CRISPR (pronounced “crisper”) as a genome-editing tool is currently one of the hottest topics of research in the biological sciences. This is very much evident in the fact that in 2011, there were fewer than 100 published papers on CRISPR; but in 2019, there were more than 30,000 and counting, including various applications, refinements to CRISPR, new techniques for manipulating genes, improvements in precision, and more. So, let us today take a look at Science’s choice for Breakthrough of the year 2015.

History and Origin

The discovery of clustered DNA repeats occurred independently in three parts of the world. The first description came from Osaka University researcher Yoshizumi Ishino and his colleagues while studying Escherichia coli in 1987. In 1993, researchers of Mycobacterium tuberculosis in the Netherlands published two articles about a cluster of interrupted direct repeats (DR) in that bacterium. They recognized the diversity of the sequences that intervened the direct repeats among different strains of M. tuberculosis. At the same time, repeats were observed in the archaeal organisms of Haloferax and Haloarcula species, and their function was studied by Francisco Mojica at the University of Alicante in Spain. By 2000, Mojica performed a survey of scientific literature and one of his students performed a search in published genomes with a program devised by himself. They identified interrupted repeats in 20 species of microbes as belonging to the same family. In 2001, Mojica and Ruud Jansen, who were searching for additional interrupted repeats, proposed the acronym CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) to alleviate the confusion stemming from the numerous acronyms used to describe the sequences in the scientific literature. In 2002, Tang et al. showed evidence that CRISPR repeat regions from the genome of Archaeoglobus fulgidus were transcribed into long RNA molecules that were subsequently processed into unit-length small RNAs, plus some longer forms of 2, 3, or more spacer-repeat units. A major addition to the understanding of CRISPR came with Jansen's observation that the prokaryote repeat cluster was accompanied by a set of homologous genes that make up CRISPR-associated systems or cas genes. Researchers have also discovered that there are numerous CRISPRs. When people talk about CRISPR, they are usually referring to the CRISPR/Cas9 system. In recent years, researchers have found other types of CRISPR proteins that also work as gene editors.

Function in the Wild

The function of these CRISPR sequences was mostly a mystery until 2007, when food scientists studying the bacteria Streptococcus thermophilus used to make yogurt showed that these odd clusters actually served a vital function: They’re part of the bacteria’s acquired immune system.

It works like this: The bacteria are under constant assault from viruses; so, they produce enzymes to fight off viral infections. Whenever a bacterium’s enzymes manage to kill off an invading virus, other little enzymes will come along, scoop up the remains of the virus’s genetic code and cut it into tiny bits. The enzymes then store those fragments in CRISPR spaces in the bacterium’s own genome. The CRISPR spaces act as a “most wanted” gallery for viruses, and bacteria use the genetic information stored in these spaces to fend off future attacks. When a new viral infection occurs, the bacteria produce special attack enzymes, known as Cas9, that carry around those stored bits of viral genetic code like a mug shot. When these Cas9 enzymes come across a virus, they see if the virus’s RNA matches what is in the mug shot. If there is a match, the Cas9 enzyme starts chopping up the virus’s DNA to neutralize the threat.

CRISPR in the Lab

For a while, CRISPR was not of much interest to anyone except microbiologists, who marvelled at the sophistication of the system in the humble bacteria. But soon enough, scientists discovered that they could fool the Cas9 protein by feeding it artificial RNA, i.e., a fake mug shot. Now, the enzyme would search for anything with that same code, not just viruses, and start chopping. In a landmark 2012 paper, Jennifer Doudna of the University of California Berkeley, Emmanuelle Charpentier of Umeå University in Sweden , and Martin Jinek showed they could use this CRISPR/Cas9 system to cut up any genome at any place they wanted. The unbelievable implications opened the floodgates of research in this field. Further advances followed when Feng Zhang, a scientist at the Broad Institute in Boston, co-authored a paper in Science in February 2013 showing that CRISPR/Cas9 could be used to edit the genomes of cultured mouse cells or human cells. In the same issue of Science, Harvard’s George Church and his team showed how a different CRISPR technique could be used to edit human cells.

