Gene scissors and gene editing

The 2020 Nobel Prize in Chemistry was awarded to Jennifer Doudna, a professor at the University of California, Berkeley, and Emmanuel Carpentier, a French professor working at the Max Planck Institute for Infection Biology in Germany (Emmanuelle Charpentier) in recognition of their invention of genetic modification method CRISPR-Cas9.

Principles of gene scissors and gene editing
The full name of CRISPR is a bit confusing, it is “Clustered regularly interspaced short palindromic repeats” (Clustered regularly interspaced short palindromic repeats). Cas is the abbreviation of Caspase, and its full name is cysteine-containing aspartate proteolytic enzyme. This is a group of proteases, Cas9 is one of them. In addition, there are other enzymes that function with CRISPR, such as the nuclease protein Cpf1, so it can also be called the CRISPR/Cas system, including CRISPR-Cas9, CRISPR-Cpf1, CRISPR-C2c1, and CRISPR-C2c2, etc., all of which are Gene scissors, but the most widely used is CRISPR-Cas9.

CRISPR-Cas9 is currently a more accurate and fast cutting gene scissors. With this scissors, the DNA of animals, plants and microorganisms can be edited and processed (cutting, deleting, shifting, adding, etc.) in a targeted manner. , So as to treat diseases, especially genetic diseases, and obtain crops and biological products that people want.

Researchers have long discovered that CRISPR is the same as many phage DNA sequences that can invade bacteria. After these sequences of phage DNA are transcribed into RNA, they become targeted RNA (gRNA), which can form a complex with the Cas protein produced by bacteria. And guide the Cas protein. When the complex detects that the invading DNA is consistent with the target RNA sequence, the Cas protein can cut the invading DNA, so that the bacteria can protect themselves against the invasion of phage. It can be said that this is a natural immune defense mechanism of organisms.

When CRISPR and Cas9 are combined to form the CRISPRCas9 system, it becomes a targeted and precisely controlled gene scissors that can cut a certain gene more accurately.

Dudna and Carpentier won this year’s Nobel Prize in Chemistry for proving the accuracy and effectiveness of CRISPR-Cas9 gene scissors. Carpentier first discovered a previously unknown molecule-tracrRNA in the study of Streptococcus pyogenes, which causes great harm to humans. Studies have shown that tracrRNA is part of CRISPR/Cas, the ancient immune system of bacteria, which disarms phage DNA by cutting it to resist the invasion of phage DNA. In 2011, Carpentier published the results of her research.

In the same year, Carpentier collaborated with Dudner, a senior biochemist with extensive RNA knowledge, and achieved breakthrough results. In their paper published in June 2012, they stated that their team purified the Cas9 protein and discovered that it is a dual-RNA-guided DNA endonuclease, and for the first time demonstrated in vitro (in test tube) that the CRISPR system using Cas9 can cut any DNA strand , Which shows that CRISPR has the ability to modify genes in living cells. At the same time, they simplified the molecular components of gene scissors and created CRISPR-Cas9 gene scissors, so they are easier to use. They were the first to transform the bacterial natural immune system into CRISPR-Cas9 gene scissors, and enable them to cut genes at will. Later, Carpentier and Dudner used CRISPR-Cas9 gene scissors to successfully edit the genes of E. coli. The results showed that CRISPR-Cas9 was more effective and accurate than other gene scissors before.

Discovery history and controversy
CRISPR is regarded as a tool and achievement that can change the entire biological field, and has great application potential in disease treatment and agricultural production. However, the discoveries and inventions of CRISPR and CRISPR-Cas9 are not only the contributions of the two winners, but the results of continuous research and mutual confirmation by a large number of scientists.

The American “Cell” magazine has published an article reviewing the early development process of CRISPR technology, and at the same time, Eric Lander, director of the Broad Institute of Massachusetts Institute of Technology-Harvard University, also summarized the invention process of CRISPR gene editing technology. . Combining these two and other studies can briefly summarize the invention process of CRISPR and CRISPR-Cas9.

In 1987, the team of Japanese scientist Yoshizumi Ishino accidentally discovered a 29-nucleotide repetitive sequence at the 3 end of which is highly homologous when analyzing the E. coli iap gene and its surrounding sequence. They are separated by a sequence of 32 nucleotides. Open, thus unveiling the veil of CRISPR.

In 1993, Spanish scientist Francisco Moika discovered palindromic repeats (CRISPR) with a length of approximately 30 base pairs (bp) in the genome of halophilic archaea. These sequences are separated by 36 base pairs. The sequence is separated. In 2005, Moika discovered that two-thirds of the spacer sequence in the CRISPR sequence was the sequence of a virus or foreign plasmid, so it was determined that the CRISPR system was related to the acquired immunity of bacteria.

