The global population is expected to increase from 6.3 billion in 2015 to more than 9 billion by 20501. As the human population continues to grow, the challenges of feeding us grows2. It has been suggested that total crop production must double by 20503. The productivity of crops, livestock, seafood and microbes must improve2. Unfortunately, an article published in 2013 reported that none of the most important crops produced in the world have been meeting the necessary annual increase of 2.4%4. There are several factors that contribute to the inability to meet the growing demand2. There is not enough arable land. However, clearing more land (especially in tropical rain forests) is not desirable. In addition, many crops could suffer from environmental stress and diseases while the global climate continues to change. At the same time, some crops are being grown to produce cleaner energy instead of food2. This increases the demand for improved yield and finding or creating better sources of biofuels, such as microalgae5,6.
Scientists are addressing the looming food shortage2. They are trying to breed and develop crops with higher yield, better resistance to pests and disease, as well as drought and other types of stress. Previously, this has been a slow process. Only one trait can be improved at a time using conventional breeding techniques. However, a stunning new technology called CRISPR is being used to change many traits simultaneously, quickly and easily2,7-12. In addition, CRISPR is being used to improve livestock and seafood production, create better animal models of diseases, help in the development of improved vaccines and new prescription drug, and possibly eventually eradicate malaria8,13-16. It can also be used in the field of synthetic biology to make entirely new organisms and may be able to bring back extinct species, such as the wooly mammoth8,17,18. CRISPR has been predicted to become one of the key technologies that will be part of the fourth industrial revolution (along with artificial intelligence, robotics, nanotechnology, information technology (IT) and big data)17,19,20.
CRISPR is a naturally-occurring defense mechanism that bacteria use to keep them from being infected by viruses2,8,10,11,14,21. To do this, bacteria use the parts of their genomes that contain base sequences that are repeated many times, with unique sequences in between the repeats21. They are called “clustered regularly interspaced short palindromic repeats” or CRISPR. They keep pieces of viral genomes in the bacterial DNA so they can recognize viruses and defend themselves against future infections22. Invading DNA from viruses is cut into small fragments and incorporated into a CRISPR location with a series of short repeats (around 20 base pairs). The DNA is transcribed and processed to produce small CRISPR RNAs (crRNAs) that bind to the targeted DNA and guide effector endonucleases that hydrolyze (destroy) the invading DNA being targeted22. The second part of the defense mechanism is a set of enzymes called Cas (CRISPR-associated proteins), which can cut out the viral DNA sequence, hydrolyze it and destroy the invading viruses. Fortunately, the genes that code for Cas enzymes are always located near the CRISPR sequences. The best known and most useful Cas enzyme is Cas9, which comes from Streptococcus pyogenes, which causes strep throat. When combined with CRISPR, it becomes the CRISPR/Cas9 system. This is often simply abbreviated as CRISPR. So, Cas9 catalyzes the hydrolysis of DNA, while CRISPR shows it where to do the hydrolysis. The same system can be used by scientists to cut and paste almost any desired DNA sequence. Faulty genes can be cut out and replaced with the desired, functioning gene, or to insert new genes that give the recipient better qualities21,22.
Agronomists have used CRISPR to make strains of wheat that are not affected by deadly fungi, such as powdery mildew9,21. Others produced low-gluten, nontransgenic wheat23. CRISPR has also been used to increase the productivity of maize during stress caused by droughts. It can produce tomato plants with much higher yields9. CRISPR has produced varieties of rice that are more productive12. The panicle number per plant was increased, which increased the number of grains per plant12. The startup company Arvegenix is using CRISPR to improve the quality of meal and oil produced by pennycress (Thlapsi arvense L.)9, which is considered to be a weed24. Recent work has shown how CRISPR can be used to convert this weed into a cash cover crop that will produce edible oilseed25. Other researchers are removing defective genes from pigs, chickens and cattle and replacing them with healthy genes2. CRISPR can produce improved varieties of sorghum, maize, corn, cassava, soybeans, tomatoes, potatoes, sunflowers, strawberries and apples2. It has also been used to make eggs that are less likely to cause allergies (hyperallergenic)13. This is important to the approximately 2% of the children who are allergic to eggs. They can’t receive many of the routine childhood vaccines that are made using chicken eggs. By making eggs hyperallergenic, such children will be able to receive potentially life saving vaccines. Others are trying to produce ‘hygienic’ bees. They clean their hives so thoroughly that they are no longer as susceptible to mites, fungi and other pathogens. Others are trying to produce pigs that are resistant to viral diseases. Still others are produced genetically altered domesticated animals that can produce prescription drugs. The FDA and the EU approved the production of goats that produce an anti-clotting protein in its milk and chickens whose eggs contain a drug to treat diseases caused by high cholesterol. More recently, the FDA approved the first transgenic animal for human consumption – fast-growing salmon13.
