The development history of CRISPR/Cas9 technology: it set off a genetic revolution as soon as it appeared

Introduction

In the last issue, we mainly talked about heredity and genes. In this issue, we will mainly talk about gene editing technology, including its development history, classification, principles, and issues of concern, such as the application of gene editing technology.

In this issue, we invited Professor Chao Yanjie, a researcher at the Shanghai Pasteur Institute. During his doctoral studies, Professor Chao participated in the foundational experiments of the Nobel Prize-winning results, published more than 20 high-impact papers, and has been cited more than 6,000 times. He is an "academic rising star" in this field.

01 Development History of Gene Editing Technology

Ye Shuisong: Genes determine our traits . Many scientists have been thinking about how to fix genes when they go wrong . Professor Chao, can you share the development history of gene editing technology?

Chao Yanjie : Gene editing technology actually has a long history. Before the advent of CRISPR/Cas9, a lot of scientific research had been carried out in laboratories.

Before this, there have been many attempts and tool development, especially in lower organisms such as E. coli or yeast, but the efficiency is not high and is far from reaching the level of application in the human body.

The first generation were large zinc finger nucleases with very low efficiency. The second generation were transcription activator-like effector nucleases.

Before CRISPR, the most useful and efficient generation was the "zinc finger protein" gene editing tool, which uses proteins to recognize DNA or perform gene editing. Although the accuracy is high, the efficiency is still low. It was not until the invention of CRISPR/Cas9 nearly 10 years ago that the latest generation of gene editing technology was available.

Because of the discovery of CRISPR/Cas9, the efficiency of gene editing technology has been improved to a very high level, which can be truly applied to human research, mammalian research, and even clinical research and treatment.

Previously, gene editing technology had a long history and foundation. It was not until recent years that the discovery of CRISPR/Cas9 achieved new breakthroughs, making it a "star technology."

02 CRISPR/Cas9 Technology Principle

Ye Shuisong: After the CRISPR/Cas9 technology came out in 2012, many people were concerned about "why this technology would have such a big impact". Can you introduce the principles of this technology?

Chao Yanjie: The advent of CRISPR/Cas9 technology can actually be attributed to a paper published by Jennifer Doudna and Emmanuelle Charpentier in Science in 2012.

Emmanuelle Charpentier

Jennifer Doudna

The two female scientists demonstrated that gene editing could be performed in vitro or in E. coli, providing the scientific community with a completely new technology that subsequently attracted a lot of attention.

However, later, two scientists, Zhang Feng and George Church, published two papers in Science, using CRISPR/Cas9 technology to perform large-scale gene editing in human cells or mammalian cells for the first time. This is actually a real application of the technology, and it has attracted more attention.

Chinese scholar Zhang Feng

The reason why this technology has such a great impact is that it can achieve efficient and large-scale gene editing in human cells in a very short period of time (a few months or less than a year), which makes people see the huge potential and value of the application of gene editing technology.

The principle of this technology is the use of a new type of nuclease, the Cas9 protein.

This nuclease has a characteristic that it cannot cut randomly, but must rely on a nucleic acid as a guide. This guide is CRISPR or guide RNA. When this RNA and protein form a complex, it can guide the protein to accurately cut a specific site.

Only in this way can gene editing achieve high efficiency and accuracy, thus achieving potential clinical therapeutic applications. Therefore, it can be said that Doudna and Charpentier pioneered this field.

03 Advantages of CRISPR/Cas9 Technology

Ye Shuisong : Compared with other gene editing technologies, what are the advantages of CRISPR/Cas9 technology and what are its shortcomings?

Chao Yanjie: CRISPR/Cas9 has many advantages. It can achieve a "unified world" situation in the shortest possible time, mainly because it is simple, feasible and efficient. The guide of CRISPR/Cas9 uses a nucleic acid or RNA. This RNA has infinite possibilities and can be synthesized in vitro with very high synthesis efficiency and low cost.