Progress Using CRISPR Gene-Editing

I. Disease models – Cas9 genomic modification has allowed for the quick and efficient generation of transgenic models within the field of genetics. Successful in vivo genome editing using CRISPR-Cas9 has been shown in numerous model organisms, including bacteria (Escherichia coli), yeasts (Saccharomyces cerevisiae, Candida albicans), nematode (Caenorhadbitis elegans), plants (Arabidopsis spp.), fish (Danio rerio), and animal (Mus musculus). CRISPR has also been utilized to create human cellular models of disease, polycystic kidney disease and focal segmental glomerulosclerosis.

II. Treatment of disorders with genetic causes – Early research in animal models suggest that therapies based on CRISPR technology have potential to treat a wide range of diseases, including cancer, beta-thalassemia, sickle cell disease, hemophilia, cystic fibrosis, Duchenne's muscular dystrophy, Huntington's disease, heart disease, and deafness.

III. Gene activation/ inactivation – Cas9 was used to carry synthetic transcription factors that activated specific human genes. The technique achieved a strong effect by targeting multiple CRISPR constructs to slightly different locations on the gene's promoter.

IV. Reversing diabetes in mice

V. Elimination of cardiovascular disease in embryo

VI. Development of molecular recorder

VII. Killing superbugs

VIII. Successful targeting of cancer – It targeted the “command center” of cancer – called the hybrid fusion – which leads to abnormal tumor growths. It also helped to slow the spread.

IX. Creating plants more resistant to disease and other stresses

X. Editing countless genes at once

Concerns with CRISPR

Despite all the promise, CRISPR is not a perfect tool, at least not yet. Scientists have recently learned that the approach to gene editing can inadvertently wipe out and rearrange large swaths of DNA in ways that may imperil human health. That follows recent studies showing that CRISPR-edited cells can inadvertently trigger cancer. That’s why many scientists argue that experiments in humans are premature: The risks and uncertainties around CRISPR modification are extremely high. However, this has only made scientists more determined to improve this invaluable tool. One way of doing that is through editing RNA instead of DNA. Another technique, discovered recently, is prime editing. It does not rely on the ability of a cell to divide to help make the desired changes in DNA. Also, it does not cut both strands of the DNA double helix, minimizing the chances of making unintended changes that could be dangerous.

However, there is something else that makes CRISPR controversial – its potential for human germline modification. This aspect raises several questions with respect to the ethics of human enhancement and that of heritable genetic modification. The waters have been muddled further by the shocking news declared in November 2018 about the world’s first CRISPR-edited babies by a Chinese scientist. The work was widely condemned as unethical, dangerous, and premature, and an international group of scientists called for a global moratorium on genetically editing human embryos.

Future Applications

Even amidst the legal battles regarding the ownership of CRISPR, one thing is clear from the current speed of progress in CRISPR research – despite the misgivings, CRISPR will continue to impact our world more and more in the upcoming days. It is presently thought to have the potential to help big issues like climate change (with genetically engineered phytoplanktons), energy crisis (with more sustainable biofuels), eradication of vectors and rodents (through gene drive), food security (with more nutritious crops), antibiotic resistance, genetic diseases, other diseases like AIDS, diagnostics (yes, it may be used to scale up coronavirus testing!), and even bringing back extinct organisms! Other prospects exist with respect to freedom from allergies, pet breeding, more nutritious fish, faster racehorses, and much more. For biologists in general, the most exciting prospect, however, probably is exploring how genomes work, that can in turn open up unending avenues for critical discoveries.

Although dreaming in the lines of Gattaca right now would be a stretch to say the least, with the ongoing improvements in the field (e.g., the recent proof of safety of CRISPR’d cells in humans, the invention of Cas-CLOVER – a cleaner, more precise alternative of CRISPR/Cas, and better targeting using light-controlled guide RNA), it would not be an exaggeration to say humanity is standing at a crossroads in the history of time.

