Gene genius

C&I Issue 10, 2015

In the early 2000s, Rudolphe Barrangou and Philippe Horvath, at Danish bio-based food company Danisco, made a surprising discovery about bacterial immunity. Sequencing the genomes of Streptococcus thermophilus, a bacteria important in yoghurt and cheese production, revealed stretches of DNA referred to as CRISPR sequences, later found to play a role in conferring resistance again bacteriophage attacks. Phage infection of starter cultures is a big problem for dairy manufacturers, slowing down production capacity.

The Danisco researchers’ work led to the first direct evidence for adaptive immunity in bacteria, and showed how CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) evolve, by acquiring spacers or fragments of DNA from phages.1 Bacteria with new spacers then become phage-resistant, and these resistant strains are highly desirable for dairy cultures in food production.

In fact, cheese and yoghurt manufacturers have been using CRISPR to make starter cultures for around a decade to deter bacteriophages and avoid wasted food. Anyone eating dairy products will almost certainly have eaten something with a hint of ‘added CRISPR’.

In 2012, DuPont, which now owns Danisco, launched a blend of S. thermophilus strains for commercial applications in pizza cheese. Interestingly, the cheese is not classed as a GMO, because food scientists have produced them by repeatedly exposing the bacterial population to phages and harvesting the resistant cells, rather than by direct genetic modification. DuPont’s ‘added value’ is to identify whether the bacteria have gained new CRISPR genetic sequences.2

The CRISPR process, then, was first identified as the defence mechanism used by bacteria against invading viruses. But this area of study collided with targeted genome engineering when scientists led by Jennifer Doudna at the University of California, Berkeley, US, showed that CRISPR can be used to target any region of a genome with great precision, with the aid of a cutting enzyme called CAS9.

The big potential of CRISPR technology is as a new genome editing tool – to cut paste and edit selected DNA targets. CRISPR technology relies on the CRISPR-associated protein 9 (Cas9) enzyme. Doudna and her colleagues showed how to redirect Cas9 to target almost any DNA sequence using a short RNA molecule called a single guide RNA or sgRNA. Like a pair of molecular scissors, Cas9 then edits the DNA by making a double-strand break, to disrupt genes or insert a desired replacement sequence.3

CRISPR-Cas9 has the potential to target and repair naturally-occurring genetic defects; add new pieces of foreign DNA with new functions at a target location; or simply remove specific DNA fragments. The Cas9 enzyme can easily be matched with ‘guide’ RNA (gRNA) sequences that lead to DNA targets. Tens of thousands of such gRNA sequences have already been engineered. CRISPR-Cas9 can also target multiple genes at the same time, another advantage over rival gene-editing tools.

The research from Doudna’s team in 2012 was rapidly followed up with researchers using CRISPR for editing mouse and human cells by 2013. Research around the globe accelerated: the results of a CRISPR gene drive to rapidly spread edited genes through a population – for example, through harmful organisms like mosquitos in a bid to combat malaria – were reported in March 2015.

Alternative gene editing methods use zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Both of these proteins are effective, but because each new target requires a new nuclease enzyme to be engineered they are more involved and costly. As a result, many labs have now turned to use CRISPR in their projects.

In April 2015, for example, geneticists in China reported editing the genomes of human embryos, by modifying the HBB gene, which if mutated leads to the blood disorder β-thalassaemia. This germ line editing, which the team at Yat-sen University in Guangzhou, carried out on non-viable embryos, prompted ethical debate over whether enzyme editing technology is moving too fast for safety. Editing DNA in the human germline could provide complete cures for deadly genetic diseases — but could equally introduce changes to the human genome that would be passed from generation to generation.

The researchers injected embryos with the CRISPR-Cas9, to find out if the procedure could replace a gene in a single cell fertilised human embryo. Of the 54 embryos the researchers tested, 28 were successfully spliced and only a fraction contained the correct replacement genetic material. The rate of mutations was much higher than had been seen in similar studies with mouse embryos or adult human cells. They concluded that the technology was still too immature; although the fact that abnormal embryos were used could be a factor.4

Nevertheless, medical applications for CRISPR are well under way. In the US, Cambridge, Massachusetts-based Editas Medicine was launched in 2013, with initial venture capital of $43m. The company’s aim is to develop therapies to directly modify disease-related genes. In May 2015, Editas announced a collaboration with Juno Therapeutics. Juno’s approach involves engineering T-cells to find and destroy cancer cells, and the company reported promising results in shrinking tumours in leukaemia and lymphoma in trials at the same time as the deal announcement.

