CRISPR – clustered regularly interspaced short palindromic repeat – refers to unique repeated DNA sequences found in bacteria and other single-celled organisms. In bacteria, CRISPRs bind to, and slice up, foreign DNA, preventing it from establishing an infection in cells. Researchers are now working on exploiting this defence mechanism for people and plants.
Although CRISPR arrays were first identified in the bacteria Escherichia coli in 1987, their biological function was not understood until 2005. It took a bit longer for the transition from biological phenomenon to genome engineering tool, which came about in 2012.
Work since then has confirmed that the system, being small and easy to reprogramme, offers a flexible, easy-to-implement and relatively cheap method for genome editing.
When a bacterium is attacked by an invader such as a virus, it captures some of the virus’ DNA, chops it up into pieces and incorporates a segment of the viral DNA into its own genome. In this way, it accumulates a bank of past infections in a special part of its genetic code – CRISPRs – which act as a kind of immune system to protect against future invasions. CRISPRs create a small non-coding strand of RNA, known as the single-guide RNA (sgRNA), which targets the gene of interest by matching DNA sequences specific to a given invader. When those CRISPR RNAs find a match, they unleash proteins (Cas9) that chop up the invader’s DNA, preventing it from replicating.
‘CRISPRs provide a molecular memory of previously encountered viruses, which is used to rapidly mount an immune response upon subsequent encounters with an infectious agent,’ explains Blake Wiedenheft, assistant professor of microbiology and immunology at Montana State University, US. ‘This is conceptually similar to our own immune systems.’
In recent research, Wiedenheft and his colleagues, at Cornell and John Hopkins Universities in the US, report on how the immune system in E. coli differentiates between its own genetic material and that of a virus.1 Without this ability, the antiviral measures would damage the host cell. To understand how it does this, the researchers used X-ray crystallography to create a detailed molecular blueprint of the CRISPR complex as it recognised a piece of foreign DNA. ‘Knowing how they work is a critical first step in reprogramming them for novel applications,’ says Wiedenheft.
CRISPR systems have been transplanted into human cells and programmed to cut out and repair defective DNA, providing great potential for treating genetic diseases, such as muscular dystrophy and cystic fibrosis. For plant scientists, CRISPR technology is another useful and potentially very efficient way to adapt crops to the challenges of disease and a changing climate.
The first applications of CRISPR or RNA-guided Cas9 in plants were described in 2013. Then in 2014, a team at the Shanghai Center for Plant Stress Biology, China, showed for the first time that induced mutations could be inherited in the plant model Arabidopsis.2 Since then, researchers have shown that heritable changes are possible in rice and wheat, and have used the CRISPR system to mutate genes in wheat to make the crop more resistant to powdery mildew.3
Wendy Harwood, a senior crop genetics scientist at the John Innes Centre in Norwich, UK, comments: ‘The beauty of the CRISPR technique is that it can create small changes in specific genes, sufficient to stop them working. This is one way to develop disease resistant crops, for example, with resistance to mildew, or to produce crops without unwanted compounds, such as toxins. The final plants produced in this way have no additional DNA inserted so they are essentially the same as plants with naturally occurring changes to genes or plants that have been bred using conventional mutation breeding methods.’
Recently, Harwood was involved with a team of UK scientists who used CRISPR to edit specific genes in barley and a broccoli-like Brassica. The team from the John Innes Centre, Norwich, and The Sainsbury Laboratory, also in Norwich, targeted the gene in barley that is thought to affect grain dormancy, where genes don’t germinate even under favourable conditions. In Brassica, they targeted the gene associated with how easily seed pods break open.
In the editing process, the team used the CRISPR system to make a cut in the DNA sequence of the targeted gene. The small changes in the sequence occurred when the cut was repaired using the plants’ own repair process: this is the edit they wanted. In both cases, these changes were small, involving just 1–6 bases of the DNA sequence in the target gene, they report, but they were enough to prevent the target gene from working.4
Significantly, the researchers found that these edits were passed down to subsequent generations of plants. They also showed that it is possible to produce next generation plants that contain the edit but not the genetic material used during the editing process (the sgRNA), confirming their genetic make-up was the same as conventionally bred plants.
In both plants, the team found that they had produced ‘off-target edits’ in a very closely related gene as well as the edit to the target gene. This presents both potential difficulties and specific opportunities, they say. The effect could be useful in crops if scientists want to edit several members of a gene family at the same time, but it also provides insight into how to use the CRISPR system to ensure only a target gene is edited.
Meanwhile, researchers from Saudi Arabia are investigating whether the CRISPR system could be used to give plants immunity against DNA viruses, a major cause of crop disease. The team from King Abdullah University of Science and Technology (KAUST), led by Magdy Mahfouz, studied the tomato yellow leaf curl virus (TYLCV), a significant scourge of tomato crops.
They used the CRISPR system to engineer tobacco plants so that they contained a RNA sequence designed to match part of the DNA sequence of TYLCV. When the plants were infected with TYLCV, the RNA sequence directed the Cas9 protein to attack the viral DNA, preventing it from replicating and significantly reducing disease symptoms.5 The researchers next engineered plants with several identical RNA sequences. This process generated even stronger resistance to TYLCV, they report, an effect called ‘multiplexing’. The team also found that it was possible that a single RNA sequence could protect against more than one virus from the same family – in this case, TYLCV, beet curly top virus and merremia mosaic virus.
