With the birth of semi-artificial life, how far are we from becoming "God"?

Synthetic biology is a magical subject that allows us to design and create completely new organisms using engineering thinking. Today, scientists have successfully synthesized all 16 chromosomes of yeast and put half of them into a cell to create a semi-artificial yeast. They also designed a new chromosome for this yeast to make its genome more stable while ensuring the integrity of its functions.

These achievements are a milestone in synthetic biology and a major step forward for humanity towards fully artificially synthesized eukaryotic cells.

Written by Gu Shuchen

On November 8, 2023, the famous scientific journals Cell, Molecular Cell and Cell Genomics focused on the same topic and published 10 research papers at once, introducing the international cooperation project "Synthetic Yeast Genome Project" (Sc2.0). This batch of results was completed by scientific research teams from the United States, China, the United Kingdom and other countries. The article reported in detail the comprehensive synthesis of 8 new yeast chromosomes and the innovative design and synthesis of a transfer RNA (tRNA) chromosome.

So far, Sc2.0 has completed the artificial synthesis of all 16 yeast chromosomes, including the 6 previously synthesized and the 2 completed chromosomes that have not yet been published. Scientists have also successfully integrated 7.5 chromosomes into a natural strain of Saccharomyces cerevisiae. This semi-artificial yeast has similar survival and replication capabilities to wild yeast strains [1] . In other words, a yeast cell that is half natural and half artificial has been created! Scientists have also written and designed a brand new chromosome for yeast cells - the tRNA chromosome [2], which is a new step towards creating the world's first fully artificially synthesized eukaryotic cell.

These exciting results all belong to a brand new discipline - synthetic biology. Today, this series of breakthroughs means that humans have developed from tinkering with a few genes to being able to design and build an entire genome from scratch.

What is synthetic biology?

Synthetic biology is a new frontier interdisciplinary subject that has emerged in recent years. It is considered the third biotechnology revolution after the discovery of the DNA double helix and the human genome sequencing project. In 2004, MIT Technology Review rated synthetic biology as one of the top ten technologies that will change the world, and in 2010 Science also ranked it second among the top ten scientific breakthroughs.

It is based on multiple disciplines such as genetic engineering, systems biology, and computer engineering, and adopts engineering design concepts to design, transform, and even synthesize the genetic material of organisms, thereby breaking the boundaries of species and creating artificial life. Synthetic biology not only has the potential to help us solve many challenges facing human society, but also allows us to uncover the mysteries of basic life sciences from a new perspective of "creation."

The essence of synthetic biology lies in design and creation. Whether it is "transforming existing natural biological systems" or "designing and building new biological components, devices and systems", it is all based on DNA. The general process of synthetic biology is a research cycle of "design-build-test-learn-rebuild".

Scientists will first use computer software to assist in the design of the DNA construct to be synthesized. The designed DNA will be divided into synthesizable fragments (synthons) of 1–1.5 kb. The synthetic fragments can be assembled and spliced ​​by single-stranded oligonucleotides to assemble larger DNA elements. The assembled DNA needs to be verified in two steps: sequence verification and functional verification after it is transformed into cells. Based on the verification results, modifications are made and the test cycle is repeated until the DNA construct with the required function is obtained [3].

However, the assembly of life components is not as simple as the assembly of circuits. Although we know that all life components in living organisms are translated from DNA, we lack a deep understanding of many components. The functions of these components may also change due to time, location, conditions, etc. Therefore, even if we know the function of each component, when we assemble these different biological components, their functions may be missing or "incompatible".

Figure 1 Synthetic biology testing cycle [3].

From 2010 to 2020, with the development of biotechnology and bioengineering, synthetic biology has made rapid progress. In 2010, researchers from the J.Craig Venter Institute (JCVI) in the United States announced the construction of the first artificial cell - "synthetic mycoplasma" JCVI-syn1.0[4] . In 2021, researchers on the project announced that they had artificially synthesized a "simplified cell" JCVI-syn3.0 with only 473 genes based on JCVI-syn1.0 [5]. Although mycoplasmas are prokaryotic cells and their structure is much simpler than that of eukaryotic cells, the artificial synthesis of mycoplasmas still inspired scientists' ambitions, and the Sc2.0 project was launched.