It can synthesize tens of thousands of guides in a few days and can edit countless sites, any human gene, and any species' gene. The efficiency of this technology is unmatched by any other method.

At the same time, the time and economic costs of CRISPR/Cas9 are very low. Guide RNA can be synthesized in just a few days and editing can be completed in mammalian cells. The editing efficiency is also very high, and rough screening can be performed in a short time to obtain edited cells and products.

Another advantage is that CRISPR/Cas9 can edit many genes at the same time. Since guide RNA can be infinitely combined, multiple guide RNAs can be used to target multiple genes in a cell at the same time. This advantage is also unmatched by other methods.

There are advantages but also disadvantages. In clinical practice, the disadvantage of CRISPR/Cas9 is that the editing has a certain off-target effect, and the desired editing is not completed at 100% of the sites. This is a problem with all editing technologies.

Despite these shortcomings, technical developers are currently trying to improve this technology, and many scientists are constantly optimizing and developing this technology.

04 The creator of CRISPR/Cas9

Ye Shuisong : We know that you also collaborated with Nobel Prize winner Emmanuelle Charpentier at the time, and even had your name on several of her pioneering works. Can you share the stories of the pioneers of this technology? What was Charpentier doing before publishing the Science paper ? Did it receive as much attention as it does now?

Chao Yanjie: I am quite familiar with the Nobel Prize winner Emmanuelle Charpentier. We both belong to the smaller field of "prokaryotic RNA biology", and she started collaborating with our laboratory before she became successful.

In fact, the article that first discovered CRISPR/Cas9 was the one in which I co-authored.

Researcher Chao Yanjie is among them

Before she became famous with this article, she was a low-level research group leader who had a hard time leading her research group in Vienna.

At that time, one of her areas of interest was Streptococcus among pathogenic bacteria. She hoped to discover some new types of non-coding small RNA in Streptococcus.

Later, she started to cooperate with our laboratory, which has RS sequencing (single molecule real-time sequencing) technology and non-coding small RNA research foundation. Then, we found non-coding small RNA in her pathogenic bacteria species.

At that time, this basic research work was driven purely by interest. We never thought that this work could be published in Nature, or that it could produce a major world-changing research like CRISPR/Cas9.

At that time, we did our best to do experiments for this work and found a new type of non-coding small RNA in Streptococcus. Coincidentally, this small RNA is related to CRISPR and interacts with CRISPR/Cas9.

Because of this job, Charpentier's academic development became very smooth. She left the University of Vienna and went to Sweden to establish a new laboratory, and later returned to Germany to establish a larger laboratory. During this process, we communicated with each other.

Charpentier is still doing outstanding work in the field of RNA biology of pathogenic bacteria and has founded a new research unit at the Max Planck Institute in Berlin.

05 Major contributions of Chinese scholars

Ye Shuisong : So, an original innovative technology is often difficult before it is born. Interest is the best teacher. We know that Chinese scientists have made very important contributions in this field. For example, Zhang Feng has won important awards such as the Gairdner Award. Can you introduce his and several other Chinese scholars' main contributions in this field?

Chao Yanjie: In fact, our Chinese scientists have made very great contributions in this field, especially in the development and growth of domestic life sciences. In this field, our Chinese contributions have been reflected.

One of the representatives is Zhang Feng, who has been involved in the field of gene editing. Before CRISPR/Cas9 was discovered, he had already been engaged in gene editing in the United States, probably in the Church laboratory. He used zinc finger proteins for gene editing.

When CRISPR/Cas9 appeared, or even when he was writing the article, he heard about this tool and was very interested and shocked, thinking it was the direction of the future. Therefore, he may be one of the earliest scientists in the world to engage in CRISPR research.

And because of this, when Doudna and Charpentier published their paper in Science in 2012, they had used these tools to achieve gene editing in mammalian cells within a year.

