Written by Divik Dodeja, Arshnoor Kaur Chadha, & Ekanshh Praveen
The Nobel Prize - an international award which is given out by the Royal Swedish Academy of Sciences each year, for outstanding contribution in the fields of Chemistry, Physics, Medicine, and Literature. On 5th October 2020, it was awarded to researchers around the world for their breakthrough novel research in their respective fields. In this article, the authors take a deeper look into the 2020 Nobel Prize and try to explain the research performed in the fields of Chemistry, Physics, and Medicine, and the future prospects of them.
Nobel Prize in Chemistry
Emmanuelle Charpentier of Max Planck Unit for the Science of Pathogens, Berlin, Germany, and Jennifer A. Doudna from the University of California, Berkeley, USA were honored with the Nobel Prize 2020 in Chemistry. It was announced by the Royal Swedish Academy of Science on Wednesday in Stockholm, Sweden. As said by the academy, the two were awarded, ‘for the development of a method for genome editing tool.’
How It All Started
The two laureates met at a café. A colleague of Doudna introduced them and on the next day as suggested by Charpentier, they explored the old parts of the capital city together wherein the idea to collaborate sprung up. After a long discussion of their results, they suspected that 12, clustered regularly interspaced short palindromic repeats (CRISPR), CRISPR-RNA needed to be identified as a virus’ DNA and that Cas9 is the scissor that cuts off the DNA molecule. After multiple failed attempts, they decided to add tracrRNA. The history of genetic scissors could have stopped when they had discovered a mechanism in a bacterium could cause great suffering for humanity. That discovery was amazing but they decided to continue the collaboration.
Theory and Execution
It all began with the J.D. Watson and F.H.C. Crick stated the molecular structure of DNA in 1953. Since then, scientists have tried to formulate technologies that could alter cells and genetic material of organisms.
A method for genome engineering has now become a reality due to the findings of the RNA-guided CRISPR-Cas9 system. The technology has enabled scientists to alter DNA sequences in a variety of cells and organisms. Genomic manipulations are now used widely in biotechnology, basic science, and in the development of future therapeutics. By 2011, it was evident that CRISPR-Cas systems acted as adaptive immune systems in prokaryotes to withstand the invading bacteriophages and plasmids.
It was also evident that Cas proteins operated at three levels:-
(1) Integration of new spacer DNA sequences into CRISPR loci
(2) Biogenesis of crRNAs
(3) Silencing of the invading nucleic acid
The discovery of CRISPR-Cas9 as a genomic editing method came from class studies—2, CRISPR-Cas system Type-II in streptococcus thermophilus, and the associated human Streptococcus pathogen pyogenes. There are four genes in this system (cas1, cas2, csn2) are involved in spacer acquisition whereas the fourth one cas9 is required for interference.
Cleavage of target DNA was prevented due to the inactivation of the cas9 gene. To describe parts of the immunity, the S. thermophilus CRISPR-Cas system was introduced into E. coli making it evident that Cas9 protein alone was enough for the CRISPR-encoded interference step, and that two nuclease domains in protein, HNH, and RuvC, were both needed for this effect.
Fig 1.4 depicts the general scheme for the function of the CRISPR-Cas adaptive immune system as presented in three stages is identified. Adaptation: Short fragments of double-stranded DNA from a virus or plasmid are incorporated into the CRISPR array on host DNA. crRNA Maturation: Pre-crRNA is produced by transcription and then further processed into smaller crRNAs, each containing a single spacer and a partial repeat. Interference: Cleavage is initiated when crRNA recognize and specifically base-pair with a region on an incoming plasmid or virus DNA. Interference can be separated both mechanistically and temporally from CRISPR acquisition and expression.
In 2011, Emmanuelle Charpentier and colleagues documented the mechanisms of crRNA maturation in S. pyogenes. Differential RNA sequencing was used to characterize small, non-
coding RNA molecules. Astonishingly, the sequencing efforts identified RNA species from a region 210 bp upstream of the CRISPR locus. The transcript had trans-encoded RNA and contained 25 nucleotides. The RNA duplex could form included processing sites for both pre-crRNA and tracrRNA, pointing towards the possibility of being co-processed after pairing.
Later, the researchers found— processing also involved the Cas9 protein, since deletion of the cas9 gene in bacteria damaged tracrRNA and pre-crRNA processing. It was also suggested that Cas9 protein should act as a molecular anchor between tracrRNA and pre-crRNA.
