CRISPR Takes Center Stage

It’s been nearly a decade since CRISPR captured public imagination. As the technology matures, we’ve rounded up some of CRISPR’s most interesting use cases to emerge in recent years.

AsianScientist (Oct. 7, 2020) – In 1986, a mysterious repetitive DNA sequence in the Escherichia coli genome baffled a team of Osaka University scientists. While studying the iap gene, they noticed something odd: found near the iap gene were five identical DNA segments, each separated by a ‘spacer’ region.

This was unusual as repetitive sequences are typically arranged consecutively. Even more unusual was the fact that unlike the DNA repeats, each of these spacers had a unique sequence.

It would take another 25 years for CRISPR’s tremendous potential as a gene editing tool to become apparent. Today, CRISPR-Cas9 is recognized as a staple in every life scientist’s tool kit. It has since been used for a variety of applications, ranging from practical and sensible (drought-tolerant plants) to somewhat bizarre (resurrecting extinct animals). Here, we take a look at some of the most interesting CRISPR research to emerge from Asia and the world in recent years.


Rice blight’s SWEET solution

From fluffy sushi rice to fragrant basmati, it’s no secret that Asians love their rice. After all, billions of people in Asia and Africa depend on the crop as a food staple. One of the biggest threats to the staple crop, however, comes in a microscopic package: the bacteria Xanthomonas oryzae pathovar oryzae (Xoo), which causes a devastating infection called bacterial blight. Up to 75 percent of crop loss in rice has been attributed to bacterial blight, with farms in Southeast Asia particularly hard hit.

In rice, sugar is transported by molecules encoded by the appropriately named SWEET genes. When Xoo infects rice, it secretes molecules called transcription activator-like effectors (TALE) that bind to the SWEET genes and turn their transcription off. This makes sugar available for Xoo to feed on, allowing them to further multiply and wreak havoc on the plant.

In an attempt to stop bacterial blight in its tracks, Dr. Ricardo Oliva at the International Rice Research Institute in the Philippines and his colleagues used CRISPR-Cas9 to modify three SWEET genes found in rice varieties grown in Asia and Africa. Following genome editing, the team found that Xoo’s TALE molecules were unable to bind to the SWEET genes, making the edited rice plants resistant to at least 95 Xoo strains. Sweet!

Towards fever-proof swine

Although everyone’s eyes may be on COVID-19 for now, it’s worth remembering that humans aren’t the only organisms that can be devastated by viral outbreaks. For instance, around 11 million pigs in the Netherlands were slaughtered in 1997 due to the classical swine fever virus (CSFV), which causes a contagious and often fatal disease that leads to fever, lethargy and even convulsions. While CSFV does not affect humans, outbreaks could still lead to significant economic losses for the animal industry.

In hopes of curbing future outbreaks, scientists from the Jilin Provincial Key Laboratory of Animal Embryo Engineering in China have engineered pigs resistant to CSFV. They did this by introducing small loops of antiviral RNA, known as short hairpin RNAs, into the pig genes using CRISPR-Cas9. These RNAs protect against viruses by triggering the degradation of viral genetic material.

The modified genes were then transferred to a pig’s egg cell, after which CSFV-resistant pigs were produced in a process known as somatic cell nuclear transfer (SCNT). If SCNT sounds familiar to you, it’s the same technique that was used to create Dolly the sheep back in 1996. To confirm the pigs’ resistance against CSFV, the researchers alternately injected the pigs with the virus or housed several resistant pigs with a CSFV-infected member. To the researchers’ delight, the pigs resisted infection, with the same resistance passed on to the next generation.

Sumo-sized sea bream

Ever heard of the red sea bream? Though fishes like tuna, mackerel and salmon are more well-known, the red sea bream takes the top spot as Japan’s most beloved seafood. This is partly because its Japanese name, madai, sounds like the word medetai, which means auspicious. Accordingly, the red sea bream has become a ubiquitous part of Japanese celebrations—even being brandished by winning sumo wrestlers and politicians as a sign of victory.

In 2018, researchers from Kyoto University and Osaka University revealed a more muscular variety of sea bream, with up to 16 percent more edible meat. They did this by using CRISPR-Cas9 to knock out, or inactivate, the myostatin gene in fertilized fish eggs. Normally, myostatin restrains muscle growth, preventing muscles from growing too large.

Like most CRISPR-Cas9 experiments, the first generation of fish was born with a mosaic of edited and unedited cells. By breeding the first-generation fish together, the researchers were able to generate new fish with purely edited cells and meatier flesh. Amazingly, the entire breeding process took only two years. Previously, it took over 20 years to produce supersized sea bream through selective breeding methods, pointing to the potential of genome editing in accelerating aquaculture operations

Detecting the coronavirus in real-time

Currently, the gold standard for COVID-19 diagnosis is reverse transcription polymerase chain reaction (RT-PCR). In this technique, specific sequences of the coronavirus are detected in genetic material collected from the patients’ nasal swabs. RT-PCR’s high accuracy, however, comes at the cost of speed. Since it requires specialized equipment and reagents, samples to be tested are often shipped in bulk to centralized laboratories. This creates a backlog with a turnaround time of at least 24 hours.

On the other side of the spectrum are antibody-based tests, which can detect the presence of antibodies against the virus in less than thirty minutes. But with antibodies only forming several weeks after infection, such an approach could miss people at the earliest stages of the disease—which is precisely when they’re most infectious. New tests from CRISPR companies Sherlock Biosciences and Mammoth Biosciences, however, could harness the best of both diagnostic approaches.

Both tests operate on the same principle: Binding to the coronavirus’ genetic material causes a nearby reporter sequence to be cleaved by the Cas enzyme, triggering a reaction that changes the color of a coated lateral flow strip or dipstick—similar to store-bought pregnancy tests. Given that the whole process takes less than an hour and uses common equipment and reagents, these CRISPR-based tests have much potential for deployment in high-risk, low-resource settings.

Bio-editing blood disorders

One of the most common genetically inherited diseases in India is sickle cell anemia, a blood disorder caused by a mutation in the beta-globin gene. This singular mutation distorts round red blood cells into a stiff, sickle-like shape and causes deformed cells to stick together, thereby blocking small blood vessels and impeding the movement of oxygenated blood. If both parents carry the sickle cell trait, then their child risks inheriting the disease.

Scientists from India’s Institute of Genomics and Integrated Biology are now tapping into CRISPR-Cas9 to correct the debilitating mutation in patient-derived induced pluripotent stem cells (iPSCs). However, the team harnessed a Cas9 enzyme derived from a different bacteria, Francisella novicida, due to its high specificity compared to the conventional Streptococcus pyogenes-derived Cas9.

In this case, the complex creates a cut in the mutated gene, after which the cells’ repair machinery whirrs into action, fixing the cut based on a DNA template with a normal gene sequence. Because the edited iPSCs come from the patient, chances of rejection upon transplantation back into the patient are low. Still, it is early days yet. While proof of concept has been established, mouse models and human trials are needed before their approach could be approved as a therapy for patients with sickle cell anemia.

This article was first published in the July 2020 print version of Asian Scientist Magazine.

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Copyright: Asian Scientist Magazine.
Disclaimer: This article does not necessarily reflect the views of AsianScientist or its staff.

A molecular biologist by training, Kami Navarro left the sterile walls of the laboratory to pursue a Master of Science Communication from the Australian National University. Kami is the former science editor at Asian Scientist Magazine.

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