Therefore, Zhang Feng can be said to be a pioneer scientist in the application of CRISPR/Cas9. After that, he not only won the first place with this work or application, but also a series of works took root in this field and made many new contributions. For example, Charpentier and Doudna used CRISPR/Cas9 protein to edit genes. But Zhang Feng believes that there should be many proteins of this kind. There are all kinds of biodiversity in nature, and there can be other better, more efficient and more application-worthy proteins.

Then, he worked with a person who was doing bioinformatics gene discovery at NCBI in the United States, and found many functionally specific Cas proteins in various microorganisms that were difficult to culture. Then, using these Cas proteins, he discovered more gene editing technologies or more sophisticated editing technologies. Not only can he do gene editing, he found that he could also do gene diagnosis or testing.

06 Excellent work in the field of gene editing in China

Ye Shuisong : Teacher Chao, can you briefly talk about some of the highlights of work in this field in China?

Chao Yanjie: There are actually many outstanding scientists in China. In our country, CRISPR/Cas9 is used to transform plants and animals in agriculture, and several agricultural colleges are doing well in animal breeding. Gene editing has also been done in monkeys, providing many models for clinical research or disease research.

In addition, some scholars at Peking University are able to use CRISPR in disease treatment, such as trying to treat HIV or infectious diseases, and have also made very good contributions.

07 Optimization of gene editing technology

Ye Shuisong : At present, there are some possibilities of off-target effects of CRISPR/Cas9 gene editing in humans , as well as some issues such as AAV vector delivery and immunogenicity . If this technology is to be applied clinically in the future, which aspects do you think need to be optimized?

Chao Yanjie: I think the delivery system is an area that needs to be optimized, because CRISPR/Cas9 is still a substance of bacterial origin, or a substance exogenous to the human body. It takes some time to deliver CRISPR/Cas9 to human cells and perform ideal editing in ideal tissue cells.

The current application is limited to local editing, especially for retinal and optic nerve diseases. This is a method of editing by local injection into the eye cells. This cannot be applied to all types of diseases, such as blood diseases, which can only be treated by extracting blood cells, editing them in vitro, and then re-injecting them into the body.

Therefore, in order to truly enable CRISPR/Cas9 to edit arbitrarily in ideal organs and cells, relevant research on delivery is still needed to enable it to achieve targeted entry into ideal target cells.

Another is gene off-target. This research is the direction that all scientists in the world are working on. As a tool for human treatment or gene editing, if there is any off-target phenomenon, it will be integrated into the human genome, and then it will be inherited to the next generation and the next generation, and will exist permanently in the human genetic history. Therefore, the potential off-target possibility will have a far-reaching impact.

Now all scientists in the world working in this field hope to further optimize the efficiency and accuracy of gene editing.

08 How will gene editing develop in the future?

Ye Shuisong : What do you think will be the future development direction of gene therapy?

Chao Yanjie: People who work in biology will appreciate or understand the diversity of nature. The emergence of these different tools is actually a development of biotechnology. Each tool has its most ideal or most suitable application scenario.

I don't think gene therapy will be replaced by other treatments in the future. Instead, we should think about which diseases and which specific scenarios must be treated with gene therapy. I can think of some research on rare diseases or genetic diseases, because these diseases are caused by DNA defects or genome defects. Therefore, editing at the DNA level can completely solve the problem with one edit. This is something that gene therapy cannot replace and is the most suitable treatment scenario.

Then, mRNA vaccines and protein degradation experiments are still a developing technology. Although it has been used as a precedent for vaccines in the COVID-19 process, it is still not mature enough, especially in the treatment of genetic diseases.

As for what scenarios they are most suitable for or what problems they solve, further research is needed.

This article is supported by the Science Popularization China Starry Sky Project Team/Author: Deep Science Review: Tao Ning, Associate Researcher, Institute of Biophysics, Chinese Academy of Sciences

Produced by: China Association for Science and Technology Department of Science Popularization Produced by: China Science and Technology Press Co., Ltd., Beijing Zhongke Xinghe Culture Media Co., Ltd.