Most of the international agricultural companies are doing research on how CRISPR can help them improve the yields of their foods. For example, Syngenta acquired intellectual property to begin using CRISPR in some of their crops. DuPont plans to release waxy corn by 202026. They are also collaborating with some of their competitors, such as Bayer and BASF2.
CRISPR is also being used to improve food processing done by microorganisms2,14,27. It has been used to make genetically modified bacteria (Streptococcus thermophilus) that are used to make yogurt and cheeses29. CRISPR has been used to produce yeasts that can consume plant matter and excrete ethanol, which could help end our addiction to fossil fuels28. This technique has been called revolutionary, but like many revolutions, it may become dangerous. This was not the first time that genetic engineering has been called potentially perilous28.
In 1975, geneticists, biochemists and others went to Asilomar, California to discuss the potential problems that could be caused by research on cloning and recombinant DNA28. Many people were afraid of what some called a “God-like power” to insert new genes from one organism into another. If used safely and wisely, it could produce bacteria and other genetically-modified organisms that could manufacture new, lifesaving prescription drugs. However, it could also be used to possibly make genetically modified humans and even ‘super babies’ or ‘super soldiers’ with blond hair and blue eyes or any other trait that parents or society might find fashionable or useful in warfare. There was also the fear that such genes could be transferred from one bacterial species to another through horizontal transfer. So, after long discussions, the scientists at the Asilomar conference wrote a consensus statement that described the kinds of experiments that should not be done, such as creating dangerous pathogens or pathogenic organisms. Few, if any worried about modifying human embryos to make a ‘super race’ with traits that could be passed on to future generations. They thought that it would be too difficult to be done in the foreseeable future28.
Well, the future has arrived. Viable human preimplantation embryos were modified by an international team using the CRISPR technique to correct pathogenic gene mutations29,30. They targeted a mutation in the MYBPC3 gene that causes myocytes (muscle cells) to thicken (hypertrophic cardiomyopathy), which is a leading cause of death in young athletes. It is a dominant mutation, so a child only needs one copy of it to suffer from this condition. Scientists also overcame two safety problems that had limited previous efforts: off-target mutations and mosaicism. No unintended genetic targets were modified and no genetic mosaicism (different types of genes) occurred. They also went around current regulations in the USA that prevent federal money from being used for research on human embryos by using private funding. Such regulations don’t exist in several other countries, so some Chinese scientists have already reported using CRISPR to alter genes in human embryos. So, CRISPR technology has advanced far enough for people to consider using it to correct inheritable mutations in human embryos after they are discovered in preimplantation genetic analysis29,30.
Still, just two years before the journal article that described using CRISPR to modify a human embryo was published, some of the same scientists that met at Asilomar subsequently met at the Carneros Inn in Napa Valley28. All the attendees had access to the CRISPR technology. They discussed the moral and legal implications of genetic engineering28. After the conference, an article was published in the prestigious journal Nature entitled “Don’t edit the human germ line” 31. Many countries (but not the USA or China) have laws that specifically forbid genetic engineering in humans, including 15 of 22 nations in Western Europe.
