by favoring cellular repair of U:G to U:A or T:A over the switch back to C:G. In tests in cell culture, Liu and coworkers observed that conventional CRISPR-Cas9 corrected 14 point mutations about 1% of the time, while causing about 5% indels. Their second base editor corrected about 10% of the same mutations and caused almost no indels, whereas the third was about 30% efficient and caused about 1% indels. Both the second and third base editors are useful, Liu notes, depending on whether mutation efficiency or avoiding indels is the more important goal of a particular experiment. The researchers demonstrated the power of the technique by using it to reverse point mutations associated with Alzheimer’s disease and cancer in mouse and human cell lines, with minimal indel formation. Emmanuelle Charpentier of the Max Planck Institute for Infection Biology, who helped discover the CRISPR-Cas9 technique, comments that “the study shows once again the power and versatility of the CRISPR-Cas9 mechanism.” The work “predicts future exciting applications in tailored genome and epigenome base editing.” This article has been translated into Spanish by Divulgame.org and can be found here.
News Article | April 22, 2016
Melbourne researchers have uncovered the genes responsible for the way the body fights infection at the point of 'invasion' - whether it's the skin, liver, lungs or the gut. Research led by Dr. Axel Kallies and Dr. Klaas van Gisbergen at the Walter and Eliza Hall Institute of Medical Research, and Dr. Laura Mackay from the University of Melbourne at the Peter Doherty Institute for Infection and Immunity has identified the genes Hobit and Blimp1 and found that these genes control a universal molecular program responsible for placing immune cells at the 'front lines' of the body to fight infection and cancer. The presence of these organ-residing cells, which differ strikingly from their counterparts circulating in the blood stream, is key to local protection against viruses and bacteria. Walter and Eliza Hall Institute's Dr. Kallies said the human body was fighting disease-causing pathogens every minute of its life. Dr. Kallies said identifying how immune cells remain in the part of the body where they are needed most was critical to developing better ways to protect us from infections such as malaria or HIV. "Discovering these 'local heroes' and knowing how the localised immune response is established allows us to find ways to ensure the required cells are positioned where they are needed most," Dr. Kallies said. "This research will help us understand how immune cells adapt, survive and respond within the organs they protect. This is critical to rid the body of pathogens even before they are established and may also have implications for understanding how the spread of cancer could be prevented." The Doherty Institute's Dr. Laura Mackay, who is also an associate investigator with the Australian Research Council Centre of Excellence in Advanced Molecular Imaging, said the factors that control the 'tissue-residency' of immune cells - their ability to locally reside in different organs of the body - was previously unknown. "These results have major implications for developing strategies to induce immune cells in tissues that protect against infectious diseases," Dr. Mackay said. "It's a crucial discovery for future vaccine strategies - Hobit and Blimp1 would be key to placing immune cells in the tissues, which we know are really important for protection." The findings have just been published in the journal Science.
