Han Chunyu's genetic scissors have been resurrected?
April 30, 2019 Source: China News Weekly
Window._bd_share_config={ "common":{ "bdSnsKey":{ },"bdText":"","bdMini":"2","bdMiniList":false,"bdPic":"","bdStyle":" 0","bdSize":"16"},"share":{ }};with(document)0[(getElementsByTagName('head')[0]||body).appendChild(createElement('script')) .src='http://bdimg.share.baidu.com/static/api/js/share.js?v=89860593.js?cdnversion='+~(-new Date()/36e5)];Worldwide, there is still new progress in the exploration of NgAgo as a “genetic scissorsâ€.
Han Chunyu’s “genetic scissors†is resurrected?
On April 4th, bioRxiv, a website operated by the Cold Spring Harbor Laboratory in the United States, published a paper saying that researchers at Purdue University discovered that a protein called NgAgo can cleave DNA (deoxyribonucleic acid) and improve the gene expression in E. coli. The efficiency of source reorganization. bioRxiv is a free pre-printed literature library open to biologists. That is to say, the articles published above are originals of authors who have not yet received peer review.
In the process of gene editing, homologous recombination is regarded as a common repairing method for cells. The behavior of gene scissors to disconnect DNA strands will stimulate the occurrence of homologous recombination. If an exogenous donor is provided, the DNA sequence can be exchanged. . Three months ago, the research team of the State Key Laboratory of Agricultural Microbiology at Huazhong Agricultural University also published a paper in the international journal "Nucleic Acid Research". It is also said that NgAgo can promote homologous recombination of genes in several bacteria, which can be regarded as potential gene editing. tool.
NgAgo, just over two years ago, let Han Chunyu, an associate professor at Hebei University of Science and Technology, get involved in the whirlpool of great public opinion. Now, with the publication of two new papers, NgAgo, which was first discovered and named by Han Chunyu, can be used as a new scissors for gene editing, and has once again become the focus of attention.
Said NgAgo can't get around Han Chunyu
On May 2, 2016, Nature's magazine, Nature and Biotechnology, published an article entitled "DNA-guided NgAgo protein can be used as a gene editing tool" by Han Chunyu, Hebei University of Science and Technology. This is the first time NgAgo has been proposed. According to the article, NgAgo can effectively cut the target gene under the guidance of the guide DNA, and can be used for gene editing of human cells.
After the publication of this article, under the impetus of various external factors, it immediately caused a sensational effect in the country, and some domestic experts and the media called it the discovery of “Nobel Prizeâ€. At the same time, NgAgo is regarded as a new gene editing technology, comparable to the world's most widely used gene editing tool CRISPR/Cas9.
Gene editing refers to the artificial transformation of DNA fragments by humans, including insertion and knockout of genes, and changes in their sequences. Gene editing is similar to editing a document on a computer, except that it operates on the ultimate password, DNA, which directly affects the traits of the organism. Generally, gene editing is divided into two steps of cutting and repairing. Therefore, in order to carry out genetic editing, we must first have a good "scissors".
At present, the most convenient genetic scissors used by scientists are CRISPR/Cas9. The developers are Zhang Feng, a Chinese scientist at the Massachusetts Institute of Technology, and Jennifer Dudna, a professor at the University of California at Berkeley. In fact, CRISPR/Cas9 was originally a defense system against bacteria against foreign enemies: when a virus or foreign DNA first invades, CRISPR/Cas9 cuts a piece of DNA (ribonucleic acid) with an invader identity and records it. In the case, when the foreign enemy re-offends, the CRISPR/Cas9 will transcribe a DNA that plays a guiding role according to the previously recorded DNA information, guiding itself to accurately identify and accurately strike the enemy. It is such a system that is used by scientists as a tool for gene editing.
However, CRISPR/Cas9 also has the disadvantage that its clipping point must be located upstream of a PAM sequence at a specific location, which means that the working range of CRISPR/Cas9 is not without dead angle and full coverage. The RNA that guides its shearing is also easy to transform, which also affects the accuracy of shearing. Therefore, the development of more reliable and accurate new scissors has become the goal of scientists. In this context, a family of proteins called Argonaute (Ago) has begun to appear.