The thoughts of another pandemic while already in the grips of one sends chills down our spines! So, without further ado, let us see if we can nip it in its bud. Although not contagious, unlike COVID-19, there is indeed reason to tout it as a pandemic in the foreseeable future, at least according to the leading Dutch neurologist Dr. Bastiaan R. Bloem. Researchers explain that this condition is communicable via new types of vectors – namely, social, political, and economic trends.

Parkinson’s disease (PD) is a progressive neurodegenerative disorder, mainly affecting the aged population. Although primarily a movement disorder, patients also suffer from non-motor symptoms, such as depression, anxiety, fatigue, and sleep disorders. The cause of this disorder is mostly unknown, and a subject of active research. The current mode treatment primarily involves medication like levodopa and others (e.g., dopamine agonists, monoamine oxidase-B (MAO-B) inhibitors, catechol-O-methyltransferase (COMT) inhibitors, anticholinergics, glutamate antagonists, and medication against non-motor symptoms), most often in combination with levodopa. Along with these, supportive therapy, including physiotherapy, occupational therapy, speech and language therapy and changes in diet, are frequently prescribed. In cases where medication is ineffective, surgery (mostly deep brain stimulation, and rarely, lesioning surgery) is prescribed.

PD is the fastest growing neurological disorder globally, and the spike has been attributed to the increasing human lifespan, the reduction in the number of smokers, and an increase in the number of industrial pollutants like the herbicide paraquat in the recent decades. Researchers believe that the key to transforming this seemingly inevitable rise in PD is activism, e.g., raising awareness, amassing funds, improving treatments, and changing policy. Stopping the production and use of the chemicals that may increase the risk of PD is essential. Also, crucial, as ever, is financial backing. More research is needed to understand why the condition appears and how it progresses, and this type of scientific investigation is never cheap. The most effective therapy remains levodopa, which is 50 years old and not without its issues, including both psychological and physical side effects.

Two hundred years since its first description, the scientists’ understanding of PD has made unimaginable progress, but there is hardly much in terms of treatment to show for it. PD is driven by the loss of dopamine-containing neurons in the brain, particularly those in the substantia nigra. For decades, researchers mimicked this pattern of cell death in animals using toxins, but the insights gleaned did not translate to humans, leading to a raft of failed clinical trials. Many failed clinical trials later, pharmaceutical company investment has withered as PD is deemed too high risk. In response, researchers are getting back to basics: re-examining their animal models, monitoring PD symptoms over years to better understand the different ways the disease unfolds, looking for early signs of the disease, and refining clinical trials so that effective therapies will not be missed. With a better understanding of the challenges, a new generation of treatment ideas is now in clinical trials, some of which aim to stall progression of the disease. A new study casts PD as an autoimmune disorder, with evidence that the immune system mistakenly attacks neurons and the alpha-synuclein protein (the normal physiological function of the α-synuclein protein involves roles in compartmentalization, storage, and recycling of neurotransmitters, and mutations in the SNCA gene that encodes it facilitates its aggregation and the Parkinson pathology). The emerging complexity of PD biology suggests that future treatment may involve multiple targets.

To quote Dr. Bloem, “From 1990 to 2015, the number of people with Parkinson disease doubled to over 6 million. Driven principally by aging, this number is projected to double again to over 12 million by 2040. Additional factors, including increasing longevity, declining smoking rates, and increasing industrialization, could raise the burden to over 17 million. For most of human history, Parkinson has been a rare disorder. However, demography and the by-products of industrialization have now created a Parkinson pandemic that will require heightened activism, focused planning, and novel approaches.” This year, a promising molecule has offered hope for a new treatment that could stop or slow Parkinson's, something no treatment can currently do. Also, the link with environmental toxicants is a trending topic of research.

So, worrying as the recent analysis is, there is reason to keep the hope alive in our hearts that the Parkinson pandemic is preventable, not inevitable.

