No other technology like the recently developed “genome editing” has taken the world by storm in the annals of modern biotechnology. Genome editing tools are being applied to alter specific gene(s) and also are being refined at such a rapid rate that it is really hard to predict its advancement on a day-to-day basis.
Genome editing offers highly precise methods of snipping unwanted gene (DNA) sequences of any organism, just like a manuscript editor can use an eraser to get rid of any stretch of a sentence. It is also used to drive the transmission of desired gene(s) in a given population.
This is not just one technology or one technique, but a family of techniques used to alter the genome of an organism by sharp and crisp editing. The techniques include CRISPR-Cas9, Site-directed nucleases (SDNs), Zinc finger nuclease (ZFNs) and Transcription activator-like effector nucleases (TALENs), Oligonucleotide-directed mutagenesis, RNA interference, Cisgenesis, Intragenesis, reverse breeding, RNA-dependent DNA methylation, Rapid trait development system (RTDS) and SpiX technology.
CRISPR-Cas9 stands for Clustered Regularly Interspaced Short Palindromic Repeats-Endonuclease9. It works like a self-vaccination in a bacterial immune system by targeting and cutting off viral DNA sequences acquired from invading phages (virus particles).
The cell uses two RNA elements—CRISPR RNA (crRNA) and trans-activating RNA (tracrRNA)—that together guide the Cas9 nuclease to the target DNA, and then the target DNA is cut off from the genome or edited out of the genome. The genome is the total complement of all DNAs in the nucleus of a cell. The CRISPR-Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements such as those present within plasmids and phages and provides a form of acquired immunity.
Cas9 was the first nuclease discovered. Subsequently, the Cpf1 nuclease was discovered in the CRISPR/Cpf1 system of Francisella Novicida. Without a doubt, other such systems exist. Cas9 is a nuclease, an enzyme specialised for cutting DNA. Editing is achieved by transfecting a cell with the Cas9 protein along with a specially designed guide RNA (gRNA) that directs the cut through hybridisation with its matching genomic sequence. When the cell repairs that break, errors can occur to generate a knockout of that gene, or additional modifications can be introduced.
The powerful CRISPR-Cas9 is a revolutionary tool in today’s molecular biology. This was first described in 2012 by Jennifer Doudna of UC-Berkeley and Emmanuelle Charpenteir—now the director of the Max Planck Institute of Infection Biology, Berlin. Kevin Esvelt of MIT has really worked hard since his post-doctoral days at Harvard to refine this technique to make precise edits at multiple locations in the genomes of all kinds of living organisms.
George Church and his colleagues at Harvard were the first to separate CRISPR from Cas9 to edit human cells. Chidananda Kanchiswamy and his colleagues at Genomics and Biology of Fruit Crop Department of the Fondazione Edmund Mach, San Michele all’Adige, Italy are also fine-tuning gene editing techniques to nullify off-target mutations that limit its applications in crop plants.
Scientists led by Jennifer Doudna have launched a public opinion campaign to prohibit the use of gene editing technology to humans for now, although the UK’s health authority has approved its use and the Chinese have already published research papers on altering human germ cell lines using CRISPR-Cas9.
The US is balking at granting permission to use gene editing techniques on human cell lines.
CRISPR is poised to revolutionise research and innovation in the biotech industry. It was selected as the breakthrough invention/innovation of 2015 by Science magazine. Researchers have used the technique to disable retroviruses threatening organ transplantation from pigs to humans. It has also been used as a potent gene drive to allow rapid transmission of introduced genes throughout insect populations.
CRISPR has made it easy to study human genetic diseases for testing and screening of drugs. Genome editing of somatic cells can lead to effective cure of certain diseases. Using CRISPR, scientists are attempting to cure autism, cancer and HIV. CRISPR is a game changer in biotechnology.
These technologies will no doubt replace the arsenal of techniques that are presently available in the biotechnology toolbox.
The beauty of genome editing is that without adding an extra piece of DNA, the genetic make-up can be altered, thereby avoiding the sobriquet “transgenic”. As such, leading regulatory authorities in the US and the EU have declared that genome-edited organisms are not transgenic organisms and, therefore, do not come under any biotech regulation. The first group of organisms that have escaped the regulatory dragnet are mushroom and maize in the US and canola in Germany.
Critics of biotechnology assert that all products of these technologies are nothing but “hidden GMOs”, examples of extreme genetic engineering and are pushing to bring them under the GMO umbrella. Developers of these technologies and their products claim that regulations will cripple innovation as it happened with GMOs. India is the worst sufferer of anti-biotech activism, whereas neighbouring Bangladesh has gone ahead with a variety of GM crops to improve its agriculture. Bangladesh serves as the best example of how to tackle anti-tech activism in South Asia.
