Gene Magic Scissors: A new tool to rewrite the code of life

  On October 7, 2020, the 2020 Nobel Prize in Chemistry was awarded to E. Charpentier, a biochemist at the Max Planck Laboratory of Pathology in Germany, and Dudner, a biochemist at the University of California, Berkeley, USA (JA Doudna), in recognition of the two for “developing a genome editing method.” They are the 6th and 7th women to win the Nobel Prize in Chemistry.
  Born in France in 1968, Chapentier received his doctorate from the Pasteur Institute in Paris in 1995. In the past 20 years, he has worked in 9 different universities in 5 different countries and has won 10 prestigious scientific awards. He is currently Germany Director of the Max Planck Institute for Etiology. Dudner was born in the United States in 1964. He graduated from Harvard Medical School with a PhD in 1989. He won the World Outstanding Female Scientist Achievement Award in 2016. He is currently a professor at the University of California at Berkeley and a researcher at the Howard Hughes Medical Institute.
  When bacteria and viruses invade the body, the body’s immune system will fight back. Similarly, in the long process of evolution, bacteria have also formed a set of “immune system” to defend against virus invasion-CRISPR/Cas system. CRISPR is clustered regularly interspaced short palindromic repeats (clustered regularly interspaced short palindromic repeats). The abbreviation for CRISPR-associated gene. Cas is the abbreviation for CRISPR-associated gene. After the virus invades the bacteria, it integrates its own genes into the bacterial genome, and uses bacterial cells to replicate its own genes. The CRISPR/Cas system of the bacteria can recognize the virus’s genes and remove them from its own genome.
  The CRISPR/Cas gene editing technology uses a guide RNA equivalent to GPS to accurately locate the target sequence of the genome, directing the “God’s scalpel” endonuclease Cas9 to the specific target sequence, and the genomic DNA is converted by the endonuclease Cas9 Cut the gap, and then use the body’s own DNA double-strand break repair mechanism to generate insertion (or) deletion mutations to achieve gene knockout, or use foreign template sequences for homologous recombination repair.
  Chapentier discovered a previously unknown molecule-tracrRNA, which is part of the ancient bacterial immune system CRISPR/Cas, which can deal with virus invasion by cleaving virus DNA. In cooperation with Dudner, Chapentier succeeded in recreating bacterial gene scissors, simplifying the molecular composition of the scissors, and reprogramming the gene scissors so that they can be controlled and cut DNA molecules at a predetermined position.
  The CRISPR/Cas9 technology can be used to modify the DNA of animals, plants and microorganisms with extreme precision, and it has had a revolutionary impact on life sciences. For example, building animal disease models provides new ideas for cancer treatment, making the dream of curing hereditary diseases a reality, and even providing the possibility of creating new species.
  CRISPR course of study
  is generally believed that CRISPR system was first discovered in 1987. In the process of analyzing genes related to phosphate metabolism in the E. coli genome, the research group of A. Nakata in Japan accidentally discovered that there is a length of Short sequences of 29 base pairs will appear repeatedly. At that time, they did not pay much attention to the characteristic sequence itself. Even the paper only mentioned this sequence, but did not name it [1].
  The CRISPR system was actually used as a research object in 1993. Mojica (F. Mojica) discovered during his PhD study at the University of Alicante in Spain: Haloferax mediterranei, an extremely salt-tolerant archaeal found in the port of Santa Pola, Spain, its limitations Endonuclease will be affected by the salt concentration in the medium and cut its own genome in different ways. When studying this phenomenon, Mojca discovered a sequence of approximately 30 bases that is different from the known sequence in microorganisms, which is a repeating approximate palindrome. The fixed sequence is separated by approximately 36 bases. . After that, Mojca successively discovered that Haloferax volcanii, which is closely related to the Mediterranean halophile, and Haloferax volcanii, which are closely related to the halophilic archaea, also have similar conserved sequences. Soon, Mojca realized the connection between this sequence and the characteristic sequence of bacteria in the literature published by the Nakata research group in 1987. He believed that such a sequence exists in different kinds of microorganisms, and they play a role. Important role, and finally named it short regularly spaced repeats (short regularly spaced repeats). By 2000, Mojca had discovered this repetitive sequence in 20 microorganisms. In the next two years, researchers quickly expanded the study of this locus to specific genes related to this sequence. In 2002, R. Jansen’s laboratory published a paper, naming this sequence CRISPR for the first time, and naming the specific gene related to it as the Cas gene.
