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2020 Nobel Prize in Chemistry Awarded to CRISPR-CAS9 Gene Editing Technology

On October 7, 2020, the Royal Swedish Academy of Sciences has decided to award the 2020 Nobel Prize in Chemistry to Dr. Emmanuelle Charpentier of the Max Planck Institute for Pathogenesis in Germany and Dr. Jennifer A. Doudna of the University of California, Berkeley, in recognition of them contributions in the field of genome editing.

Figure 1. Emmanuelle Charpentier and Jennifer A. Doudna. (Image source: NobelPrize.org)

Wonderful Encounter
The discovery and application of CRISPR-CAS9 gene editing technology has become a major revolution in the field of modern gene editing. The emergence of this technology has greatly simplified the process of gene editing. Its discovery process is also full of accidents like other scientific discoveries.
In an occasional conversation with a colleague who is engaged in microbiological research, Dr. Doudna learned that the same code of repeated DNA sequences in the genetic material of extremely different bacteria and archaea appear repeatedly, but they are separated by different sequences. These repeats are called clustered regularly interspaced short palindromic repeats, abbreviated as CRISPR. Because the unique non-repetitive sequences in CRISPR seem to match the genetic codes of various viruses, researchers believe that it is part of the ancient immune system of bacteria, which can protect bacteria and archaea from viruses. If the bacterium successfully resists the virus infection, it will add part of the virus’s genetic code to its genome as a memory of the infection. Although no one knew the molecular mechanism at the time, the basic hypothesis of the scientists at the time was that bacteria used the mechanism of RNA interference to neutralize viruses.

Complex Molecular Mechanism Map

If bacteria are proven to have an ancient immune system, it will become a very important discovery in the scientific community. For this reason, Dr. Doudna began to learn about the CRISPR system out of curiosity. It turns out that in addition to the CRISPR sequence, there is also a special gene called CRISPR-related inside the bacteria, abbreviated as Cas. Dr. Doudna discovered that these genes are very similar to those that encode proteins that are specifically used to melt and cut DNA. Therefore, it has become a new problem to prove that Cas protein has the same function of cutting viral DNA.

A few years later, the research team led by Dr. Doudna succeeded in revealing the functions of several different Cas proteins. At the same time, the system has been discovered by other research groups. The bacterial immune system can take very different forms. The figure below shows the working mechanism of different types of CRISPR / Cas systems. The CRISPR/Cas system studied by Dr. Doudna belongs to Class I; this is a complex mechanism that requires many different Cas proteins to clear the virus. Interestingly, Type II systems are very simple, they require less protein. At the same time, Dr. Emmanuelle Charpentier happened to encounter a Type II system.

Dr. Emmanuelle Charpentier is a scientist with a wide range of interests. While working on pathogenic microorganisms, she is also very interested in small RNA molecules involved in gene regulation. In collaboration with researchers in Berlin, Charpentier et al. located small RNAs inside Streptococcus pyogenes. There is a large number of small RNA molecules that have not been reported before in this bacteria, and its genetic code is very close to the CRISPR sequence in the genome. By carefully analyzing their genetic code, Charpentier discovered that part of this new type of small RNA molecule partially matched the repetitive sequence in the CRISPR gene. Although Charpentier had never been exposed to the CRISPR system before. But her research team used a series of thorough microbiological tests to locate the CRISPR system in Streptococcus pyogenes. According to existing research, this system is known to belong to the class II CRISPR/Cas9 system, that is, only one Cas protein-Cas9 is needed to achieve the purpose of targeted lysis of viral DNA. Charpentier’s research also showed that an unknown RNA molecule (called trans-activated crisp RNA (tracrRNA)) is of decisive importance for the realization of CRISPR’s function. It can help the long RNA molecules produced by the transcription of CRISPR sequences in the genome to be processed into mature, active forms.

Figure 2. Streptococcus’ natural immune system against viruses: CRISPR/Cas9.(Image source: NobelPrize.org)

Dr. Emmanuelle Charpentier is a scientist with a wide range of interests. While working on pathogenic microorganisms, she is also very interested in small RNA molecules involved in gene regulation. In collaboration with researchers in Berlin, Charpentier et al. located small RNAs inside Streptococcus pyogenes. There is a large number of small RNA molecules that have not been reported before in this bacteria, and its genetic code is very close to the CRISPR sequence in the genome. By carefully analyzing their genetic code, Charpentier discovered that part of this new type of small RNA molecule partially matched the repetitive sequence in the CRISPR gene. Although Charpentier had never been exposed to the CRISPR system before. But her research team used a series of thorough microbiological tests to locate the CRISPR system in Streptococcus pyogenes. According to existing research, this system is known to belong to the class II CRISPR/Cas9 system, that is, only one Cas protein-Cas9 is needed to achieve the purpose of targeted lysis of viral DNA. Charpentier’s research also showed that an unknown RNA molecule (called trans-activated crisp RNA (tracrRNA)) is of decisive importance for the realization of CRISPR’s function. It can help the long RNA molecules produced by the transcription of CRISPR sequences in the genome to be processed into mature, active forms.

After in-depth and targeted experiments, Dr. Charpentier published his findings on tracrRNA in March 2011. Although she has many years of experience in microbiology, she hopes to cooperate with more professional scientists in continuing to study the CRISPR-Cas9 system. Dr. Jennifer Doudna therefore became a natural choice. When Charpentier was invited to a conference in Puerto Rico, the two scientists had a historic meeting.

Alliance Between Giants
After further communication, they hope to cooperate to complete the follow-up research. They speculate that bacteria need CRISPR-RNA to recognize the DNA sequence of the virus, and Cas9 is the scissors that ultimately cut the DNA molecule. However, when they tested in vitro, they did not get the expected results. After a lot of thinking and failed attempts, the researchers finally tried to add tracrRNA to their system. Previously, they believed that tracrRNA was only needed to cut CRISPR-RNA into its active form. When Cas9 obtained tracrRNA, the result everyone was waiting for finally appeared: the DNA molecule was cut into two parts. After that, the researchers simplified the “genetic scissors”. Using their new discoveries of tracr-RNA and CRISPR-RNA, they successfully fused the two into one molecule and named it “Guide RNA”. Using a simplified version of this genetic scissors, they successfully achieved the goal of cutting DNA at any position.

Figure 3. The CRISPR/Cas9 genetic scissors. (Image source: NobelPrize.org)

Upheaval in Life Sciences
Shortly after Emmanuelle Charpentier and Jennifer Doudna discovered the CRISPR/Cas9 gene scissors in 2012, several other research groups demonstrated that the tool can be used to modify the genomes of mouse and human cells, leading to their explosive development. Using CRISPR gene editing tools, researchers can in principle cut any genome they want. After that, it is easy to use the cell’s natural system to repair DNA, thereby realizing the “redefinition” of genes. Through the new discoveries of Emmanuelle Charpentier and Jennifer Doudna, life science has successfully entered a new era.