Similar to CRISPR/Cas9, Ago protein is also the defense system of the organism. Initially, it was discovered that Ago proteins in eukaryotes can degrade foreign RNA, and it has been found that Ago proteins derived from prokaryotes can destroy invading DNA. Morphologically, the difference between prokaryotic and eukaryotic organisms is that there is no nuclear membrane in the nucleus. Prokaryote refers to the original single-celled organism surrounded by a nuclear-free membrane, and eukaryotes cover all animals and plants including humans. Waiting for life.
In 2014, Professor van der Oster of Wageningen University in the Netherlands discovered in the Ago protein family of prokaryotes a protein called TtAgo that binds to DNA with a guide, at 65 ° C, for cutting purposes. DNA. But obviously, high temperatures limit its potential to play a bigger role.
The industry once believed that Han Chunyu's research solved this drawback in a breakthrough way. By comparison screening, he found another Ago protein derived from Alcaligenes grisea and named it NgAgo. It was confirmed by experiments that this protein can be combined with the guide DNA at 37 ° C at room temperature to achieve gene editing. This means that NgAgo will have broad application prospects in the fields of human disease treatment and bio-pharmaceuticals. Compared with CRISPR/Cas9, NgAgo has a series of advantages that are not dependent on PAM sequences, DNA-guided failure and off-target, low fault tolerance, and high efficiency.
On May 8, 2016, the new media platform "intellectuals" reported on Han Chunyu's research and his work with "Han Chunyu: "The Chinese scientists invented world-class new technologies". "Intellectuals" added an editorial note in front of this article entitled "Special Feature", which wrote: "What excites the Chinese people is that Han Chunyu is in poor condition, lack of funds, and few people in Hebei University of Science and Technology. The research done in the case is better than the world-class MIT, Harvard, and Stanford. The editor of "Intellectuals" and the professor of Peking University Rao Yi introduced Han Chunyu to the editorial department. Here we specially published an exclusive interview. reader."
Since then, the domestic media has swarmed up and reported on the research results of Han Chunyu. Han Chunyu has been named as the vice chairman of the Hebei Provincial Association for Science and Technology, the most beautiful teacher in Hebei Province, the national “Young and middle-aged science and technology innovation leader†candidate, the “Changjiang Scholars Award Program†candidate, and the 2016 college and university special government allowance. In August 2016, the Hebei Provincial Development and Reform Commission also approved and agreed in principle to the project construction project of the Genetics and Technology Research Center of Hebei University of Science and Technology, which estimated a total of 224 million yuan.
Questioning and praise are almost at the same time. Less than 20 days after the publication of his paper, Yan Ning, a professor of molecular biology at Princeton University and a structural biologist at Tsinghua University, first proposed the reproducibility of Han Chunyu's papers on Weibo.
Since then, similar questions have appeared in forums such as Unnamed Space, Knowing, and Baidu Post Bar. By September 2016, Rao Yi and the Academician of the Chinese Academy of Sciences, Shao Feng, jointly sent a letter to the president of Hebei University of Science and Technology, saying that “it should be treated with caution and its research results†and suggested the establishment of an expert committee to verify the results of Han Chunyu’s research. In October of that year, Wei Wensheng, a professor of Peking University Health Science Institute, and other 13 domestic biologists jointly voiced in the media, saying that the experiment results could not be repeated. He hoped that Han Chunyu would disclose all the original data and called on schools and relevant parties to intervene in the investigation.
In November 2016, the editorial department of Nature·Biotechnology said that the paper was being investigated. Later, Han Chunyu added relevant experimental data to the editorial department of Nature·Biotechnology.
In January 2017, the State Intellectual Property Office withdrew the patent for the gene editing technology of the Han Chunyu team with Ago as the core. According to reports, at present, the team resubmitted the application in the form of international patents and is in a public state. In August of the same year, Han Chunyu team withdrew the paper because the experimental results were not repeated. The editorial board of Nature·Biotechnology published an editorial entitled “The Time When the Data Speaks.†We are now convinced that Han Chunyu’s decision to retract is the best way to maintain the integrity of published scientific records.
After that, the Han Chunyu team continued to search for reasons why the results of the paper could not be repeated. After a lapse of one year, Hebei University of Science and Technology issued a document saying that Han Chunyu’s team did not have subjective fraud. However, in accordance with the relevant regulations, the honorary title obtained by Han Chunyu was cancelled, the research project undertaken by the Han Chunyu team was terminated and the research funding was withdrawn. Han Chunyu responded that there are flaws in the experimental design and there are some rigorous problems in the research process.