First attempt to synthesize a eukaryotic genome

Yeast is a type of single-cell eukaryotic microorganism that is closely related to human life. Because of its clear genetic background, yeast has become one of the most commonly used model organisms in scientific research. As early as 1996, scientists completed the whole genome sequencing of Saccharomyces cerevisiae and found that the genome has a total of about 6,000 genes, of which 5,000 are not necessary to maintain yeast life activities and can be deleted and rewritten.

In 2007, Professor Jef D. Boeke of New York University initiated a global eukaryotic research collaboration project, the Sc2.0 Project. The project is distributed in many countries around the world, with the United States and China accounting for 28% and 39% of the total synthesis, respectively. In 2011, the Sc2.0 Project was officially launched in the United States, China, the United Kingdom, Singapore, Australia and other countries. The project aims to complete the design and chemical reconstruction of all 16 chromosomes of Saccharomyces cerevisiae, thereby providing an application platform for systematic research on eukaryotic chromosomes. This is the first attempt by humans to design and synthesize the genome of a eukaryotic organism from scratch [6].

To achieve this goal, the researchers will synthesize the yeast genome from scratch, remove all transposons and repetitive elements, recode stop codons, and move transfer RNA genes to completely new chromosomes, while avoiding adaptive defects and adding features that facilitate chromosome construction and manipulation. During the design process of the Sc2.0 project, although bases were deleted, inserted, and replaced in the gene sequence, in principle, the synthetic strain must maintain the same phenotype as the natural strain, while also ensuring the stability of the genome. In order to enhance genetic flexibility, scientists have also optimized the wild-type genome sequence[6].

In 2014, a research team led by Professor Boyke created the first artificial yeast chromosome (chromosome 3, the smallest of all yeast chromosomes) [7] . In 2017, the Sc2.0 project international collaboration announced that it had completed the design and synthesis of one-third of the yeast genome, and Science magazine reported it in a special issue [8]. This marked a major step forward for the Sc2.0 project.

Scientists have now completed the synthesis of 16 chromosomes and created 16 partially synthetic yeast strains, that is, each cell contains 15 natural chromosomes and 1 synthetic chromosome [1] . Scientists also hybridized yeast cells containing different synthetic chromosomes and searched for individuals carrying two synthetic chromosomes in their offspring, gradually combining the artificially synthesized chromosomes to form a completely synthetic new cell. After a long hybridization process, 6 complete chromosomes and 1 chromosome arm have now been integrated into the same cell. The resulting yeast strain with 6.5 artificial chromosomes has a synthetic DNA ratio of more than 31% and a normal morphology, with only slight growth defects compared to wild yeast [1].

Figure 2. SEM images of yeast cells with 6.5 synthetic chromosomes showing normal appearance and budding behavior [1]

In order to increase the efficiency of chromosome replacement, scientists have also developed a new and efficient chromosome replacement method. They used this method to transfer the largest chromosome in yeast chromosomes (chromosome 4, synIV), resulting in a yeast cell with 7.5 chromosomes, with synthetic DNA accounting for more than 50%. Although the chromosomes of the yeast underwent major changes, it can still survive and replicate [1].

One of the main goals of Sc2.0 is to improve the stability of the yeast genome. However, natural yeast cells contain a large amount of repetitive DNA that does not encode anything, but can recombine with each other through natural processes, causing major structural changes in the genome and becoming unstable. In order to better control yeast cells, the Sc2.0 team used a computer program to comb through the yeast genome, find highly repetitive DNA regions and delete them, including all DNA fragments encoding tRNA. Although these DNA sequences are unstable, the tRNAs they encode are essential for the function of the cell. Therefore, scientists concentrated all the genes encoding tRNA on a new chromosome - the tRNA new chromosome, and then added it to a completely artificial yeast cell [2]. This also provides researchers with a new way to better control synthetic yeast and explore the limits of biology.

Of course, such a large-scale scientific research project is not all smooth sailing. In the early stages of the project, the scientific research team experienced setbacks in the project's technical research and development. Not only did they encounter complex sequences that made synthesis difficult, but they also encountered problems with the coding of the genes themselves that made conventional synthetic cloning impossible. In addition, as the genome becomes larger and larger, how to further improve efficiency and reduce costs is also a problem that scientists need to solve. The most time-consuming and labor-intensive part of the project execution also includes the location and repair of synthetic defects, which is also one of the biggest difficulties in synthesizing yeast genomes. Compared with other synthetic genomes, the number of sequences involved in synthetic yeast is large and complex, and its defect troubleshooting is like looking for a needle in a haystack, requiring a lot of verification work. But in the end, through joint efforts, scientists from many countries ushered in the smooth progress of the Sc2.0 plan, and putting all artificial chromosomes into the same yeast will be a new challenge in the future.