In 2005, French scientist Gillis Vignord and Russian scientist Alexander Bolotin also proved that CRISPR is related to the acquired immunity of bacteria.

In 2007, the French molecular biologist Philippe Horvat, who worked for DuPont, was studying the resistance of Lactobacillus producing yogurt to phage. They discovered the role of Cas7 and Cas9 proteins in CRISPR. Cas7 produces intervals and repetitions. Sequence, Cas9 is a nuclease.

In August 2008, Dutch scientist Van der Oost and others identified a series of Cas proteins and found that these proteins need to cut 61 base pairs of precursor RNA (crRNA) to be functional, thus clarifying the role of crRNA. Based on this, they artificially designed the corresponding crRNA sequence, so that the bacteria acquired the characteristics of resistance to bacteriophages. This is the first time that humans have edited the CRISPR system.

In December 2008, the team of Argentine scientist Luciano Maravoni confirmed that the substrate of the CRISPR system is DNA, and proposed that the system may be used for DNA editing and is an effective gene scissors.

In December 2010, Sylvain Moino of Laval University in Canada confirmed that Cas9 can cut DNA with a specific sequence under the guidance of crRNA.

In July 2011, the Lithuanian scientist Virginias Sxnis recombined the CRISPR system of Streptococcus thermophilus in E. coli, and proposed that CRISPR-Cas9 needs at least Cas9, crRNA and anti-virus to become a precise and effective gene scissors. Type activation of crRNA (tracrRNA).

In August 2012, Carpentier and Dudner published a paper revealing that CRISPR-Cas9 gene scissors can perform accurate gene editing. They simplified the CRISPR-Cas9 system by chimerizing tracrRNA and crRNA into a single-stranded guide (sgRNA). Only two components, sgRNA and Cas9 protein, can target specific sequences. So far, CRISPR-Cas9 is efficient and simple. Genome editing technology (gene scissors) came out.

In January 2013, MIT professor and Chinese scientist Zhang Feng and Harvard Medical School professor George Qiu Qi simultaneously published papers in the journal Science, proving that CRISPR-Cas9 can edit mammalian cell genes and successfully Edited the genes of mouse and human cells. A few weeks later, Dudner’s laboratory also published similar results.

Therefore, some people think that Zhang Feng may also win the Nobel Prize for discovering that CRISPR-Cas9 can be applied to human cells, and Zhang Feng, Dudner and others have also filed multiple patent lawsuits regarding the application of CRISPR-Cas9 technology in the past. Both sides have their own winners and losers. And Ryosumi Ishino might win the prize for his discovery of CRISPR first. In addition, Moika of Spain was the first to recognize the importance of CRISPR and made outstanding contributions to revealing its structure and function, and should also be awarded. However, in the view of the Nobel Prize Committee, in the process of discovering CRISPRCas9 gene scissors, only the achievements of Carpentier and Dudner were the most critical core contributions.

Practical application
As a more accurate and efficient gene scissors, CRISPR-Cas9 has a wide range of applications in various fields, such as biology, medicine, agriculture, and chemistry. This kind of gene scissors can be used to delete, add, activate or inhibit the target genes of other organisms, including genes in the cells of humans, mice, zebrafish, bacteria, fruit flies, yeast, nematodes and crops. Therefore, it is a kind of A very versatile biotechnology.

In terms of crops, people can use this gene scissors to produce crops that are resistant to mold, pests and drought, and many results have been achieved. In 2019, the Jaindara Tripas team of the Kenya International Institute of Tropical Agriculture used gene scissors CRISPR-Cas9 to cut the viral DNA in the genome of a large banana, so that it no longer causes stripe disease in bananas. Compared with large bananas that have not been genetically edited, 75% of edited large bananas have no symptoms of banana streak virus. Banana production and increase.

In terms of modifying animal genes, gene scissors have also shown important value. The research team of Nanjing Medical University State Key Laboratory of Reproductive Medicine and other institutions used CRISPRCas9 gene scissors to create and breed two targeted gene-editing monkeys for the first time in the world. The purpose of creating targeted gene-editing monkeys is to establish monkey disease models to simulate humans, test drugs and treat genetic diseases, thereby reducing the risk of drug research and humans as test subjects.

