CRISPR/Cas9 - The Complete Story So Far

Author: James Severin, Research Feasibility Officer, Imperial College London Healthcare NHS Trust (other works).

James explores the origins, development, scientific background and subsequent business activities surrounding the emergence of this tremendously powerful gene editing technique.

ORIGINS

In 2011 a conference was held in Puerto Rico in which two Microbiologists had a discussion which quickly led to a huge revelation. Jennifer Doudna, a researcher at the University of California, and Emmanuelle Charpentier, of the University of Umea in Sweden were discussing their research into the immune system of Bacteria, and found that their work was similar enough to justify a collaboration.

The object of their research was a curious genetic system which was originally noted in a paper published in 1987 simply as an "unusual structure" in the DNA code the authors were investigating. Strangely, the sequence they observed was identical both forwards and backwards along the sequence, and on either side of these palindromes were sections of code that couldn't be identified. It wasn't clear at the time what the purpose of this sequence was, and so their observation led to many further projects in the following decades. The observed 'structure' eventually gained the descriptive title of 'Clustered, Regularly Interspaced, Short Palindromic Repeats' commonly known, as CRISPR.

By the time of Charpentier and Doudna's meeting in 2011, studies had already shown that the symmetrical repeats were in fact interspaced with DNA code that matched viral DNA. It was clear that the system must therefore be used as a kind of database which the bacterial cell use to identify invading viral DNA. Once found, the threat could then be removed using specialised DNA cutting proteins.

Doudna and Charpentier, who were independently investigating the DNA cutting enzymes known as the CRISPR Associated proteins 9 (Cas9), aimed to identify how the CRISPR system could direct them towards the threat so that it could be silenced.

Their collaboration led to a paper, written and published in the same year of the conference, which outlined how the CRISPR/Cas9 system functioned. They showed that the system is able to provide adaptive immunity against viral infection by transcribing RNA copies from the integrated viral DNA, and attaching a processed copy to the cutting enzymes. The palindromic sections seemed to act as markers during this process. Once armed with this sequence, the complex would be specific to that type of virus. Now, if this Cas9 complex found the corresponding sequence in the cell, it would bind to it and cut it into two pieces. In this way a defence can be rapidly mounted against any previously registered infection. Since the CRISPR system is integrated directly into the cell genome, all further progeny are also beneficiaries of this defence.

The discovery was an amazing achievement, however Doudna and Charpentier knew immediately that this revelation had significance beyond their stated aims. They realised that system they had described could be used as a reprogrammable genome modification system, where the DNA of the cell could be targeted in place of the virus. Furthermore, it seemed capable of outperforming existing solutions in many ways. If this was true, it was thought that CRISPR/Cas9 could be the first system to be feasibly used by individual researchers or big pharma alike.

 

THE SCIENCE

The two prevalent gene editing technologies, Zinc Finger Nuclease (ZFN), and TALEN, differs from CRISPR in several ways. While these solutions can be modified to identify and silence specific DNA sequences, the process of reprogramming ZFN and TALEN is comparatively expensive and complex, as the DNA cutting protein itself must be reengineered to specify a target sequence. Since the CRISPR/Cas9 system is able to confer specificity to an unmodified enzyme using sequences of RNA, the process is more simple and cost effective.

This simplicity is reflected in the amount of DNA necessary to code the system - only 2 or 3 plasmids (loops of DNA) are required to describe the entire CRISPR/Cas9 system to any cell, allowing the Cas9 proteins to be manufactured in the cell and directed to the gene of choice. ZFN or TALEN on the other hand, typically requires dozens.

Furthermore, CRISPR is also usually more predictable and effective than the alternatives. Jennifer Doudna recalls a colleague trying seven times to silence a gene in mouse models using TALEN, before she offered the newly discovered CRISPR system as an option. Within four weeks, her colleague had “seven for seven” successful experiments.

Finally and perhaps most importantly, CRISPR is effective in many cell types, allowing it to modify yeast, plant, bacterial or human DNA. CRISPR/Cas9 therefore has the additional significance in offering a platform for collaboration in research between groups specialising in Healthcare, Biotech, Agriculture, or perhaps even more distant disciplines. The field of Biosynthesis, a merging of Engineering and Genetics, is now rapidly growing due to the improvements made in the ease and versatility of CRISPR/Cas9.

 

APPLICATIONS

CRISPR/Cas9 therefore offers a comparatively cheap and highly effective tool to carry out gene modification experiments in a wide range of cell types, and this gives the technology a huge amount of potential. In the near future the benefits it offers may allow an emergence of start-up companies creating engineered cells that can produce a range of medicines, animal feed or biofuel, as well as fulfil numerous other purposes. CRISPR/Cas9 could even be used to create DNA 'devices' that function as a proxy to electronic devices, functional at the molecular level.

