EMBL’s GeneCore steps up to discover the facts and settle disputes
Munich, the Bavarian state capital, is the quintessential German town. From lederhosen to Oktoberfest, it’s filled with tradition and antiquity. It was here, however, that a curious group gathered to discuss one of the most revolutionary biological techniques of the 21st century: CRISPR-Cas, the latest tool for gene editing (see explainer: What is gene editing?). Its convenience and affordability make CRISPR-Cas particularly accessible, but who should be using it, and what for? For most attendees sitting in the sunlit seminar room, this was a topic for discussion. But at the close of that day’s meeting, a Bavarian government official asked one of the organisers to hand over the CRISPR-Cas kit sitting unopened at the front of the room. No explanation was given.
In the lab
CRISPR-Cas is already being used in research labs around the world. It allows biologists to cut strands of DNA in specific places to insert or delete particular genes (see infographic: How does CRISPR-Cas work?). At EMBL, CRISPR-Cas is found almost everywhere, both in research and as an educational tool. It’s used to insert genes to fluorescently label and track proteins in zebrafish, or to delete genes in lab-grown brain cells to mimic neurodegenerative diseases, allowing scientists to study these conditions. EMBL is co-organising a CRISPR-Cas course in March 2019 which will teach scientists how best to use the technique, through theoretical and practical sessions. Researchers at EMBL Rome have visited a local school to introduce and discuss the concept and its implications to pupils.
What is gene editing?
Genes are sections of DNA that usually act as code for building a specific protein within a cell. Different proteins might affect how a cell looks or functions, including its susceptibility to disease.
Genes can be edited in the lab to change a cell’s characteristics. A gene can be deleted so that the protein it encodes won’t be made. Deleting a gene and studying how the cell or organism changes can give researchers an idea of what cell characteristics the deleted gene normally controls. Deleting a gene can also mimic a genetic disease, so that the disease and its potential treatments can be further researched. Alternatively, a gene that encodes a new protein can be inserted into the DNA, potentially altering the cell’s characteristics.
In Germany, CRISPR-Cas technology is strictly limited to certified labs, but people’s curiosity extends much further. Those particularly interested in its application are members of the do-it-yourself biology (DIYbio) community, whose scientific backgrounds are as broad and varied as their aims. Their portrayal in the press has been mixed, with journalists alternately highlighting DIYbio’s potential dangers or educational possibilities.
At the DIYbio space in Munich, one group of enthusiastic undergraduate students have set their sights on the International Genetically Engineered Machine (iGEM) science competition. Others are more interested in improving the taste of their homemade kombucha – a type of fermented tea – by creating automated monitoring systems. Whatever their interests, these groups share a common curiosity and fascination with biology.
Rüdiger Trojok is one such DIY biologist. Today he works alongside industry partners to translate creative biological ideas into start-up companies. Prior to this, he completed his formal biology training to master’s level and for almost five years he worked in Berlin as a policy consultant for the German parliament. There, he advised on changes to the laws surrounding genetically modified organism (GMO) regulations and applications. In Berlin, Trojok set up a home laboratory and would regularly call government officials to clarify the legality of certain techniques he wished to use. “Techniques such as making green fluorescent protein in bacteria are relatively safe and have been known about for a long time,” says Trojok. “But for DIY biologists, there are a lot of grey areas concerning the current GMO laws.”
As new biological techniques such as CRISPR-Cas surface, Trojok believes that people’s curiosity about biology will grow. In light of this, he set up the CRISPR.kitchen in 2017 – a week-long event in Munich to explain what CRISPR-Cas is and how it works. Discussions about the ethical issues and legal framework surrounding the technique were a key part of the event. To solidify the concepts, Trojok bought a DIY CRISPR-Cas kit online from a US company. “From its description I knew that it was legal to own it, but not to use it,” says Trojok. “That’s why I invited officials from the Bavarian government department for health and food safety [LGL] to the event – I wanted to discuss the legality surrounding the kit.”
The event took a new turn, however, when the LGL asked – without explanation – for the DIY CRISPR-Cas kit to be handed over. Trojok and other DIY biologists were keen to know what was going on. “The government didn’t want to discuss why they took away the kit,” says Trojok. “We didn’t know why they were acting so strangely.” A week later, a leaked government document surfaced, revealing that several potentially pathogenic bacteria had been discovered in three independently obtained kits. This was closely followed by an official press release from the LGL, but Trojok was still unable to discuss the situation with government officials. “I asked the Bavarian agency to reveal the original data, but they refused,” says Trojok. “If they had really found contamination, we didn’t know why they wouldn’t share this information.” Trojok contacted the company who manufactured the kits, asking them to run their own tests. These came back clean, showing only a safe strain of E. coli bacterium, which was listed as part of the kits.