Previously a collaboration between Charpentier and Jennifer revealed the significance of Cas9 for interference in contrast to what had been assumed in Charpentier’s report a year earlier, the addition of crRNA to purified Cas9 could not stimulate Cas9-catalysed target DNA cleavage.
The two scientists made a critical report on the addition of tracrRNA to the in vitro reaction accelerated Cas9 to the target DNA molecule. Two important functions of tracrRNA were triggering the pre-crRNA process by the enzyme RNase III and later activating crRNA-guided DNA cleavage by Cas9.
In a series of experiments, it was researched that the biochemical mechanisms of the reaction, the nuclease domains in Cas9, HNH, and RuvC, were each shown to cut one strand of marked DNA. As estimated, target recognition and cleavage were shown by mutations in the PAM sequence. The Cas9 cleaves within the protospacer, that cleavage specificity is directed by the crRNA sequence, and that the two nuclease domains within Cas9, each cleave one strand was also demonstrated in their reports. In their study, they had also worked to delineate the regions of tracrRNA and crRNA that were important for Cas9-catalysed cleavage of target DNA leading to the identification of an active domain in tracrRNA and the realization that 10 nt of seed region in the PAM-proximal region of the target strand was important for target recognition.
In an experiment, they also showed the possibility that the RNA components (crRNA and tracrRNA) of the Cas9 complex could be weaved together to form an active,
chimeric single-guide RNA molecule (sgRNA) furthering the scope of research in this field.
Conclusion and Future Prospects
In 2012, the two laureates reported that the Cas9 endonuclease can be programmed
with guided RNA engineered as a single transcript to cleave any double-stranded DNA sequence. Their discovery has led to widespread applications of the CRISPR-Cas9 system as a powerful and versatile tool in genome editing. With the introduction of engineered sgRNA and vector encoded the Cas9 nuclease, scientists can create accurate single-base-pair changes or larger insertions giving the scope of future developments, improvements, and researches in the field.
Especially holds great potential in the field of Cancer research and therapy, providing remarkable discrete cancer-associated mutations in the genome contradicted with the actual ZFNs or TALENs technologies. Also, providing an effective gene-editing method to the cancer biologists enabling them to modify the genetic make-up of cells in various remarkable ways. Moreover, TMEM135-CCDC67 and MAN2A1-FER fusion genes have been recognized as cancer-derived genes in hepatocellular carcinoma and human prostate cancer. Further, the HSV1-TK death-promoting gene, which—as a suicide gene; is a phosphotransferase that blocks DNA synthesis was used to replace the previous genes via the CRISPR-Cas9 technology.
Personalized therapy is another application of the CRISPR/CAS9 system requiring organized screening which would be efficient to identify the genotype-specific changes in a patient’s genome. Therapeutic strategies are constantly being developed based on the results of the gene screening. An example of personalized therapy is the treatment of EGFR-mutant lung cancer. The CRISPR technology is suspected to open the door to effective personalized cancer treatment from specific applications of genome screening to therapeutic strategies with results being achieved in basic research to the development of potential therapies against various diseases. The CRISPR-Cas9 system has the potential to fully revolutionize scientific research providing tremendous resources for greater awareness of cancer biology and treatment.
Nobel Prize in Medicine or Physiology
This year’s Nobel Prize is awarded to three scientists who have made a decisive contribution to the fight against blood-borne hepatitis, a major global health problem that causes cirrhosis and liver cancer in people around the world.
Harvey J. Alter, Michael Houghton, and Charles M. Rice made seminal discoveries that led to the identification of a novel virus, the Hepatitis C virus. Prior to their work, the discovery of Hepatitis A and B viruses had been critical steps forward, but the majority of blood-borne hepatitis cases remained unexplained. The discovery of the Hepatitis C virus revealed the cause of the remaining cases of chronic hepatitis and made possible blood tests and new medicines that have saved millions of lives.
Foundation of the Idea to Success
The viruses Hepatitis A and Hepatitis B had been discovered by the mid-1960s.
But Prof Harvey Alter, while studying transfusion patients at the US National Institutes of Health in 1972, showed there was another, mystery, infection at work. Patients were still getting sick after receiving donated blood. He showed that giving blood from infected patients to chimpanzees led to them developing the disease. The mysterious illness became known as "non-A, non-B" hepatitis and the hunt was now on.