Through human cell modification, new research avenues in therapeutics through ‘genome surgery’ are much more accessible to researchers using CRISPR/Cas9. In the future it should be possible for example, to treat genetic conditions such as Beta Thalassaemia in the clinic, or to remove viral DNA from the T-Cells of HIV infected individuals to cure latent infection. In fact, any disease that originates from the DNA sequence could be directly treated using CRISPR/Cas9. Along with the rapid development of genome sequencing, the new technology would therefore have a key role in the era of personalised medicine.

 

BIOTECH AND BUSINESS

For bioentreprenuers, the discovery of CRISPR/Cas9 is clearly very significant. It was recently announced that a group of investors, including the Bill and Melinda Gates foundation, have committed to a $120m investment in Editas, a company founded less than two years ago at the time of writing, to support their development of therapeutics. Although there are already a number of well financed start-ups specialising in the use of CRISPR/Cas9, now there seems to be at least one with the backing to match the discovery. The general consensus, is that is it unlikely to be the first.

However, currently, there are many obstacles to overcome before the potential of CRISPR can be fully realised and some of them are significant enough to bring into question whether CRISPR/Cas9 really is the progressive force that the facts seem to indicate.

For therapeutic use, the first major challenge is in modifying the system to mitigate the risk of patient harm. Although several improvements have already been made, CRISPR/Cas9 is too prone to errors in DNA targeting to be suitable for clinical purpose. The system could introduce DNA breaks in unintended places, causing serious damage to the cell and individual. 'Off-target' cutting is therefore is a key area of research, and although cutting errors are not expected to be a permanent barrier to clinical use, it is not yet clear how long it might take to solve the problem.

 

INTELLECTUAL PROPERTY

Meanwhile, there is also a fierce ongoing dispute over who has the rights to licence the technology for developing therapeutics. While Doudna and Charpentier published the first paper describing the mechanism, Feng Zhang, co-founder of Editas, was the first to inconclusively prove that the CRISPR/Cas9 system is effective in Eukaryotic (ie. mammalian, plant and yeast) cells. After filing for a fast-tracked patent application, Zhang subsequently secured the first patent for use in humans in April 2015. The validity of this claim is currently contested, and therefore companies seeking to acquire permission for use of CRISPR may need to apply to multiple groups, making the process complex and expensive. There is also no guarantee at present that any of the main contenders for the intellectual property will have the right to licence CRISPR for commercial use once the dust has settled, which is predicted to take several years.

Although this legal dispute may be restrictive to the use of the technology itself, start-up companies that operate in parallel to CRISPR/Cas9 research are growing in size and number rapidly. Some online services are able to suggest effective guidance RNA sequences to carry out a given task using the CRISPR system, while providing a platform for researchers from a range of disciplines to collaborate online. Other website based start-ups are able to receive the engineered DNA sequences for lab synthesis, or carry out other bench-work tasks, before shipping the product to the customer. To a large extent these services are therefore able to bypass many of the hurdles faced by those seeking to use CRISPR/Cas9 itself for business purposes, although the major industry growth expected may be delayed until a decision is reached in the courts.

 

ETHICS

While these technical and legal issues are undoubtedly significant, the most discussed and perhaps most important long-term challenge concerns the ethical use of CRISPR, especially in cases where inheritable modifications are made to human cells. Recently, an opinion piece written by a group of scientists including Jennifer Doudna, steps were recommended to “strongly discourage” researchers creating inheritable modifications on human cells while the ethics surrounding gene modification is discussed. In response to recent news of investigators using CRISPR/Cas9 to edit human embryonic cells, other notable groups have called for a complete moratorium on germ line usage, or else risk a public backlash against CRISPR that may slow or halt research entirely. Meanwhile, in February this year the UK government voted in favour of allowing modification of mitochondrial DNA for clinical use. Although mitochondrial DNA may be inherited, it does not form part of the cells main sequence of genes and was therefore seen as ethically feasible to be modified. This seems to suggest that in the UK at least, tentative steps are being made toward acceptance of reasonable clinical use of CRISPR/Cas9. It is therefore important that the use of the technology is carefully managed as the public become familiar with it.

What the latest genome editing technology has made clear to researchers and to society as a whole, is that genome modification is set to become a hugely significant and wide reaching agent of change. Accordingly there are numerous hopeful, if tentative, opinions on the future of CRISPR/Cas9, and these views are actively encouraged by the stakeholders of the technology - which we may all become soon.

 

CONCLUDING REMARKS

In the meantime, while the dust is settling many may wait to invest in CRISPR/Cas9. Though, it is safe to assume that any restraint is not likely to last. The revolution that CRIPSR/Cas9 offers is in its accessibility. It has made genome modification a much more feasible task, particularly for those with the resource typically available to start-up companies, or for those that don't have the background normally required for DNA modification. The number using the technology will ultimately reflect that. As a result, the diversity of ideas generated by researchers and entrepreneurs using CRISPR/Cas9, or whatever technology comes next, will contribute to a new rate of evolution in the Biotech and Pharmaceutical industries, as well as in an ever increasing number of related fields. Though we can't yet predict how that might unfold, Doudna and Charpnetier were certainly right in thinking that there was something hugely important about this system.