A versatile technique for editing genomes has been called the biggest biotechnology advance since the polymerase chain reaction (PCR), and the US Patent and Trademark Office (USPTO) is set to determine who will reap the rewards. On 11 January, the USPTO granted a request to review a key patent awarded for the technique, known as CRISPR–Cas9. The outcome of the ensuing proceedings, called a patent interference, could be worth millions to the research institutions that are at war over the relevant patents. It might also influence who is allowed to use the technology — and under what terms. “This is an absolutely humungous biotech patent dispute,” says legal scholar Jacob Sherkow of New York Law School. “We’re all waiting with bated breath.” CRISPR–Cas9 is a bacterial defence system that uses the enzyme Cas9 to snip DNA at sites determined by the sequence of a ‘guide’ strand of RNA. Scientists can disable, replace or tweak genes by using the technique to rewrite snippets of DNA sequences. Use of the technology in research has exploded, thanks to CRISPR–Cas9’s relative simplicity and versatility compared to other gene-editing methods. Several companies have sprung up to harness the technique for generating improved crops, research reagents and therapies for human genetic diseases. The roots of the CRISPR–Cas9 dispute date back to 2012, when researchers reported that they had reprogrammed the system to cut strands of isolated DNA at sites of their choosing1. The team, led by biologists Jennifer Doudna at the University of California, Berkeley, and Emmanuelle Charpentier, now at the Max Planck Institute for Infection Biology in Berlin and Umeå University in Sweden, filed a patent application on 15 March 2013. By then, publications had emerged from other groups showing that the method works in human cells2–4 and bolstering dreams of CRISPR-based gene therapies — the basis for several companies that have sprung up to capitalize on the technique. One of those groups, led by synthetic biologist Feng Zhang of the Broad Institute and the Massachusetts Institute of Technology, both in Cambridge, filed a patent application for the CRISPR–Cas9 technique in October 2013. The institutions filed the patent under a special expedited review programme, and it was granted in April 2014. Zhang has since been awarded additional patents on the technology. The original Doudna–Charpentier patent remains under review. In April 2015, the Berkeley team asked the USPTO to begin an interference proceeding to determine which team was the first to invent the technique. The proceedings will be much like a court case, with both sides presenting evidence culled from publications and laboratory notebooks. “Once the [USPTO] declares an interference, that’s really when the fur is going to fly,” Sherkow predicted in a June interview. The patent interference is also a testament to the high stakes involved: companies aiming to use CRISPR–Cas9 for gene therapy have raised hundreds of millions in venture capital and other funds in under three years. One company, Editas Medicine in Cambridge, Massachusetts, has already filed to go public. Arti Rai, a legal scholar at Duke University in Durham, North Carolina, says that it is unusual for academic research institutions to battle so intensely over a patent. Instead, such institutions usually come to an agreement to share rights to the invention. “This seems more bitter than disputes I’ve heard of in the past,” she adds. The two patents in question make broad claims to 'foundational' intellectual property thought to be necessary for most lucrative CRISPR–Cas9 applications. But many patents have been filed on CRISPR–Cas9 technologies, and there is still the chance that the winner of the interference will face additional challenges in court. Zhang's group has also reported another enzyme, called Cpf1, that could provide an alternative to Cas9. Researchers expect other alternatives to emerge with time. As for the various CRISPR–Cas9 companies, Zhang remains involved in Editas, which was founded by both Zhang and Doudna, among others, in 2013. Doudna has since severed ties with Editas and thrown her support behind Intellia Therapeutics, also in Cambridge. Charpentier, meanwhile, co-founded CRISPR Therapeutics of Basel, Switzerland. For now, it is unclear how the dispute will affect researchers who use CRISPR–Cas9, if it does so at all. Academics who might use the technology for basic research make unattractive targets for patent lawsuits, says Rodney Sparks, a biotechnology patent counsel at the University of Virginia in Charlottesville. “Patent holders might send out a few cease-and-desist letters, but they probably won’t sue academic researchers,” he says. Doing so would take time and money with little reward: the spoils in a patent lawsuit are typically damages or a share of royalties from a marketed product. That leaves little to gain from suing academics who are not selling anything. But those who intend to use their research as the basis for a start-up company will need to be wary, Sparks says. Some patent holders do ask that even scientists doing basic research take out a licence on a patented technology, typically for a fairly small fee. Such was the case for PCR, says Warren Woessner, a lawyer at Schwegman Lundberg and Woessner in Minneapolis, Minnesota. Woessner recalls how, during his previous career as a scientist, his institution decided to patent a method he developed. Officials at the institution later noticed that someone had published a paper that used the technique without a licence. “They sent the professor a little note,” recalls Woessner. “‘We have a patent on this. Pay up.’” The professor did. The Broad Institute has noted on its website that it will continue to make CRISPR–Cas9 reagents available to the community, and has given no indication that it will pursue licensing fees from academics. But Sherkow warns against assuming that the spirit of academic camaraderie will prevail: licensing revenue has become increasingly important, particularly for major research institutions, he says. “We’re just living in a brave new world these days.”