Finally, CRISPR-Cas is being used to produce completely new organisms and to try to bring back extinct species18. For example, CRISPR-Cas is being used to make mutants of the mosquitoes that causes malaria (Anopheles genus), yellow fever, dengue and chikunguya and Zika (Aedes aegypti), as well West Nile and encephalitis (Culex quinquefasciatus). Together, these mosquitoes have killed more people than any other animal on Earth – including us humans with all our wars18. CRISPR-Cas9 is being used to identify genes in human hosts that can reduce viral infection when they are edited32. Researchers from China and the USA did a genome-wide CRISPR-Cas9 screen that targeted 19,050 genes. The goal was to identify those human host genes that could reduce flavivirus infection. This included the West Nile, Zika, Japanese encephalitis, Dengue serotype 2 and yellow fever viruses. They found nine genes that flaviviruses needed to be infective. Genes coding for signal peptidase complex (SPC) proteins were found to be required for the proper breakdown (hydrolysis) of flavivirus structural proteins and the secretion of viral particles. When the expression of these genes was suppressed, it not only reduced viral infections, but also had no adverse side effects on the human host cells. One or more of these SPC proteins could become effective therapeutic targets for new prescription drugs to protect against infections by flaviviruses32.
To eliminate mosquitoes, the gene modified by CRISPR must spread throughout the environment. This is done using gene-drive systems33. These gene-drive systems can enable super-Mendelian inheritance of a transgene that could potentially reduce mosquito populations without having to create millions of genetically modified mosquitoes and releasing them into the environment. Instead, the gene drive technology can simply cause a desired gene or genes to be released. The idea is that they will spread on their own. Gene drives are ‘selfish’ genetic elements that spread through wild populations even though they confer no survival or reproductive advantage in the individuals that carry them. They can even make those individuals and their offspring less fit33. So, CRISPR-Cas9 was used to produce a gene drive system in A. gambiae, the carrier of malaria34. It targeted the reproduction of female mosquitoes. The goal is to reduce mosquito populations to such low levels that they can’t spread malaria effectively34.
However, some people may question the wisdom of using gene drives that cause genes to spread relatively uncontrollably in the environment. Such concerns already exist on the use of genetically modified crops, like soybeans that are more commonly referred to as genetically modified organisms (GMOs)35. However, unlike these GMOs that can spread their seeds and generate many new offspring, the goal of producing genetically modified mosquitoes is NOT to produce more offspring and possibly even eradicate them completely21. Still, some may question the wisdom of possibly driving any species into extinction – even the deadly mosquito. In contrast, the attempt to resurrect currently extinct species like the woolly mammoth may not raise such concerns. Instead, the goal is to produce enough woolly mammoths and other large but extinct animals to convert the frozen tundra in Siberia from forests to its former grasslands that will be more environmentally friendly and reduce the emission of methane gas as the frozen tundra melts. However, the woolly mammoths and other extinct species will not be exactly the same as the animals that existed in the Pleistocene epoch. They will be different holobionts with different microbiota because they will be grown in utero in currently existing species, such as elephants. Still, if they can adapt to their new microbiota they could grow to be big enough to convert the vegetation in the Siberian tundra to grass, which will reflect more sunlight than the trees that are there now. However, there is so much that we don’t know about the Siberian ecosystem that some people are saying we should be cautious or maybe not even allow such things to be done. Still, people who say it shouldn’t be done are often being passed by people who are doing it21.
One of the safety concerns about CRISPR is that it the results of gene editing can be irreversible38. Moreover, there is a concern that CRISPR technology can be accidentally or purposefully misused. So, scientists have looked to nature to find a way to control CRISPR-mediated gene editing. This will produce cells that are “write protected”, so they can’t be edited38.
The addition of this ability to control gene editing, very important applications in food and biofuel production can take place. Perhaps the most important example is the production of improved microalgae. They are single cell photosynthetic organisms that could be a sustainable and environmentally friendly source of food and fuel21. Microalgae have been used as food for hundreds of years37. They were consumed by the Aztecs in Mexico, as well as in China, Korea, Japan, Burma, Thailand, Vietnam and India. Moreover, Spirulina was called the best food for the future in the United Nations World Food Conference of 1974. During the 60th session of the United Nations General Assembly, a resolution was issued stating that Spirulina can be used to fight hunger and malnutrition and help achieve sustainable development. Chlorella and Spirulina are sold in health food stores because they are some of the most nutritious foods known. They contain not only protein, polysaccharides, sterols and lipids, but also many vitamins, carotenoids and polyphenols37.