This simple microbial system can provide crop plants with a powerful defence against multiple invading viruses, Mahfouz notes. The system can even be fine-tuned to defend against newly emerging viral strains simply by tweaking the RNA sequences used, he adds. The next step for the KAUST team is to modify the system to work on RNA-based as well as DNA-based viruses. Mahfouz is confident that this more difficult challenge is also surmountable. ‘It is only a matter of time,’ he says.
Trees could also benefit from the power of this genome editing tool, according to researchers at the University of Georgia, Atlanta (UGA), US. By mutating specific genes in Populus – a genus of deciduous trees that includes poplar, aspen and cottonwood – the researchers reduced the concentrations of two naturally occurring plant polymers: lignin and condensed tannins (CTs or proanthocyanidins). A lower lignin content makes the plant a better food source for animals as it is more digestible; and is also beneficial for paper production since lignin must be removed when processing wood fibre to make paper or composite products. A lower CT content would also be good for trees destined for food sources for animals because this polymer, when present in leaves and bark, deters ruminants, such as deer, cattle, goats and sheep, from feeding.
‘CRISPR could improve our ability to produce novel varieties of food crops, animal feeds and biofuel feedstocks,’ comments the study’s lead researcher Chung-Jui Tsai, director of UGA’s Plant Center. ‘Compared with some other gene editing techniques, this is incredibly simple, cost-effective and highly efficient, and it could serve as the foundation for a new era of discovery in plant genetics.’ Tsai says it’s like using ‘a pair of scissors with GPS tracking to locate and snip out tiny bits of DNA, enough to nullify the gene you don’t want, while leaving everything else unchanged’.
When they targeted the lignin gene, the team found that every poplar plant produced had red-coloured wood.6 Red stem is a known side-effect of lignin modification found in natural mutants of maize, sorghum and pine, she explains. The modified plants were uniformly discoloured rather than having patchy red areas, showing how efficiently the process had worked, and they were found to contain about 20% less lignin. In the CT experiments, the modified trees contained 50% less CTs than wild trees but this reduction was less uniformly distributed. Tsai says this could be because CTs have a more complicated biosynthetic pathway than lignin.
‘We thought we knew which genes control lignin and condensed tannin production, and we did target the right genes, but the work showed us that there are other genes with overlapping roles,’ Tsai says. ‘The CRISPR system can now guide researchers seeking to identify these previously unknown gene family members.’ For woody perennials with long generation cycles, it also means that accelerated breeding is finally within reach.
The European regulatory framework for genetically modified crops focuses on the process and not the product so two identical plants produced by conventional breeding and genetic engineering would be regulated differently under the current guidelines. This means that it’s possible that plants altered using genome editing tools such as CRISPR would not be classified as genetically modified organisms (GMOs).
Researchers in South Korea make this point in their work on food crops. The genetic modifications resulting from CRISPR techniques look just like genetic variations resulting from the selective breeding that farmers have been doing for millennia, comments IBS director of the Center for Genome Engineering in South Korea, Jin-Soo Kim. ‘The targeted sites contained germline-transmissible small insertions or deletions that are indistinguishable from naturally occurring genetic variation.’ Since the technique does not use DNA, it may be able to avoid being in violation of EU GM rules.
Kim’s team targeted specific genes from tobacco, lettuce and rice. In lettuce, for example, they disrupted a gene (BIN2), which regulates the signalling of brassinosteroid, a class of steroid hormones responsible for a wide range of physiological processes in the plant life cycle, including growth. They found that, after cell division, almost half of the lettuce cells maintained the disrupted gene.7 Importantly, there were no off-target edits, they add, and they grew full plants from the seeds of the genome-edited plants, which had the mutation from the previous generation.
Jin-Soo Kim believes CRISPR ‘paves the way for the widespread use of RNA-guided genome editing in plant biotechnology and agriculture,’ adding that it could be revolutionary for the future of the seed industry as it produces plants that are more robust and more suited to climate change. In addition, the technique is cheaper, faster and more accurate to apply to plants than previous breeding techniques such as radiation-induced mutations, he adds. This means that it’s not just large agribusiness companies that will be able to afford to develop it – as is the case for genetically modified food – allowing for a more ‘decentralised’ gene-edited seed production industry.
Overall, genome editing is particularly attractive because it can accelerate plant breeding by allowing the direct introduction of precise and predictable modifications. CRISPR is particularly beneficial because several traits can be modified simultaneously, which is difficult to achieve by classical breeding or even conventional genetic engineering.
1 B. Wiedenheft et al, Nature, 2016; doi: 10.1038/nature16995]
2 J-K Zhu et al, Proc. Natl. Acad. Sci. USA, 2014 Feb 18; doi: 10.1073/pnas.1400822111].
3 J.L. Qiu et al, Nat. Biotechnol., 2014, 32, 937.
4 W. Harwood et al, Genome Biology, 2015; doi: 10.1186/s13059-015-0826-7.
5 M. Mahfouz et al, Genome Biology, 2015; doi: 10.1186/s13059-015-0799-6.
6 C. J. Tsai et al, New Phytologist, 2015; doi: 10.1111/nph.13470.
7 J. S. Kim et al, Nature Biotechnology, 2015, 33, 1162; doi:10.1038/nbt.3389