While political leaders across the globe have been closing their borders, scientists have been transcending theirs, creating an unprecedented global collaboration. Never have so many experts in so many countries focused simultaneously on a single topic and with such urgency. Nearly all other research has come to a grinding halt. Usual imperatives like academic credit have taken a backseat. Online repositories are making studies available months ahead of journals. Researchers have identified and shared hundreds of genome sequences of the viral pathogen. More than 200 clinical trials have been launched, amassing global endeavours. It is thought that the closest comparison to this moment might be the height of the AIDS epidemic in the 1990s, when scientists and doctors locked arms to combat the disease. But the two can hardly be compared considering today’s technology and pace of information-sharing three decades later. This openness is very much reflected on the servers of medRxiv and bioRxiv, two online archives that share academic research before it is reviewed and published in journals. They have been flooded with coronavirus research from across the globe. And, despite the nationalistic tone set by the Chinese government, even Chinese researchers have contributed a sizeable portion of the coronavirus research available in the archives. So, nearly five months into the pandemic, with over 1.5 lakh people already dead and no signs of slowing of the spread, let us look at the round-up on some of the vital aspects of COVID-19 research.

Therapy

• The combination of lopinavir-ritonavir features prominently in the SOLIDARITY trial launched by the World Health Organization (WHO), both alone and in combination with interferon-β.

• To date, over 380 trials for COVID-19 have been posted on ClinicalTrials.gov, ranging from repurposed antiviral drugs to novel diagnostic imaging techniques.

• Antibody- and convalescent plasma-based approaches have dominated the news. The FDA just approved a plasma therapy trial at Johns Hopkins University. Takeda has announced a polyclonal hyperimmune antigen-purified antibody concentrate. The process used to recover antibodies from patients, already approved for the treatment of other infectious diseases, could lead to fast-track approval. Regeneron is pursuing a monoclonal antibody strategy using its humanized mouse antibody screening platform to produce an antibody cocktail for both therapy and prophylaxis.

• Although hopes for antibody-based immunity are high, currently, there is hardly any data available on whether human populations develop immunity to SARS-CoV-2. The WHO has announced a large-scale effort (named SOLIDARITY II) to collate serological data from different countries and post results from the initiative within the next few months.

• Some studies have found a correlation between the serum levels of interleukin-6 (IL-6) and severity of COVID-19 symptoms. Additionally, a preprint suggests that treatment of 20 severe or critical COVID-19 patients with the anti-IL-6 receptor drug tocilizumab could have been effective. Roche announced the launch of a trial of tocilizumab, recruiting 330 participants with severe COVID-19. Preliminary results are expected in the summer. Sanofi and Regeneron have expanded the testing in an existing clinical trial of their own anti-IL-6 receptor monoclonal antibody in rheumatoid arthritis to include severe or critically ill COVID-19 patients.

• Novartis announced plans for a phase III trial of its Janus kinase 1 (JAK1) and JAK2 inhibitor ruxolitinib in patients suffering from COVID-19-associated cytokine storms.

• Gilead reported the outcomes of patients who received a repurposed RNA polymerase inhibitor, remdesivir, on a compassionate basis in the New England Journal of Medicine. 36 out of 53 patients showed some sign of improvement, including 17 out of 30 patients on mechanical ventilation who were extubated. However, several limitations include the lack of a primary endpoint, a target for patient recruitment and a control group. The study also did not collect information on viral load, making it impossible to correlate the results with direct measures of the drug’s antiviral activity.

• A phase IIb trial of 81 patients in Manaus, Brazil, treated with azithromycin-chloroquine, was reported in MedRxiv. The study found no significant benefits of chloroquine–azithromycin and highlighted some safety concerns, with the high-dose arm terminated early due to the incidence of QT interval prolongation. A Chinese multicenter open-label randomized control study of COVID-19 patients treated with hydroxychloroquine alone, also in MedRxiv, found no effect on its primary endpoint, the negative conversion rate, but did exhibit a moderate reduction in lymphopenia and C-reactive protein levels.

Vaccines

• No vaccines are expected to reach the market within the next year. Currently, the WHO is curating a list of potential vaccines, of which two are currently under clinical evaluation: an adenoviral vector-based approach by CanSino Biological Inc. and the Beijing Institute of Biotechnology, and an RNA product by Moderna Inc. and the National Institute of Allergy and Infectious Diseases.