Two new discoveries and the future of genetic scissors
Although the "Han Chunyu incident" came to an end, whether NgAgo first proposed by him can be used as a gene editing tool is still inconclusive.
As early as November 2016, Liu Dong, an associate professor of the Key Laboratory of Neuroregeneration of Nantong University, and Wang Yongming, a researcher at the School of Life Sciences of Fudan University, published the second article on NgAgo in the journal Nature of the journal Nature. The article said that NgAgo can "knock down" the zebrafish gene, causing defects in its eye development. However, since "knocking down" only reduces the gene expression and does not change the gene sequence, it is not possible to talk about "editing." Because of the different research objects, Liu Dong and others said that their research also "cannot confirm or falsify the conclusion of the former Han Chunyu."
In contrast, two recent new studies on NgAgo seem to give people more hope.
In the study of Purdue University, NgAgo was first confirmed to have the ability to cleave DNA in vitro. Further, the researchers found that two sites on the NgAgo called PIWI and RepA can cut DNA independently. However, unlike Han Chunyu's conclusion, in the in vitro experiment conducted by Purdue University, NgAgo was not ligated with the guide DNA paired with it. Kevin Solomon, assistant professor of agriculture and bioengineering at Purdue University, who was in charge of the study, speculated in an interview with China News Weekly that it is possible that NgAgo has previously combined other fragmented DNA fragments generated by DNA during replication. Act as a guide.
In vivo experiments with E. coli, NgAgo has been shown to bind to guide DNA and rely on its ability to cleave to increase the efficiency of E. coli homologous recombination. In January of this year, the research of Huazhong Agricultural University also proved that NgAgo can improve the efficiency of gene homologous recombination in Pasteurella and E. coli. However, unlike the research of Purdue University and Han Chunyu, the author of the paper, Zhang Anding and Jin Meilin, professors of the State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, observed that NgAgo promotes homologous recombination through NgAgo. The interaction of the PIWI site with an enzyme that promotes homologous recombination, RecA. "Of course, we do not rule out that both principles are working." Zhang Anding said in an interview with China News Weekly.
However, it should be noted that whether it is Huazhong Agricultural University or Purdue University research, the focus is now on the cutting role of NgAgo in prokaryotes. "We didn't intend to repeat the research conclusions in Han Chunyu's paper, that is, genetic editing in mammalian cells." Solomon said, "It is too early to say whether NgAgo can be applied to human cell gene editing. What we can say is This is another tool that can improve gene editing in bacteria, and can play a role in biofuel production and pharmaceuticals."
In fact, at the beginning of the publication of Han Chunyu's papers, China once again set off a trend of follow-up research. According to an industry insider who did not want to be named, six or seven domestic research institutes with strong capabilities in this field have been interested in NgAgo. However, after Han Chunyu’s papers were enthusiastically pursued and strongly questioned, these researchers gradually withdrew and basically no longer involved in the field of research. In contrast, research on NgAgo and even a wider range of Ago protein families continues to progress worldwide.
In February, Alexei Alawin, a professor of bioengineering at the California Institute of Technology, published a paper on bioRxiv that CbAgo from Clostridium butyricum is highly active and can be used to cleave single-stranded and double-stranded DNA at moderate temperatures. . Alawin further stated that CbAgo may be active in eukaryotic cells at 37 °C. Van der Oster, a professor at Wageningen University in the Netherlands mentioned above, recently published similar research results in bioRxiv. However, he replied to China Newsweek, "So far, we have not used CbAgo to edit human genomes, and if applied, more optimization is needed."
A long-term researcher of the Ago protein family at a leading research institute in China said that Han Chunyu's research was only one of many scientific research achievements in the field. Even if the conclusion of the article is established, there is no particular concern. The reason why his research caused a sensational effect was mainly the hype of the media. In her opinion, at the moment, Ago protein is not suitable for use as a gene editing tool. “It’s like taking a very blunt knife to cut meat.†“But it’s not ruled out what kind of way to think about protein in the future. A very large transformation, or two or three proteins work together, researchers should think of some more useful methods. Once the basic research has made a breakthrough, the application breakthrough will soon follow. So where is the breakthrough? I think still I have to give everyone more time."
The aforementioned industry insiders, who are unwilling to be named, said that from the current research results at home and abroad, NgAgo has its scientific research value. "There are many effects that are worthy of our study." He believes that compared with foreign continued follow-up, if Han Chunyu is no longer concerned about this research because he was "killed" in the past, he will be in a backward and passive position in this field.
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