  However, the function of this particular sequence has not been clarified for a long time. In 2003, as a new leader in the CRISPR field, Mojza began to focus on the spacer that separates the repeating sequence. Because the spacer sequence has the characteristics of intraspecies conservation, Moitsa performed a large number of comparisons of different spacer sequences, and finally found that the sequence of a spacer was consistent with a certain segment of the genome of the P1 phage infected with E. coli. After that, he found through a series of comparisons that the spacer sequence in CRISPR came from an exogenous phage or plasmid. It is worth noting that A. Bolotin first proposed that the CRISPR locus would inhibit the expression of phage genes through antisense RNA. Although this hypothesis was later proved to be wrong, it was the first to give CRISPR its immunological aspects. Meaning.
  The role of CRISPR in the bacterial immune system was revealed by P. Horvath. In overcoming the phage infection that plagues industrial production, Horvath is committed to developing DNA-based strain identification technology and using CRISPR to genotype strains. In 2004, he also noticed the link between spacers and the resistance of bacteria to phage. In 2005, Horvath and colleagues tried to prove that CRISPR is a bacterial adaptive immune system. In 2007, Horvath described a Streptococcus thermophilus (Streptococcus thermophilus) after being invaded by a phage, the streptococcus genome will integrate a spacer sequence from the phage genome. When the streptococcus is invaded by the same phage again, Streptococcus can develop resistance, and if this spacer sequence is changed, the resistance of the streptococcus will be affected. At the same time, it was also discovered that the Streptococcus needed the participation of the Cas7 protein to obtain this resistance (but the Cas7 protein was not required to maintain resistance), and the Cas9 protein-whose sequence contains two types of nuclease domains (HNH and RuvC)-and The product may cleave nucleic acid, which is necessary to resist bacteriophages. Therefore, the Cas9 protein is considered to be the active component of the bacterial immune system.

  Scientists soon began to further study the mechanism of CRISPR/Cas, the first key research came from the team of J. van der Oost. By inserting an E. coli CRISPR system into another E. coli strain that lacks an endogenous CRISPR system, they biochemically verified that the system is a complex composed of 5 Cas proteins, and named it Grade Joint complex (cascade). Then by knocking out each component of the cascade complex in turn, it is proved that the cascade complex is necessary to cut the precursor CRISPR RNA (crRNA) transcribed from the CRISPR locus, and the final cleavage product has 61 bases. crRNA, which consists of the last 8 bases of the previous repeat sequence, the complete spacer sequence and the beginning of the next repeat sequence, can guide the Cas protein to the target DNA. Subsequently, Van der Ooster constructed two CRISPR sequences in the antisense direction (complementary to the sequence of mRNA and coding strand DNA) and cissense direction (complementary only to the other DNA) so that they could be targeted to the essential genes of λ phage. . Their results suggest that CRISPR does not act on RNA, but on DNA.
  In the same year, L. Marraffini and E. Sontheimer confirmed that the target of CRISPR is DNA. Two years later, in 2010, S. Moineau et al. discovered that CRISPR/Cas9 cuts 3 base pairs upstream of the protospacer adjacent motif (PAM) of the target DNA. A double-strand break (DSB) is produced.
  At the same time, Charpentier is focusing on studying how the pathogen Streptococcus pyogenes achieves gene regulation. In 2011, Chapentier et al. conducted a localization study on a large number of small RNAs in Streptococcus and found that its base sequence was very close to the revealed CRISPR locus. A series of studies confirmed that an unknown RNA molecule can achieve the function of the CRISPR system. It has an important role, so it is named trans-activated CRISPR RNA, referred to as tracrRNA (trans-activating crRNA). TracrRNA guides Cas9 to the target by forming a duplex with crRNA.
  Since he had never been in contact with the CRISPR system before, Chapentier met Dr. Dudner from the University of California, Berkeley at a conference held in Puerto Rico. Chapentier invited Dudner to conduct collaborative research. Dana readily accepted. Dudner’s colleagues made a plan for cooperation through a digital conference. Previously, they speculated that Cas9 in bacteria would eventually perform the role of cutting DNA, but it has not been verified in vitro.
  In 2012, they combined the recombinantly expressed Cas9 with crRNA and tracrRNA transcribed in vitro, and successfully constructed a CRISPR/Cas system that can cut and purified DNA in vitro: the mature crRNA and tracrRNA form a double-stranded RNA structure to guide Cas9 protein targeting Combining the target DNA fragments, causing DNA double-strand breaks by cutting, thus making it possible for the CRISPR/Cas system to edit genes [2].
  Inspired by the work of Chapentier and Dudner, the Harvard University G. Church research group and Zhang Feng research group took the lead in realizing gene editing in mammals [3, 4]. Since then, in just a few years, this technology has become popular in laboratories around the world and has become the most popular gene editing tool in the biological world.
  In 2013, many laboratories around the world have used CRISPR/Cas9, which has been used in human cell lines and a variety of model organisms, such as zebrafish, yeast, nematodes, fruit flies, mice, rats, etc. Editing of the target gene [5]. In 2015, CRISPR technology was named the best scientific breakthrough of the year by the US “Science” weekly magazine.