Based on the smooth progress of the previous Sc2.0 research, scientists also realized that we can make deeper modifications to the yeast genome to study specific biological problems or achieve related application goals. Therefore, Chinese scientist Dai Junbiao, together with Professor Cai Yizhi of the University of Manchester in the UK and Professor Boyke of New York University in the United States, jointly initiated the Sc3.0 project. In this project, scientists will make a more streamlined and in-depth design of the yeast genome and construct the first minimal yeast genome to explore major biological issues, such as: Which yeast genes can be deleted without affecting their activity? What is their evolutionary significance? Under given conditions, what functions are required for the minimal genome to maintain eukaryotic life? Does the organizational form of the wild-type genome have significant biological significance? Can we artificially design the arrangement and regulation of genes within the genome? The initiative and project introduction of the Sc3.0 project were also published in the journal Genome Biology in 2020[9].

Are humans now able to create life?

No, at present we have only copied the answers from the textbook. In essence, this synthetic yeast cell has not deviated from the gene template of nature, but has only copied or modified the existing genes. This cannot be said to be "innovation from scratch", but only "re-optimization" based on the map. Each point of "optimization" needs to be further verified to see whether it will affect the survival activity of the yeast itself. Our understanding of life is far from reaching the stage where we can innovate from scratch. At present, although scientists have been able to artificially synthesize the genome of eukaryotic organisms, they cannot put all artificial genomes into the same eukaryotic cell. In order to directly build a cell "from scratch", or to further design a series of new genomes without relying on the gene sequence of nature, scientists also need to have a deeper understanding of the structure of cells, the function and regulation of genes, and have a clearer understanding of the nature and origin of life.

But we are still slowly "overturning" Darwin's theory of evolution. Compared with Sc1.0 (natural yeast), the Sc2.0 plan can be used as a tool to directly "create things" by introducing genes from exogenous sources. Theoretically, the artificial design and synthesis of yeast genomes and subsequent rapid genome evolution research can not only realize the functional research of the whole yeast genome, but also provide a large amount of material for studying the evolutionary history of yeast through the yeast library generated by random changes in the genome. In addition, yeast is closely related to human life. It is an important material necessary for brewing and making bread. In industry, yeast can produce many substances for us. For example, by adding genes related to the synthesis of artemisinin to yeast, the mass production of artemisinin can be achieved. In the future, we can also put some substances synthesized by other microorganisms into artificial yeast for production, such as antibiotics, monosodium glutamate and even hyaluronic acid. It can be said that artificial yeast has a lot of application potential to improve people's lives, and can be widely used in food production, drug production, bioenergy, biomaterials and other fields.

In 2010, Craig Venter, the “father of the human genome,” recombined the DNA of a microorganism called Mycoplasma mycoides, “glued” the new DNA fragments together, and implanted them into another bacterium to create the first so-called “artificial life” [10] . Although this achievement caused considerable controversy at the time, the short human text he added to the genome of this “artificial life” was impressive: “To live, to err, to fall, to triumph, to recreate life out of life.” Nature created the human world, and perhaps humans can also create a new world towards a better home.

References

[1] doi: 10.1016/j.cell.2023.09.025.

[2] doi: 10.1016/j.cell.2023.10.015.

[3] doi: 10.1101/cshperspect.a023812.

[4] doi: 10.1126/science.1190719.

[5] doi: 10.1016/j.cell.2021.03.008.

[6] doi: 10.1126/science.aaf4557.

[7] doi: 10.1126/science.1249252.

[8] SCIENCE VOLUME 355|ISSUE 6329|10 MAR 2017

[9] doi: 10.1186/s13059-020-02130-z.

[10] doi: 10.1126/science.1190719.

This article is supported by the Science Popularization China Starry Sky Project

Produced by: China Association for Science and Technology Department of Science Popularization

Producer: China Science and Technology Press Co., Ltd., Beijing Zhongke Xinghe Culture Media Co., Ltd.

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