The common white button mushroom (Agaricus Bisporus) has been modified to resist browning by Yinong Yang, a plant pathologist at Penn State University. Yang targeted a family of genes that encodes an enzyme polyphenol oxidase that is responsible for browning. Yang knocked out one of six genes in the family of genes that reduced the enzyme activity by 30 percent. The reason USDA-APHIS decided not to regulate this mushroom is because, by using CRISPR-Cas9, Yang had not introduced any plant pest sequence of DNA to the mushroom from any other unrelated source organism.
Thirty years ago, when USDA formulated its regulations, it only considered sequences being introduced into the transgenic organisms as one that would have plant pest potential. But gene editing technology has supplanted the old transgenic technology. Germany’s risk assessment agency (GfR) declared that a gene-edited canola is not a GMO as defined for the purposes of regulating transgenic organisms. Similarly, the Swedish Board on Agriculture has decided that genome-edited plants do not fall under the European Union’s definition of GMO.
A new type of corn (maize) developed by Pioneer-Dupont, using CRSIPR-Cas9, has also been certified to be unregulated by USDA-APHIS. There are at least 30 other plants and animals altered by Newer Breeding Techniques (NBT), under regulatory purview, right now in North America and Europe that are likely to be certified non-regulatable—paving the way for a flood of genetically modified organisms that will not undergo any regulatory review. Since leading regulatory agencies around the world and scientific academies and organisations (not the fake academies set up by the anti-biotech lobby) have declared that genome-edited crops and animals will not be regulated, there will be no other alternative for the Indian regulatory authorities to follow suit. This will happen only if India’s anti-biotech lobby does not create another fuss as it has created with GMOs.
Flaminia Catteruccia, an Associate Professor of Immunology and Infectious Diseases at Harvard, has developed “knock out” mosquitoes by deleting genes responsible for hosting the malarial parasite ‘Plasmodium falciparum’, using CRISPR. This allows for the possibility of editing out the entire gene(s) of a variety of organisms like mosquitoes, and any other sexually reproducing plant or animal in the wild, and hopefully conquering dreaded epidemics like malaria and cholera.
Kevin Esvelt, then a post-doctoral fellow at George Church’s lab at Harvard, and now an Assistant Professor of Biology at MIT, had an idea to use CRISPR to transmit beneficial genes in the wild to control pandemics like avian influenza, taming of invasive plants, and modifying corals to resist bleaching by warming seas. Esvelt’s idea— “gene drive”—is to ensure that the desired gene variant is passed on from generation to generation by winning a lottery of life virtually every time, and will almost always be passed on. Gene drive is a molecular machinery called “meganuclease” that enables a particular gene variant to be inherited with certainty.
The CRISPR technology is so simple to use and so affordable that even a teenager can use it in his garage. That’s why Esvelt is concerned that all sorts of mischief can happen as teenagers are inclined to play around and might release altered insects that will allow the spread of unwanted gene drive in the environment. Safety issues of this technology are not trivial, but it is only experts who can deal with it in a professional way.
George Church convened a meeting of all those who had used CRISPR in insects—almost 27 of them—and thrashed out a volunteer protocol to ensure no accidental release of modified insects would occur. This was akin to what scientists had done earlier in 1970 at the Asilomar conference to deal with the gene splicing techniques, which eventually resulted in the highly regarded National Institutes of Health rDNA guidelines.
Church has suggested separating CRISPR from Cas9. Omitting the Cas9 scissors from germline DNA would prevent editing of the genome in offspring, allowing researchers to safely test a genetic change without the risk of an accidental release, causing the altered gene to spread through an entire species. Church feels that, in the case of a gene drive, safety planning must be the first necessary step as there is no such thing as “limited release” of insects. Esvelt says that if you are talking about something that alters the environment, you better be careful and get it right. He aims for a large collaborative effort to figure out what can go wrong to determine the safest possible gene drive system ready for deployment. He asks: “How can we even begin those sorts of experiments without first telling people what we are doing?” That’s how scientists think positively, but the activists think negatively to kill the technology development itself.
The limiting factor may not be the time required to build a gene drive in the laboratory. It may be the time required for society to decide whether or not it should be used. All scientists working on gene editing technologies emphasise the urgency to engage in public debate and also discuss the dire necessity for the applications of these technologies to meet human needs.