The researchers used CRISPR-Cas9 gene scissors to modify three genes, namely the gene Ppar-γ that regulates metabolism, the gene Rag1 that regulates immune function, and the gene that regulates stem cells and sex determination. The researchers targeted and edited these 3 genes simultaneously in more than 180 single-cell monkey embryos, and then sequenced the genomes of 15 embryos, and found that 8 embryos showed evidence of simultaneous mutations in two target genes. These two genes are the gene Ppar-γ which regulates metabolism and the gene Rag1 which regulates immune function. Since then, the researchers transferred the genetically modified embryos to the surrogate female monkeys, and one of the female monkeys gave birth to a pair of twin monkeys. After examining the genomes of the twin monkeys, it was found that there were indeed two mutated target genes in their DNA. Therefore, these two monkeys are called targeted gene editing monkeys.

In 2014, Zhang Pumin’s research team at Baylor College of Medicine in the United States also used CRISPR-Cas9 gene scissors to create a gene-edited pig capable of producing human albumin.

In early 2016, teams from multiple research institutions, including Zhang Feng, used CRISPR-Cas9 gene scissors to treat the genetic disease of Duchenne muscular dystrophy in animals. As a result, they improved their life expectancy and quality of life. This is obviously for treatment. Animal test on human Duchenne muscular dystrophy.

In addition, Qiu Qi’s team is also using CRISPR-Cas9 gene scissors to edit a large number of pig genes to remove harmful microorganisms in pig genes, so that they can provide organs for humans, thus solving the current situation of human organ transplantation being in short supply.

Ethics and restrictions
CRISPR-Cas9 gene scissors also have shortcomings and defects. The biggest shortcoming is that it may miss the target, that is, the gene scissors may cut the DNA in the wrong position, or deviate from the original design target, causing accidental damage. If it is used to treat human diseases, it may have unpredictable serious consequences.

In September 2018, “Nature Methods” published an article stating that in experiments on mice, CRISPR can indeed successfully correct the genes that cause blindness, thereby treating blindness in mice. However, there were more than 1500 single-nucleotide mutations and more than 100 deletions and insertions of larger gene fragments in the genomes of two mice that received gene scissors. This is the consequence of off-target, and it may endanger the life of the mouse.

There are currently more than 4,000 hereditary single-gene diseases that affect more than 1% of newborns worldwide. In theory, CRISPR-Cas9 gene scissors can be used to cut off disease-causing genes to prevent these diseases and allow every family to get healthy babies. This is obviously more advanced than the genetic testing of the fetus before birth.

However, in actual operation, CRISPR-Cas9 gene scissors have the risk of off-target, so there is a huge risk when used to treat human diseases. In 2015, Huang Jun of Sun Yat-sen University used gene scissors CRISPR-Cas9 to modify the β-globin gene that causes β-thalassemia (change the problematic guanine G into adenine A, thereby correcting the genetic root of thalassemia). Successful, but this research result was rejected by the British “Nature” and the American “Science” magazine. The reason is that this will cause unforeseen risks. Failure to impose strict restrictions on the study of human embryos may lead to unsafe and unethical use of this technology.

The actual situation also shows that the success rate of Huang Jun’s team using CRISPRCas9 gene scissors is not high, and the off-target is obvious. When they modified the genes that cause β-thalassemia in embryos, they tested 86 discarded embryonic cells. In the end, only 28 genes were successfully edited and modified, with a success rate of about 33%. This success rate is not enough to obtain the guarantee of safety and success rate, and it also makes people have doubts about this technology.

In 2018, the team of He Jiankui, an associate professor in the Department of Biology, Southern University of Science and Technology of China, used CRISPR-Cas9 gene scissors to edit the CCR5 gene related to AIDS immunity in embryonic cells, so that the baby has the ability to innately immune AIDS after birth. According to the research team, the first is to use monkey embryos and mice to carry out CRISPR-Cas9 editing experiments, and achieve the expected results, and found that the organization and behavior of the mice are not significantly different from those of normal mice. From March 2017 to November 2018, He Jiankui’s team recruited 8 couples of volunteers (male and female HIV antibody positive) to participate in the experiment. In the end, two volunteers became pregnant, and one of them gave birth to twin girls with the pseudonym “Lulu” and “Nana”.

This incident has caused great controversy. Due to the inevitable off-target consequences of CRISPRCas9 gene scissors, it may cause unexpected dangers to future generations. Therefore, this is a serious incident that violates medical ethics. On December 30, 2019, the gene-edited baby incident was publicly pronounced at the Shenzhen Nanshan District People’s Court in the first instance. He Jiankui was sentenced to 3 years in prison for illegal medical practice and fined 3 million yuan.

Although CRISPR-Cas9 gene scissors have great application prospects and have won the Nobel Prize, when applied to humans and animals, there are still huge risks and need to be supervised by human ethics and laws.