‘Editas’ disruptive genome editing technology may unlock the ability of CAR (chimeric antigen receptor) and TCR (high-affinity T cell receptor) technologies to address a much wider range of cancers, giving hope to countless patients and families waiting for treatments,’ said Juno ceo Hans Bishop.

Other Editas projects include in vitro tests on the potential of CRISPR/Cas9 to repair the haemoglobin beta gene to correct sickle cell anaemia. The company is also working to improve the Cas9 enzyme itself, using Cas9 derived from S. aureus rather than S. pyogenes, which expands the amount of DNA sites that researchers can target and gives them more options.

Editas cofounder Doudna, who jointly led the team that developed CRISPR/Cas9 at Berkeley, is also a key player in Intellia Therapeutics. Launched in 2014 with $15m in finance led by Atlantis Venture and Novartis, Intellia is focused on in vivo and ex vivo therapeutic applications of CRISPR-Cas9. Near-term targets for ex vivo gene editing – eg removing cells from blood or bone marrow, treating and then returning them – include blood disorders, therapeutic protein production and cancer.

On the in vivo side, Intellia is exploring ways to deliver gene editing treatments directly to the site of the disease, either systemically or locally. Potential targets include genes in the eyes, central nervous system, muscle, lungs and liver.

In vivo editing also has the potential to fight infections or tumours. Novartis is collaborating with Intellia, with a strong interest in CARTs and hematopoetic stem cells (HSCs). ‘CARTs and HSCs represent two of the most immediate opportunities for CRISPR therapeutic development, and Novartis, as a leader in this space, is the ideal partner with which to develop strong product pipelines in these areas,’ according to Intellia ceo Nessan Bermingham.

Elsewhere in the dairy world, meanwhile, Scott Fahrenkrug of Recombinetics, a St Paul, Minnesota, US, biotech company that focuses on genetics technologies for agriculture and biomedicine, is using TALENs and CRISPR-Cas9 to target a gene called POLLED in dairy cattle. The aim is to switch it to a version resembling one found in hornless beef cattle. The researchers have used these gene-editing tools to develop cell lines with the desired genotype, to create GM dairy cows without horns – an announcement on progress is due later in 2015.

Dehorning dairy cows is labour-intensive Fahrenkrug says.5 ‘Producers have been wanting to get rid of this trait for a long time, and it just takes too long using traditional breeding methods.’

The reaction from farmers and cattle producers has been overwhelmingly positive, the company says. ‘Perhaps the people most receptive to our technology are those doing the dehorning. The process is traumatic for all those involved, and the idea of a genetics-based solution is generally met with praise.’

The public’s wariness around GMO produce is well known, but Recombinetics thinks that animal welfare benefits will count in its favour.  ‘American consumers are overwhelmingly interested in improving animal welfare and genetically dehorning cattle is the perfect way to enhance animal welfare while ensuring the safety of the animals and their handlers,’ Fahrenkrug says.

Beyond cattle, the company is using TALEN and CRISPR for a range of projects spanning agriculture, biomedicine and regenerative medicine.

Regulatory agencies around the globe, meanwhile, are considering how best to regulate gene edited products. Several countries, including the US, Argentina and Germany, have already indicated that crop plants developed through gene editing will not require the same level of pre-market approval that transgenic crops currently face.

In the US, when the genome-edited plant contains no foreign DNA, and the resultant change cannot be distinguished from a natural mutation, it does not count as a GMO. In the EU, however, an organism has until now been considered genetically modified if it has been altered in a way that does not occur naturally by mating and/or natural recombination. Crops altered through mutagenesis using chemicals and radiation are considered exempt from these regulations, because thousands of crop varieties have been generated in this way since the 1940s with no evidence of risk.