Microalgae have high biomass productivity and are excellent sources of polyunsaturated fats, carotenoids, complex carbohydrates and protein21. They grow in water, so they do not compete for arable land or cause pollution like other foods, especially beef5. Microalgae can grow rapidly in seawater as well as in polluted industrial and domestic waters38. They do not require fertilizer or pesticides, like crops grown on land. In the USA, companies like Solazyme, Sapphire Energy and Algenol are producing diesel and jet fuel, as well as gasoline and ethanol on a commercial scale5. CRISPR has been used to modify the microalgae Chlamydomonas reinhardtii to make it produce more lipids38. CRISPR was also used to modify Synechocossus species to produce the valuable chemicals 1-butanol, limonene, lactate, succinate and ethylene38.
In addition, the microalgae commonly known as Spirulina (actually Arthrospira platensis) are an excellent source of protein and other bioactive ingredients39. They are about 60% protein with all the essential amino acids. It also has more β-carotene than any other whole food. It is an excellent source of gamma linoleic acid, phycocyanin, B vitamins, minerals, trace elements, chlorophyll and enzymes, including superoxide dismutase, an important antioxidant. Spirulina has almost twice the concentration of calcium as cow’s milk, seven times the protein as tofu, 31 times as much β-carotene as carrots and 51 times as much iron as spinach. It can protect the kidneys and liver, while also preventing anemia. It has important anticancer activities and benefits for diabetics and their cardiovascular system. It can remove heavy metals from the body and help control allergic rhinitis. It can also build immunity and help provide resistance to viral infections. In 1974, the United Nations wrote that Spirulina is one of the best foods for the future39. It also contains bioactive peptides that have antimicrobial, antiallergic, antihypertensive, antitumor and immunomodulatory properties40. The bioactive peptides can also promote cardiovascular health41.
More recently, a new version of the CRISPR system has been developed that can target not only DNA, but also RNA21,42. In contrast to CRISPR-Cas9, Cpf1 can process the pre-crRNA on its own, and then using the processed RNA to target and cut DNA specifically43. This is the most minimalistic CRISPR immune system yet. The mechanism of combining two separate catalytic modalities in one creates new possibilities for genome engineering, and enables researchers to target several sites at once or do multiplexing43.
So, CRISPR can be used in synthetic biology to produce gene circuits in living cells44. One of the goals of synthetic biology is to build regulatory circuits that control cell behavior using programmable elements. Gene circuits contain three modules: sensors, processors and actuators. Sensors detect environmental conditions or cellular status, such as being transformed into cancer cells. Processors determine the appropriate response, such as delivering anticancer therapy. Actuators transmit the signals that modify cellular function. Biological systems that regulate the transcription of DNA into RNA and logic gates have been engineered into bacteria and mammalian cells using a deactivated deactivated Cas9 enzyme, abbreviated as dCas. The CRISPR-dCas9 system no longer has endonuclease activity, but it can still be able to bind to specific DNA sequences. Using dCas9 to activate or inactivate the transcription of target genes is often abbreviated as CRISPRi and CRISPRa, respectively. CRISPR was used to build a logic circuit in bladder cancer cells44,45. It contained a promoter that was specific for bladder cancer cells. The circuit was used to express three transgenes that could be useful in cancer therapy44,45.
In conclusion, gene editing with CRISPR-Cas9 is being used to improve the production of crops, livestock, seafood and microorganisms. It’s also being used in new drug development and to help remediate pollution. Researchers are also trying to discover ways to use it to eradicate mosquitoes that cause malaria and other diseases. It may also be useful in making entirely new organisms (synthetic biology) and in bringing back extinct animals, such as the wooly mammoth. As such, CRISPR may become an important part of the fourth industrial revolution.
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