Prof Michael Houghton, while at the pharmaceutical firm Chiron, managed to isolated the genetic sequence of the virus in 1989. This showed it was a type of flavivirus and it was named Hepatitis C. And Prof Charles Rice, while at Washington University in St. Louis, applied the finishing touches in 1997. He injected a genetically engineered Hepatitis C virus into the liver of chimpanzees and showed this could lead to hepatitis.
Prof Houghton, now at the University of Alberta in Canada, told the BBC: "We had limited tools available to us then, so it was rather like searching for a needle in a haystack.
"The amount of virus present in the liver and the blood was very low, and the sensitivity of our techniques was not high enough, so we were sailing very close to the wind all the time.”
"We tried a lot of methods, probably 30 or 40 different methodological approaches over seven years, and eventually one worked."
Commenting on the announcement, Dr. Claire Bayntun, a clinical consultant in global public health and vice-president of the Royal Society of Medicine, said the discovery was an "extraordinary achievement". She said: "[In] unlocking the door to the development of effective treatment and screening of blood transfusions, and protecting populations in many regions of the world, millions of lives have been saved."
Discovery of Hepatitis-C
Despite this significant progress, the identity of the virus(es) responsible for NANBH remained frustratingly obscure. The unsuccessful search employing all the traditional methods that had allowed the discovery and characterization of HAV and HBV would continue for more than 10 years. Michael Houghton, working at Chiron Corporation, initiated his hunt for the NANBH virus in 1982 using a molecular approach based on the screening of DNA fragments, also called a complementary DNA (cDNA) library, isolated from infected chimpanzees. The initial screenings identified only genetic material from the host. Attempts to enrich viral sequences by eliminating host sequences that were also found in an uninfected control liver were also unsuccessful. Houghton, then working with Qui-Lim Choo and George Kuo, decided to try a novel immune-screening approach. A cDNA library was generated from RNA isolated from plasma of NANBH-infected chimpanzees and this was transferred to bacteria using a highly efficient lambda bacteriophage system. The expression of viral antigens was then investigated using serum from a patient with fulminant NANBH, which was presumed to contain antibodies against the unknown virus. Screening one million bacterial colonies using this approach resulted in the identification of one colony that did not contain chimpanzee or human DNA sequences. This was the viral signal they were looking for. The sequence, named clone 5-1-1, hybridized to an RNA of about 10,000 nucleotides. The RNA encoded a large open reading frame (ORF) and exhibited distant homology with the genomes of known RNA viruses. Proteins could be translated from the RNA molecule itself indicating that the virus had a positive-strand RNA genome. This allowed classification of the virus, which they named Hepatitis C virus (HCV), as a new member of the Flaviviridae family. Further experiments showed that the new viral sequence encoded protein that reacted with sera from a NANBH-infected chimpanzee, but not with sera from control HAV or HBV-infected animals (Figure 3.3).
Figure 3.3 Choo et al. Science 1989. Immunoblots with sequential serum samples from representative chimpanzees infected with NANBH, HBV, or HAV, probed against the protein encoded by the 5-1-1 ORF.
Following identification of the virus, the Houghton team rapidly developed an immunoassay for the detection of HCV-specific antibodies and showed the presence of such antibodies in a blood donor that had transmitted the disease to ten different recipients, and in NANBH patients from Italy, Japan, and the USA. These findings established a firm relationship between infection with the newly discovered HCV and the occurrence of NANBH around the world.
The Final Proof
The work of Alter and Houghton established a critical link between NANBH and HCV infection. However, it did not constitute definitive proof of causality because transmission of the disease by transfer of infectious blood could not exclude the involvement of essential co-factors. To conclusively demonstrate causality, the isolation of a virus capable of reproducing the clinical hallmarks of the disease, including chronic liver damage and persistence of the infectious virus in the blood of the infected host, was required. A first step towards achieving this goal was made when the groups of Kunitada Shimotohno, working at the National Cancer Center Research Institute in Tokyo, and Charles Rice, working at Washington University in St Louis, in close succession identified a conserved, non-coding region at the 3’ end of the HCV RNA genome, which they surmised could play an important role in virus replication.
Rice constructed viral RNA genomes containing the conserved 3’ region, injected them into the liver of chimpanzees, and looked for evidence of HCV replication but failed to observe newly produced viruses in the blood. He then took the next decisive step. Knowing that RNA virus replication is error-prone and that many viral sequences carry inactivating mutations, he engineered a set of RNA genomes that comprised both the conserved 3´ region and a consensus sequence to exclude potential inactivating mutations. He injected the engineered RNA into the liver of chimpanzees and this time the experiment was successful. Productive infection was established, the animals developed clinical signs of hepatitis, and infectious virus was found in their blood for several months (Figure 3.4).
