Gene editing finally gets the Nobel Prize in Chemistry! Two female scientists won, but why not Zhang Feng?

Written by | Fanpu

On the evening of October 7th, Beijing time, the 2020 Nobel Prize in Chemistry was awarded to Emmanuelle Charpentier and Jennifer A. Doudna in recognition of their contributions to gene editing.

About the winner:

Dr. Emmanuelle Charpentier, a French microbiologist, is currently the director of the Institute of Infection Biology at the Max Planck Institute in Germany. Her major contribution to the development of CRISPR is the discovery that the activity of the Cas9 protein depends on tracrRNA.

Dr. Jennifer A. Doudna is a professor of chemistry and molecular biology and cell biology at Berkeley University, a researcher at the Howard Hughes Medical Institute, and a member of the National Academy of Sciences. She and Dr. Emmanuelle Charpentier jointly discovered the cutting function of Cas9 and the positioning function of crRNA, and that crRNA and tracrRNA can be fused into single-stranded guide RNA (sgRNA).

Experts in related fields analyzed that Chinese scientist Zhang Feng was the first to use CRISPR-Cas9 to achieve gene editing in eukaryotic cells, but he failed to win the award. It may be because Zhang Feng's work is less original, and his contribution is more like the contribution of Cohen and Boyer in cell recombination (the 1980 Chemistry Prize was awarded to Berger for artificial recombination); and the Chemistry Prize pays more attention to in vitro experimental results, and his breakthrough is mainly reflected in cells.

CRISPR is already a very popular gene editing technology in biomedicine. In recent years, the technology has developed rapidly and has been applied to many fields such as biology, medicine, agriculture and environment, creating a batch of scientific research miracles, especially in the treatment of genetic diseases, screening and detection of disease-related genes, tumor treatment, transformation of animals and plants, prevention and control of pathogenic microorganisms, etc. It has huge potential and will profoundly affect the whole world.

1. What is CRISPRCRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats, which translates into “regularly clustered short palindromic repeats” in Chinese. The literal meaning of this word is “represents a repetitive sequence of the same type with obvious characteristics, neat arrangement, and consistent order”. It is an adaptive immune system of bacteria. When foreign viruses or plasmid DNA enter cells, specialized Cas proteins will cut the foreign DNA into small fragments and paste them into their own DNA fragments for storage. When encountering a virus invasion again, bacteria can recognize the virus based on the stored fragments and cut off the viral DNA to render it ineffective [1].

Scientists have used this function of CRISPR to transform it into a revolutionary new molecular tool. Because of its ability to accurately locate and cut any kind of genetic material, scientists can more easily crack the life code of any organism on Earth (including humans).

1. A brief history of CRISPR

Figure 1: A brief history of CRISPR development

Discovery and naming of CRISPR As early as 1987, Nakata's research group at Osaka University in Japan discovered some abnormal repetitive sequences in bacteria when analyzing Escherichia coli [2]. But at that time, people did not know what the role of these repetitive sequences was, and they had not even officially named them. It was not until the late 1980s that Francisco Mojica of the University of Alicante in Spain discovered similar repetitive sequences in an archaea species [3], which aroused his great interest. In subsequent studies, he continued to look for similar structures in microorganisms, and by 2000 he had found similar sequences in more than 20 different microbial species [4]. In 2002, Ruud Jansen of Utrecht University in the Netherlands discovered that there were huge differences in the number of bases in the repetitive sequences of different species, and that this sequence only existed in prokaryotes. In order to better standardize related research, they jointly named this repetitive sequence "CRISPR". . Multiple CRISPR-related genes were named the Cas (CRISPR-associated) family [5].

The biological role of the CRISPR-CAS system In 2005, CRISPR research ushered in an important discovery. Two research groups (Mojica and Pourcel) observed that the spacer sequences between CRISPR repeat sequences did not come from prokaryotes themselves, but from plasmids or viruses [6-7]. Therefore, Mojica proposed the hypothesis that CRISPR is an adaptive immune system. In the same year, Bolotin's research group discovered Cas9 in Streptococcus thermophilus and predicted that this huge protein has nuclease activity. They also found that the spacer sequences homologous to viruses all have a similar tail, called PAM (protospacer adjacent motif) sequence, which is crucial for the recognition of target sequences.

Inspired by this hypothesis, French microbiologist Rodolphe Barragou, who was working at the famous yogurt company Danisco at the time, decided to verify it to solve the problem of phage infection and death of thermophilic Streptococcus, which affected yogurt production. In 2007, they proved through experiments that the CRISPR system is indeed an adaptive immune system. After being invaded by a virus, thermophilic Streptococcus integrates new spacer sequences from the phage genome. When the same virus invades again, the bacteria have the ability to resist attack[8], and the Cas9 protein may be necessary for the generation of this immunity. This is the first experimental confirmation that CRISPR-Cas is a bacterial acquired immune system.

The confirmation of the biological function of CRISPR-CAS has made many research teams realize the importance of this system. Subsequently, many research teams have begun to supplement the details of the CRISPR-Cas system's interference with bacteriophages. In 2008, John van der Oost's research team found in Escherichia coli that the spacer sequence from the bacteriophage was transcribed into small RNA, becoming CRISPR RNA (crRNA), and guiding the Cas protein to the target DNA. In the same year, Marraffini and Sontheimer proved that the target molecule of the CRISPR-Cas system is DNA, not RNA. They also clearly pointed out that if the system is transferred to a non-bacterial system, it may become a powerful tool system [9-10]. This laid the groundwork for subsequent gene editing.