Christoph Then of Testbiotech, a notorious detector of traces of GMOs that other labs cannot detect, and thereby handily serve the anti-GM lobby’s efforts to cry hoarse against GMO contamination in the food supply chain, has already fired his first volley against NBT organisms, saying that the new techniques can cause radical changes and nobody has sufficient experience to declare them safe. This is a classic case of misuse of the precautionary principle.
This kind of a fight between technology protagonists and antagonists has been going on for centuries without any resolution. For the non-scientific types in the anti-biotech lobby (and they outnumber all scientific bodies), it is sufficient to kick up a storm and influence gullible politicians and leftists to join the bandwagon in India, to stop technology implementation or go on holding nationwide discussions with all sorts of useless street people to decide on cutting-edge technologies—where even many scientists cannot understand it, if they are not working closely in the field.
Leftists oppose anything that comes from the capitalist US as they believe modern technologies are exploitative and do not render any social justice. If the anti-biotechwallahs cannot get the technology banned, they will at least delay it—as they have succeeded in the case of conventional GMOs. Anti-biotech activists in India must realise that progress in science and technology is relentless, and newer and newer innovations will render older innovations obsolete. That is exactly what has happened in the case of GMOs. The 1980s technology to develop GMOs is now fast becoming obsolete, and new techniques are not going to be regulated using old laws. That is the reason why scientists and progressive nations were always demanding dynamic laws that can catch up with technological developments, whereas activists want rigid laws to kill innovation and technology implementation.
Raising ethical and safety questions about gene editing is certainly the need of the hour, but the real question is - who is best qualified to address those questions? There are very good experts in science and technology and social sciences who can address them. Instead, a bunch of anti-tech ignoramuses take centre stage just because they have the decibel power, and politicise the whole debate.
On 19 April 2016, the US National Academy of Sciences convened a meeting to discuss the regulatory regime that may be most useful for continued innovation in new biotechnologies. In the UK, the House of Commons Science and Technology Committee issued a report in 2015 titled “Advanced Genetic Techniques for Crop Improvement: Regulation, Risk, and Precaution” and unequivocally recommended that new technologies should not be regulated because of the methods used, but regulate the products depending on the environment into which they are being introduced. Most genome editing techniques introduce genetic variations indistinguishable from those obtained through natural means or conventional mutagenesis. Addressing the regulatory ambiguities is critical for the effective commercialisation of genome-edited crops and animals.
The same US National Academy of Sciences released a report on GMOs on 17 May 2016, that once again asserted that GMOs are safe and must be allowed to be commercialised using fairly non-stringent laws and regulations, which is what the US tries to do. But, in India, a minister like Jairam Ramesh rejects recommendations of science academies out of hand just to please his masters in his party and the erstwhile National Advisory Council (NAC) and NGOs.
What is more disheartening is that even the Narendra Modi government is dragging its feet on GMOs due to opposition within its own ranks, like the Swadeshi Jagran Manch and the Bharatiya Kisan Sangh. Objections of these outfits are not based on science but on imagined fears of foreign companies taking over the holy cow called ‘Indian agriculture’. What is even more detestable is that the BJP government intervened in the marketplace to determine the price of Bt cotton seeds and also the amount of royalty the technology developer would get. This reprehensible act has come under severe criticism by the likes of Swaminathan Anklesaria Iyer and Montek Singh Ahluwalia.
If Modi believes in less government, why he has allowed this kind of market intervention to prevail is beyond one’s belief. His government is sending a wrong message to the private sector. By doing so, he has thrown up obstacles to technology companies from investing in Indian agriculture. And, there isn’t any Indian seed company that has modern seed biotechnologies.
It is high time that the National Academy of Agricultural Sciences (NAAS) spearheads scientific efforts to keep the new breeding techniques out of regulations in India. It must come out strongly against those who oppose modern biotechnology. Even scientists must oppose politicians and political parties who oppose modern science and technology.
If genome editing technology is regulated strictly without any scientific basis, this too will be denied to Indian farmers, just like its predecessor technology. Indian agriculture is lagging behind by decades, and any effort to stifle technology development will make it even less competitive.
Dr Shantharam is an Adjunct Professor of Biotechnology, University of Maryland—Eastern Shore, Princess Anne. A former biotechnology regulator with the United States Department of Agriculture, served as a biotechnology consultant with the World Bank, Asian Development Bank, UNIDO and UN-FAO. He is also a Visiting Professor at the Seed Science Center at Iowa State University, Ames.
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