• Inovio Pharmaceuticals launched a phase I clinical trial of INO-4800, its DNA vaccine for COVID-19. Previously Inovio had reportedpartial positive results using a similar strategy in a phase I trial of its Middle Eastern respiratory syndrome (MERS) DNA vaccine.

• Shenzhen Geno-Immune Medical Institute (SGIMI) has begun phase I trials of LV-SMENP-DC, a cellular vaccine made up of dendritic cells (DCs) transduced with SARS-CoV-2 spike, membrane, nucleocapsid, envelope and protease (SMENP) minigenes along with immunomodulatory genes using a lentiviral vector. SGIMI also announced a second phase I trial testing artificial antigen-presenting cells modified with lentiviral vectors to express multiple SARS-CoV-2 minigenes and immunomodulatory genes.

• Pfizer announced jointly with BioNTechSE from Germany to bring its COVID-19 mRNA vaccine into phase I trials by the end of April.

Diagnostics and Serology

• A detailed analysis of 9 mild cases of COVID-19 in Germany detected SARS-CoV-2 in oro- and naso-pharyngeal samples that peaked at day 5 of symptoms. Live replicating SARS-CoV-2 was found in the throat, unlike that described for SARS-CoV. Both the viral RNA and the live virus were detected in the sputum, which may lead to simpler sample-collection protocols. Although high viral RNA concentrations were found in the stool, the authors were not able to isolate live virus from it. No urine or blood samples had viral RNA.

• Ju et al. used labeled SARS-CoV-2 spike protein receptor-binding domain (RBD) as a probe to sort antigen-specific B cells from eight infected patients in Shenzhen, China. From these sorted B cells, they produced 206 monoclonal antibodies with confirmed RBD binding. The capacity of monoclonal antibodies to compete with the receptor ACE2 for RBD binding was the best predictor of their neutralizing activity. Interestingly, the study found both germline clones and somatically mutated clones with high virus-neutralizing capacity.

• A study surveying levels of neutralizing antibodies (NAbs) to the SARS-CoV-2 spike protein in the plasma of 175 patients who had recovered from COVID-19 in one health centers in Shanghai, China, published in medRxiv, shed light on the development of natural immunity to the virus. Although patients in this study were classified as mild cases, antibody levels correlated positively with C-reactive protein and inversely with lymphocyte counts (NAb levels also tended to be higher in older subjects). A troublesome aspect of the study was that approximately 30% of recovered patients showed low NAb titers, including 10 patients with NAbs below the detection limit of the assay.

• A screen of Epstein-Barr virus-immortalized memory B cells derived from a patient who recovered from SARS in 2003 identified eight monoclonal antibodies that cross-reacted with SARS-CoV-2, and one, S309, showed potent neutralizing activity. Though S309 recognizes an epitope on the SARS-CoV-2 spike glycoprotein, it does not target the receptor-binding domain and is not predicted to block angiotensin-converting enzyme 2 (ACE2) binding. The work, led by David Veesler (University of Washington, USA) and Davide Corti (Humabs Biomedical, Switzerland), was reported as a preprint.

Pathophysiology

As the number of confirmed cases surges past 2.2 million globally, clinicians are uncovering new information about the spread of this disease in the body of an individual every day. The fast-evolving understanding of how the virus attacks cells around the body could be a crucial help for the doctors on the front lines providing treatment.

Animal Models

Establishing animal models of COVID-19 is a critical step toward understanding its pathophysiology and developing novel therapies.

• In Cell Host & Microbe, ferrets have been shown to mimic important aspects of human SARS-CoV-2 infection, including viral replication, fever and ferret-to-ferret transmission (although there were no fatalities). Infected ferrets also shed virus in nasal washes, saliva, urine and feces like humans.

• A preprint in bioRxiv also reported viral replication and transmission in domestic cats, but not in dogs, pigs, chickens or ducks.