  In 2016, Zhang Feng’s group took the lead in using an enzyme called C2c2 (also called Cas13a) in the CRISPR system and successfully edited RNA. In October of the same year, another scientist used CRISPR/Cas9 edited cells to conduct human clinical trials for the first time. Immune cells isolated from the blood of patients with metastatic non-small cell lung cancer were specifically knocked out by CRISPR/Cas9 technology. The PD-1 gene is then amplified and cultured in vitro, and then returned to the patient’s body, in order to achieve the goal of curing cancer.
  In 2017, the research team at the University of Miami in the United States designed the CRISPR-RNA-targeting (CRISPR-RNA-targeting, CRISPR-RT) system, which has made a great contribution to RNA editing. In addition, in addition to Cas13a, the discovery of Cas13b and Cas13d has also promoted the application and development of the CRISPR system. In the same year, Liu Ruqian’s team directed evolution of the protein of tRNA adenine deaminase and fused it with the CRISPR/Cas9 system, which realized the conversion from AT to GC without causing DNA strand breaks, and its editing efficiency in human cells More than 50%. It provides an effective tool for the treatment of a variety of single-base mutation genetic diseases [6].
  New tools for gene editing based on the CRISPR/Cas9 system are emerging in an endless stream. In 2018, Liu Ruqian’s team evolved a Cas9 variant that can recognize multiple PAM sequences, improving the editing efficiency of CRISPR/Cas9. In the same year, Dudner released the CRISPR/Cas14 system. In 2019, Dudna successfully modified the activity of Cas9 protein to make it active only in specific tissues. Zhang Feng also developed the CRISPR/Cas12b system, which greatly enriches the CRISPR/Cas system for genome editing.
  At the beginning of 2020, when a new type of coronavirus pneumonia broke out, scientists developed a kit for detecting and quantifying SARSCoV-2 viral RNA based on CRISPR/Cas13a technology. It does not require reverse transcription of RNA, but directly detects viral RNA. As Thierry Peng Sha said: “CRISPR / Cas9 system has exceeded the boundaries of the genetic engineering techniques to more common, effective and easy to operate its application seems to be really no limit [7]..”
  CRISPR works
  in After foreign DNA such as phage or plasmid invades the host, the foreign nucleic acid is processed into short fragments and integrated into the CRISPR repeat spacer sequence in the host chromosome as a new spacer. This process is equivalent to an “immune memory” of the host. . There is an extremely conserved motif (PAM) adjacent to the downstream of the spacer sequence on the exogenous nucleic acid, which is very important for the selection and processing of the spacer sequence. When the invader invades again, the transcription product of the CRISPR sequence is processed into mature crRNA by endonuclease. The 5’end of crRNA contains a spacer sequence integrated into the host genome, which can be complementary to the foreign sequence, while the 3’end contains a CRISPR repeat sequence that combines with tracrRNA to form a double-stranded structure. The crRNA-tracrRNA with Cas endonuclease assembles into a ribonucleoprotein complex, which is complementary to the invading nucleic acid through the spacer sequence to perform specific destruction, thereby realizing the “secondary immune response” of the host (bacteria)

  According to the different CRISPR locus, the CRISPR system can be divided into 6 types (I-VI), which have different crRNA patterns and specific Cas proteins [9]. Different from the type I and type III systems that use complex multi-Cas protein complexes for crRNA binding and target sequence degradation, the type II CRISPR system uses a single endonuclease Cas9 to identify the target site of double-stranded DNA. The two domains of Cas9 (HNH and RuvC) cut two DNA strands respectively. These two different domains splice double-stranded DNA 3 base pairs upstream of PAM. The HNH domain is responsible for cutting the DNA strand complementary to crRNA, and the RuvC domain is responsible for cutting the other DNA strand. In the meantime, tracrRNA and crRNA repetitive sequence base complementary pairing to form a unique double-stranded RNA structure. About 20 bases in this double-stranded RNA molecule are targeted to a DNA sequence complementary to its sequence, and the DNA site-specific cleavage is performed by Cas9, resulting in a blunt-ended double-strand break gap. Then, it is repaired through the body’s own DNA repair mechanism. One is the highly error-prone nonhomologous end joining (NHEJ), which generates short random insertions and/or deletions at the cutting site; the other is through high-fidelity homologous recombination repair (homology directed repair, HDR), under the guidance of the homologous repair template, the DSB site is accurately repaired. Later, by artificially combining crRNA and tracrRNA to form a single RNA for transcription and expression, a chimeric single-stranded guide RNA (singleguide RNA, sgRNA) was obtained, which greatly simplified the CRISPR gene editing method.