A similarly engineered HCV RNA was soon thereafter reported by the laboratory of Jens Bukh, also showing that productive infection could be achieved. The work of Charles Rice provided conclusive evidence that HCV alone could cause hepatitis, persist long-term, and stimulate a specific antibody response, all features of the human infection.
Figure 3.4. Kolykhalov et al. Science 1997. HCV viremia and liver enzymes measured weekly in two chimpanzees (shown in A and B, respectively) inoculated with in vitro transcribed RNA encoding the full-length HCV consensus sequence.
New Antiviral Treatments
The seminal discovery of HCV by this year’s Nobel Laureates paved the way for the development of effective antiviral drugs. While infectious in primates, the full-length clones generated by Rice exhibited poor replicative capacity in cell lines, which hampered in vitro studies of the virus life cycle and testing of candidate antiviral drugs. This obstacle was overcome thanks to the work of Ralph Bartenschlager at the University of Heidelberg in Germany who constructed the first HCV sub-genomic clones that replicated with high efficiency in transfected hepatoma cell lines. Further improvement of the technology, and the identification of virus isolates that could replicate in cell lines without the need for adaptive mutations, led to the production of sub-genomic replicons that upon transfection into hepatoma cells resulted in the secretion of virus particles that were infectious. The second obstacle stems from the very restricted host spectrum of HCV – the virus infects only humans and chimpanzees – and the consequent lack of small animal models for precise assessment of the pathological and immunological profile of the disease, and for pre-clinical testing of candidate drugs. Progress was made when T- and B-cell deficient mice with severe combined immunodeficiency (SCID) could be grafted with human hepatocytes and other models were developed reviewed in.
The availability of in vitro replication systems and the development of small animal models for in vivo studies facilitated the development of highly effective antiviral drugs that have revolutionized the treatment of HCV infection. Earlier therapeutic regiments, including recombinant type I interferon (IFN) and the nucleoside analog ribavirin, were ineffective and associated with significant side effects. Some improvement was made at the end of the 1990s with the introduction of pegylated-IFN and further improvements were made with the introduction of inhibitors of the NS3/NS4A protease, such a boceprevir, telaprevir, and simeprevir. The development of drugs that specifically target the viral RNA-dependent RNA polymerase NS5B, e.g. sofosbuvir, and the regulatory replicon protein NS5A, e.g. ledipasvir, constituted major breakthroughs in HCV therapeutics. The combination of drugs that target critical viral functions, collectively known as directly acting antivirals (DAAs), proved to be highly effective, caused only minor side effects, and strongly diminished the risk of selecting drug-resistant variants of the virus.
Figure 3.5. The discovery of the Hepatitis C virus has led to the development of sensitive blood tests and anti-viral drugs that have saved millions of lives. Extended blood screening programs and the availability of treatments at a global scale is a critical remaining goal.
Thanks to the pioneering work of Alter, Houghton, and Rice, validated tests that identify HCV carriers and allow the elimination of contaminated blood and blood products are broadly available around the world, and effective drugs have changed the fate of HCV infected patients. HCV induced hepatitis is now in many cases a curable disease and the lesions associated with infection are often reversible. Clinical studies have shown that short-term anti-viral treatment cures more than 95% of the patients, including advanced cases who failed to respond to previous therapeutic modalities. This outstanding achievement has already benefitted millions of individuals worldwide. The remaining obstacles towards the eradication of viral hepatitis are now mostly associated with the lack of broad screening campaigns (according to the WHO Global Hepatitis Reports 2017 less than 20% of people with HBV or HCV-associated hepatitis have been adequately diagnosed) and with the high cost of the most effective treatments.
Nobel Prize in Physics
The Nobel Prize in Physics was awarded to three remarkable Scientists on 5th October 2020 for their comprehensive work in understanding and discovering the Super Massive Black Hole at the center of our galaxy. This was a result of decades worth of research and intensive usage of various Scientific disciplines including Mathematics, Physics, and Astronomy.
British Physicist, Roger Penrose bagged ½ of the prize for his massive contribution in the Theoretical aspect of the project, and the other ½ of the prize was split between US astronomer Andrea Ghez and German astronomer Reinhard Genzel.