• Also, a tiger in the Bronx zoo, USA has tested positive for SARS-CoV-2.

Epidemiology

• Modelling work by Ferretti et al. suggests that digitizing contact tracing through a mobile phone app may be able to suppress the epidemic sustainably. The app would help build “a memory of proximity contacts” and eliminate the delay in notifying contacts of infected people. The authors, however, caution that their models rely on a basic reproduction number (R0) derived using the Chinese data, which may not be accurate for the fast-spreading European epidemic.

• An analysis of 1,591 patients in 72 regional hospitals in Lombardy, Italy, published in JAMA, reports that mortality in the intensive care unit was 26%. Among the patients studied, the majority were male (82%) and had extensive comorbidities, confirming previous reports that these factors may play a part in the severity of the disease. The most frequent comorbidity was hypertension (49% overall and 62% of deaths).

• Among those patients whose files had respiratory support data, 88% were put on mechanical ventilation.

• A brief communication, published in Nature Microbiology, reports evidence of community spread of SARS-CoV-2 within the city of Wuhan, China. The researchers re-analysed 640 throat swabs collected for influenza-like illness in Wuhan between October 2019 and January 2020. Nine of the swabs tested positive for SARS-CoV-2; the first positive sample was collected in the first week of 2020.

• A modeling study led by Marc Lipsitch at Harvard’s T. H. Chan School of Public Health, published in Science, concludes that “prolonged or intermittent social distancing may be necessary into 2022”. The model assumes that immunity to SARS-CoV-2 will resemble what is observed for the related human coronaviruses OC43 and HKU1 — an assumption that remains to be tested. Based on serological samples from approximately 1,000 inhabitants of the German town of Gangelt (population of 12,529 people), an early COVID-19 epicenter, Bonn University researchers estimate an infection rate of 14% and a fatality rate of 0.37% (44 reported deaths in the town) in this non-peer reviewed report (in German). Correspondence in the New England Journal of Medicine shows results from the systematic screening of 214 mothers admitted into Columbia University Irving Medical Center’s labor and delivery unit in New York City. The letter reports a 13.7% frequency of asymptomatic carriers of SARS-CoV-2 (along with four symptomatic cases, or 1.9%).

• The New England Journal of Medicine published a large-scale COVID-19 diagnostic testing effort in Iceland, which found that 43% of positive cases had reported no symptoms at the time of testing. The study also found very low rates of infection in children under 10 years of age. A brief communication in Nature Medicine reinforces the importance of asymptomatic carriers. The study analyzed 414 throat swabs from 94 SARS-CoV-2-positive mildly or moderately ill patients in Guangzhou Eighth People’s Hospital, China. They found the highest SARS-CoV-2 viral load in the first days after symptom onset, which gradually declined to the detection limit around day 21 after onset. The authors also analyzed publicly available data on 77 infector-infectee pairs and arrived at an estimate of 44% of pre-symptomatic transmission. The study estimates that peak infectivity occurs between days 0 and 2 of symptom onset.

Viral Origin and Structure

• Andersen et al. presented a detailed analysis of the origin of SARS-CoV-2. A couple of papers report additional crystal structures for the SARS-CoV-2 RBD bound to ACE2. Lan et al. inferred convergent evolution of SARS-CoV and SARS-CoV-2 RBDs, indicative of selection in the passage to humans. Shang et al. used surface plasmon resonance to show that the RBD from SARS-CoV-2 bound more strongly to human ACE2 than did the RBD from SARS-Co-V. Interestingly, Shang et al. proposed that a related bat coronavirus, RaTG13, might also use ACE2 to enter human cells – a finding with worrying implications for the ability of bat coronaviruses to directly invade human hosts. In another report, a probable link to pangolin was identified with respect to the origin of SARS-CoV-2.

• A Nature study led by Haitao Yang at Shanghai Tech University, China, has solved the crystal structure of the SARS-CoV-2 main protease (Mpro).

Here’s looking forward to many more such discoveries lighting up our gloomy hearts in the upcoming days!