  Advantages of CRISPR
  Gene editing technology is essentially an application of DNA double-strand break damage and repair mechanisms. External factors such as radiation and internal factors such as cellular metabolites can cause different degrees of damage to DNA. DNA double-strand breaks are a common type of DNA damage in eukaryotic cells. After DNA double-strand breaks, the body will pass through two types of damage. Repair mechanism for timely repair: non-homologous end repair and homologous recombination repair [10]. So far, gene editing technology has undergone three generations of changes, from zinc finger nucleases (ZFNs) technology, transcription activator like effector nucleases (TALENs) technology to CRISPR/Cas technology [11].
  ZFNs are fusion proteins composed of zinc finger protein and bacterial FokI restriction enzyme. Zinc finger proteins include tandem zinc finger domains, which belong to DNA binding domains. Each zinc finger domain recognizes 3 bases, and the tandem zinc finger domain allows ZFNs to potentially bind to long nucleotide sequences. When used, it needs to be designed in pairs, one cutting site is on the flanking sense strand, and the other cutting site is on the flanking antisense strand. When the ZFNs on both sides of the cutting site are combined, the FokI restriction enzyme dimerizes and cuts at the site, causing a double-strand break with 5’overhangs. ZFNs technology makes DSB do not have to rely on natural occurrence, thus laying the foundation for the development of genetic engineering.
  The structure of TALENs is similar to ZFNs, consisting of a tandem transcription activator-like effector (TALE) DNA binding domain and FokI restriction enzyme. Unlike ZFNs, each TALE protein recognizes a single specific nucleotide, so its structure is more complex, but its design is simpler. However, ZFNs and TALENs technologies are difficult to operate, long in construction and assembly, and are easy to accumulate in cells and cause cytotoxicity. Animal experiments have shown that they can trigger the body’s immune response.
  The CRISPR/Cas system is composed of a guide RNA and Cas nuclease. The guide RNA binds to the target gene and guides the Cas protein to cut the target sequence to produce a double-strand break. Different Cas proteins are selected according to different experimental purposes. By modifying the sequence of the guide RNA, any selected target sequence can be cleaved. Similar to the ZFNs and TALENs systems, the CRISPR/Cas system can be used for non-homologous end joining repair methods to introduce random mutations at the DNA cleavage site, or by co-injecting engineered DNA vectors that are homologous to the DNA. Recombinant repair methods introduce specific mutations or insertions. The CRISPR/Cas system makes site-specific modification of genes more efficient.
  Compared with ZFNs and TALENs, the CRISPR/Cas system has many advantages: ①Simple design. Unlike ZFNs and TALENs, the CRISPR-Cas system relies on the recognition between protein and target DNA. Instead, ribonucleotide complexes are formed between guide RNA and target DNA, and ribonucleotides are used to identify targeted genes. ②High efficiency and low cost. For example, when creating a mutant mouse, you only need to inject the RNA encoding Cas protein and guide RNA directly into the mouse embryo. Compared with the method of transfection and selecting mouse embryonic stem cells to inject the mouse and then perform homologous recombination, it can save money. Months or even years. ③Multi-gene mutation operation can be performed at the same time. Cas9 protein monomers can be combined with any number of specific guide RNAs with different sequences to easily realize multiple genome editing with CRISPR/Cas9 libraries.
  Application and Prospect of CRISPR
  in less than 10 years time, CRISPR been an unprecedented rapid development of various types of CRISPR-based technology has been widely used in many aspects of scientific research, health care and agriculture. Researchers can precisely edit the mutated genes that cause the disease to cure the previously helpless disease from the root cause. For example, the CRISPR system is delivered to specific cells through a special virus to repair disease-causing mutant genes. This has been successfully verified in sickle-type anemia, Duchenne muscular dystrophy and other diseases, and seeks clinical trials [12 ]. In agricultural genetic breeding, CRISPR technology can make more convenient and precise modification of crop genomes to obtain better genetic traits, such as enhancing crop resistance, improving the quality of agricultural products, and increasing yield.
  Although CRISPR has shown great potential in gene editing, it also has risks that cannot be ignored. Since the advent of CRISPR technology, the discussion about its off-target (that is, the risk of cutting non-target sequences) has never stopped. Although it is constantly optimized to reduce the off-target rate, the problem has not been fundamentally solved so far. Modifying other genes may cause a series of unimaginable serious consequences, which is a major obstacle to the application of CRISPR.
  The ethical issues caused by gene editing need to be further discussed and resolved in the medical field, as well as the ethical issues of genetic modification, such as strengthening certain physiological functions of people, and even creating a “customized person”. We think and treat it seriously. Today’s application on human embryos should be strictly controlled. When paying attention to the benefits it brings, everyone must also consider the risks it brings.

  It is the common responsibility of mankind to manage and control risks, avoid violating ethics and morals, and apply them to life reasonably. We must always remain vigilant and cautious on risks and ethical issues, and look forward to CRISPR bringing more unexpected surprises to mankind in the future.