In December 2010, Moineau's team demonstrated that CRISPR-Cas9 precisely cuts upstream of the PAM sequence to break the DNA double strand. As a prominent feature of the type II CRISPR system, Cas9 is the only protein required for cutting, and it mediates the interference function of CRISPR-Cas9 together with crRNAs [11].

In 2011, Charpentier's research group sequenced small RNAs in Streptococcus pyogenes and found that in addition to crRNA, there is another small RNA called tracrRNA. TracrRNA complements the repetitive sequence in crRNA through 24 nucleotides to form a double strand, guiding Cas9 to the target DNA. At this point, the puzzle of the natural CRISPR-Cas9 interference mechanism is basically complete [12].

The emergence of CRISPR-CAS gene editing technology In 2012, the Charpentier and Doudna teams collaborated to prove that Cas9 not only has the ability to cut double-stranded DNA, but also can link tracrRNA and crRNA into sgRNA (single guide RNA), and confirmed in vitro experiments that sgRNA can also guide Cas9 protein to complete double-stranded DNA cutting. They can control the targeting site of Cas9 by changing the sequence of crRNA [13]. Later, Siksnys' team also reported the same discovery. This discovery is not only a milestone in the field of bacterial acquired immune system, but also opened a new chapter in CRISPR-CAS gene editing technology. Soon, many papers in early 2013 successfully applied the CRISPR-Cas system to mammalian cells. Among them, the Church research group designed a type II CRISPR-Cas system, which successfully targeted specific sequences in human 293T cells, K562 cells and induced pluripotent stem cells by designing sgRNA, and multiple gRNAs can achieve multiple editing of target genes [14]. Zhang Feng's laboratory demonstrated that the CRISPR-Cas9 system can perform precise site-specific cutting in human and mouse cells, and mutated Cas9 into a nickase to promote the homologous repair process [15]. Qi's research group established the CRISPRi system and achieved simultaneous site-specific mutation of multiple genes (Tet1, Tet2, Tet3, Sry and Uty) by targeting multiple sgRNAs [16]. Wu et al. used the CRISPR-Cas9 system to perform gene therapy on mice with dominant mutations in Crygc and obtained healthy offspring, providing a basis for the use of the CRISPR-Cas9 system in gene therapy for genetic diseases [17].

As a result, the research and application of CRISPR system in the fields of gene site editing, genome screening, gene transcription regulation, genome imaging, gene diagnosis and treatment, ecological application, etc. of various organisms began to explode. In the following years, Zhang Feng's laboratory further expanded the gene editing system of CRISPR-CAS, and not only discovered the CRISPR-Cas system with great advantages in specificity and multi-gene editing: CRISPR-Cpf1[18], but also discovered CRISPR enzymes Cas13a (C2c2)[19] and Cas13b[20] with RNAse function. In 2017, several articles studied the mechanism of action of CRISPR-Cas13 system, its application in clinical diagnosis, and its ability to target RNA in mammalian cells.

1. Application of CRISPR gene editing technology The rapid development of CRISPR technology has led to its application in translational medicine and disease treatment. In 2015, Science magazine published a method for successfully using CRISPR to treat animal models of genetic diseases. They used the CRISPR system to edit the Dystrophin gene, which was able to repair the muscle function of Duchenne muscular dystrophy mice to varying degrees, thereby achieving the effect of treating DMD [21]. In 2018, a study confirmed that four dogs with DMD (Duchenne muscular dystrophy) were successfully treated with CRISPR technology, and the dystrophin protein in their muscle and heart tissues was restored to 92% of normal levels [22]. In addition, CRISPR technology has been combined with cell immunotherapy to improve CAR-T therapy and enhance tumor suppression in mice. The first clinical trial using CRISPR-Cas9 to knock out the PD-1 gene in T cells has been approved for the treatment of muscle invasive bladder cancer, castration-resistant prostate cancer, metastatic renal cancer, and metastatic non-small cell lung cancer, and phase I clinical trials began in 2016 [23].
2. Other applications of CRISPR Currently, CRISPR is not only used in genome editing, but also shows great potential in genetic testing. In a study published in Science in 2017, Doudna's team discovered an interesting phenomenon: when the CRISPR system cuts the targeted double-stranded DNA, the DNA enzyme activity of Cas12 will be activated [24]. This discovery provides a new idea for detecting whether a cell contains a certain target DNA: the CRISPR-Cas12a system targeting the DNA and the non-specific ssDNA fluorescent reporter gene (FQ-labeled reporter) are delivered into the cell at the same time. Once the target DNA is detected, the CRISPR-Cas12a system will be activated, and at the same time, the fluorescent reporter gene will be degraded, thereby releasing a fluorescent signal. Using this technology, Doudna's team developed the DETECTR system, which can detect HPV 16 infection with 100% accuracy within one hour, and the cost of a single test is less than one dollar. At the same time, Zhang Feng’s team also used CRISPR-Cas13a to develop the SHERLOCK system and SHERLOCKv2 system[25]. Unlike Doudna’s team, the fluorescent reporter gene designed by Zhang Feng’s team must be specific. It is precisely this advantage of “specificity” that enables SHERLOCKv2 to detect multiple sequences at the same time. Zhang Feng’s team also developed a test strip detection method similar to a pregnancy test stick. With only one test strip, SHERLOCKv2 can display the test results of viral infection, which makes detection more convenient. In the development of COVID-19 detection technology this year, SHERLOCKv2 also showed its prowess.

Although DETECTR and SHERLOCK have shown their powerful diagnostic capabilities, researchers still need to do a lot of work to ensure the accuracy of diagnosis before they can be used clinically. We believe that these new diagnostic tools will rewrite future diagnostic technologies, especially in developing countries with relatively poor sanitary conditions and high incidence of viruses, which will provide great help in diagnosing viral infections.

References

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