In July 2015, the US government announced a year-long review of the Coordinated Framework for the Regulation of Biotechnology, in part because of the advent of technologies like TALEN and CRISPR.Many believe that the barriers to modified animals like the hornless dairy cow coming to our farms will be far from trivial. But CRISPR could represent a breakthrough in GMO safety, according to others. Christopher Voigt’s team at the Synthetic Biology Centre at Massachusetts Institute of Technology has developed a new, targeted device relying on CRISPRs that can be triggered to activate under specific conditions, which he describes as ‘a delete key for DNA’.6

‘CRISPR machinery can be used to degrade specific intracellular DNA in an inducible and targeted manner,’ according to Voigt. ‘One can imagine combining these systems to create layers of redundancy to ensure that engineered organisms are viable in particular environments and if they escape, they eliminate their synthetic DNA.’

The device is non-toxic, is stable for months, and is inactive until the specific target conditions or molecules come into play. Whether GMOs need to be taken out of circulation because of accidental release, or just so that they can be safely used as fertiliser, the extracellular DNA eventually decays in the soil within around two months.

Another application to benefit from the switch would be safeguarding intellectual property. Sensitive samples primed to self-destruct under certain conditions – if exposed to light for instance – could help to protect trade secrets from theft. Voigt is also involved in research that brings the idea of a targeted device to human gut bacteria.

Researchers say the market for CRISPR RNA-guided nucleases could be $46 bn in biomedicine.7 But this figure only takes into account their use as research tools. Look as well at agriculture, biomedicine and regenerative medicine, and those in the field believe the value of gene edited products in those markets will be much greater.

Glowing results

In July 2015, US researchers reported a series of sensors, memory switches and circuits, encoded in the common human gut bacterium Bacteroides thetaiotaomicron.8 The bacteria sense, remember and respond to gut signals. And thanks to a luciferase gene inserted into the gut bacteria’s genome, the glowing faeces from mice used in the study shows researchers when it has been switched on.

 ‘We wanted to work with strains like B. thetaiotaomicron that are present in many people in abundant levels, and can stably colonise the gut for long periods of time,’ said Timothy Lu, associate professor of biological engineering, and electrical engineering and computer science, who led the research alongside Voigt at the Massachusetts Institute of Technology, US.

Sufferers of colon cancer or Crohn’s disease could benefit, the team hopes. If inflammation flips a bacterial switch to make coloured faeces, patients could seek immediate treatment. Bacteroides could even be pre-loaded with therapeutics, triggered to release by specific gut conditions. Controlling the quantity of genes the bacteria make is an important safety consideration, however, along with how long the engineered bacteria survive in the gut.8

To be useful, the bacteria need to remember information on the host organism’s gut pathology and report it externally. Bacterioides populations naturally have multiple cellular states thanks to reversible recombinases that vary the expression of their cell-surface polysaccharides. These recombinases were used as a form of externally switchable genetic memory device for B. thetaiotaomicron, as the team integrated inducible promoters to make them ‘switch’ depending on the levels of specific substances in the mouse food.

The researchers also used CRISPR mediated gene knockown to control which of the bacteria’s genes is activated, modulating B. thetaiotaomicron’s ability to consume a specific nutrient and to resist being killed by an antimicrobial molecule. When mice ate food containing specific ingredients, their designer gut bacteria remembered what they had eaten, with glowing results.

The next steps include applying the tools to other Bacteroides species, as gut bacteria vary between individuals. Other studies on switches being placed in the gut via bacteria exist, but this one is the first to get them working with the most common gut bacteria genus, Bacteroides.


1. R. Barrangou et al, Science, 2007, 315, 1709.
2 K. Grens, The Scientist, January 1, 2015.
3 M. Jinek et al, Science 2012, 337(6096), 816.
4 P. Liang et al, Protein & Cell, 2015, 6(5), 363.
5 J. Akst, The Scientist, June 1, 2014, 41.
6 B. J. Caliando and C. A. Voigt, Nature Commun., 2015, 6, 6989.
7 P. B. G. van Erp et al, Curr. Opin. Virol., 2015, 12, 85.
8 M. Mimee et al., Cell Systems, 2015, 1, 1.

Helen Carmichael is a freelance science writer based in Dorset, UK

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