EMBL steps in
Clarification was sorely needed, so the DIY biologists approached Vladimir Benes, head of the Genomics Core Facility (GeneCore) at EMBL’s Heidelberg site. “EMBL is independent of the company that produced the kit, the DIY biologists who want to use it, and the government that banned it. We just want to know the facts,” explains Benes. This independence stems from one of EMBL’s founding goals: to be a supranational research centre that is independent of the changing priorities of national governments. “It was an opportunity for GeneCore to provide a service to one of its member states and manage a conflict by being a neutral party,” Benes continues.
From the information that Trojok had gleaned, Benes knew that different identification tests had been used by the LGL and the company involved. Researchers at the LGL tested the identity of the bacterial species using a technique known as MALDI/TOF analysis. This uses mass spectrometry to identify which bacterial molecules are present and then compares them against a known set of bacterial profiles. Meanwhile the company used a method known as 16S sequencing, in which a specific DNA region in the bacterial cells is sequenced and compared against public databanks.
Both analyses are routinely used in professional research labs to identify bacterial species, but each method has its limitations. At GeneCore, whole-genome sequencing is the norm. This method – in which all the bacterial DNA is amplified, sequenced and compared against the genome that the researchers expect it to be – is currently the gold standard in DNA sequencing. According to Benes, this method is the most robust form of analysis to find out what’s really in a sample.
Aided by government agencies in Berlin – with whom Trojok has a close working relationship – the DIY biologists provided EMBL with three of the same DIY CRISPR-Cas kits, bought before the CRISPR.kitchen event. Alongside Benes, GeneCore team members Anja Telzerow and Jonathan Landry set to work: Telzerow in the lab – taking all necessary precautions for working with an unknown substance – and Landry at the computer, meticulously analysing the DNA sequences.
Bacterial colonies could be grown from only one of the kits, so it was these that were sequenced and compared with a known E. coli genome at GeneCore. Only 30% of the DNA fragments from the kit matched the E. coli genome – an unexpected result for a kit supposed to contain only E. coli. Yet the exact bacterial species present were difficult to pin down, as large portions of the DNA sequences matched four other bacterial species. Tellingly, three of those were species within the Enterobacter genus, a type of bacterium which was also identified by the LGL. But Benes had to be sure of the result – it was vital to confirm the findings with another test. Therefore, the bacterial DNA sequence in question was compared with a subspecies of Enterobacter, profiled in one of GeneCore’s databases. This gave an 83% match, indicating that the bacterium present in the kit was likely a species of Enterobacter. “Without Anja and Jonathan’s skills and tenacity, we wouldn’t have been able to get the right answers,” says Benes.
A risk assessment of the kit by the European Centre for Disease Prevention and Control deemed the risk of infection as ‘low’, and no one was thought to have been harmed as a consequence of this contamination. But some species of Enterobacter are known to cause opportunistic infections in ill people or hospitalised patients.
It’s still unknown why the company didn’t discover the bacteria in their production line. It may be that contamination of the kits occurred during or after the kit had been shipped, as indicated by the company in their official press release. Another explanation, proposed by both Trojok and Benes, is that the kits were contaminated during production, and the 16S sequencing method was not robust enough to distinguish between two bacterial genera. Either way, both Trojok and Benes believe that, in this instance, the government was right to ban the product. “I need to be able to trust the source of everything that I order into my lab at EMBL,” says Benes. “This should be true for everyone interested in learning more about biology through kits such as these.”
We hope to see DIY biologists learning in a safe environment – and doing sound science
In our interlinked societies, bacteria can travel much further and faster than they would be carried by a mere sneeze. Other industries transporting potential bacterial breeding grounds, such as food, must therefore adhere to strict regulations. But companies supplying curious DIY biologists with kits like these are new players to the game – and it’s unclear how they are regulated. Where does the freedom to explore and learn end, and the responsibility to protect begin? For Trojok, the answer is clear. Whether it’s one person tinkering around with kombucha tea, a company shipping educational biological kits across the world, or an international research organisation such as EMBL, “You have to take responsibility for what you do.”
At EMBL, there are stringent regulations in place to allow scientists the opportunity to explore innovative and sometimes off-beat research questions. Within the DIYbio community, Trojok does everything he can to ensure national regulations are put in place, to allow people the chance to safely explore the latest technologies and advances in biology. But, as this situation has shown, there can still be grey areas when sourcing educational kits from companies where the regulations are unclear, or where they differ between countries. For scientists at EMBL, the DIYbio movement provides a conundrum. “As scientists, we recognise and support the urge to explore, ask questions and tinker,” says Benes. “But we also hope to see DIY biologists learning in a safe environment – and doing sound science.”