It all started in the year 1965, when English Mathematical Physicist, Roger Penrose attempted to gain theoretical and mathematical proof for the existence of a black hole in the middle of our galaxy. Penrose tried to use “The Principle of General Relativity” developed by Legendary Physicist Albert Einstein, to explain how under the right conditions, an actual “Black Hole” can be formed. He explained if a surface can trap large amounts of light in space, it could provide physically apt conditions for the formation of a region where mass undergoes an indefinite and irreversible gravitational collapse which produces a region of infinitely dense energy which is called a “Singularity”.
Penrose worked intensively with Great Physicists like Stephen Hawking; who had a significant contribution to Penrose’s notion that all mass in a Black Hole condensed into a single point.
Roger Penrose also created the Twistor Theory which mathematically bridged the Theory of Relativity and Modern Quantum Mechanics.
He defined a term called “Twistor Space” which was a Vector Space used for conceptualizing how photons travel in light using only four complex numbers. Penrose also developed a method of mapping the regions surrounding the black hole which popularly came to be known as Penrose Diagram. This diagram allows one to properly visualize the effects of gravity in regions surrounding the Black Hole.
Fig 2.4-2.6: Illustrations of the Penrose Diagram which aimed to visualize the effects of gravity in regions surrounding Black Holes
As a Mathematical Physicist, Penrose employed advanced Mathematical and Geometrical Principles in the World of Physics and also had major contributions in Geometry where he developed Penrose Tilings which was a non-repeating 2D structure, which attracted heaps of attention when it naturally occurred in substances called “Quasicrystals”. He also worked with various artists to try and come up with ways of sketching impossible structures.
In conclusion, Penrose built up the theoretical aspect of this magnificent feat, but that is only one aspect of it. Now we move onto the execution and astronomical discovery of the Black Hole which backs up Penrose’s theory.
Now as more and more scientists heard about the possible existence of Black Holes, many groups of Astrophysicists were determined to capture evidence of such a possibility. This included American astrophysicist, Andrea Ghez, and German astronomer, Reinhard Genzel who had set up their research groups for finding evidence of Black Holes.
Initially, when these groups observed the Milky Way Galaxy, they observed energetic emissions from the Centre of the Galaxy and named the object causing these emissions as “Sagittarius A*” or “Sgr A*”. They also observed that the center was packed with stars and gas that was hurling around at high speeds.
Fig 2.7: Representation of the Milky Way Galaxy; a depiction of the exact position of the Sagittarius A* with respect to the Solar System.
Although the groups faced a lot of obstacles like boosting the resolution of the observations and constant blockage of stars due to gas and dust, the groups managed to pull off the impossible by constantly looking for better ways of recording observations. The groups led by Ghez and Genzel used some of the world’s most advanced telescopes situated in Hawaii and Chile respectively and used modern observational techniques.
Both of the groups used techniques like Speckle Imaging, which allowed the groups to avoid blurring due to the Turbulence of the Earth’s Atmosphere., and adaptive optics which uses a mirror to correct any distortion - which allowed them to gain larger exposures and also to track the motion of stars in three dimensions. During their initial observations, the resolution wasn’t ideal but it improved as more modern techniques were implemented. After decades of experimentation, the teams were able to observe thousands of stars near the Galactic Centre and also map around 30 of them.
Fig 2.8. These diagrams are based on Penrose’s paper from 1965 and show the collapse of matter into a black hole. On a trapped surface all light cones are tipped inwards, and the formation of a singularity is inevitable. Schematic diagram showing the interior of a black hole. Inside of the horizon, the radial direction is time-like.
The Nobel Prize Laureates paved a way for tons of future research opportunities and ideas with their massive contribution to the World of Physics. Now, many Astrophysicists believe that supermassive black holes reside at the center of all galaxies and played a role in the formation of galaxies from the primordial soup of matter in the early universe. Although this was a remarkable discovery, it is just the beginning. More and more research projects like NASA’s NuSTAR(Nuclear Spectroscopic Telescope Array); a spacecraft which aims to hunt and characterize more Supermassive Black Holes around the universe, and the observations made by the Laser Interferometer Gravitational-Wave Observatory (LIGO) which detected space-time ripples generated by mergers involving relatively small black holes — objects harboring just a few dozen times the mass of the sun. Few projects have also been scheduled for the future, such as the European Space Agency's Laser Interferometer Space Antenna mission, which is scheduled to launch in the mid-2030s — will aim to spot gravitational waves generated by mergers of supermassive black holes.