Eukaryotic genome synthesis

Synthesize-non-synthesize!

Svetlana Bozrova, "Biomolecule"

The creation of an artificial eukaryotic genome is a new international project of Johns Hopkins University in the USA. It sounds mysterious, but in fact it's quite the opposite. All interested universities of the world are invited to participate in the project – there are enough chromosomes for everyone. Researchers are slowly but surely recreating and improving the genome of yeast – the most studied eukaryotes in the world. Why was this project started and what difficulties do scientists face?

Synthetic biology is a fairly new direction in science, which gained a particularly loud resonance in 2010, when the genome of a living organism, Mycoplasma genitalium, was completely synthesized at the Craig Venter Institute under the leadership of Hamilton Smith [1]. Later, the genome of another mycoplasma, M. mycoides, was obtained there, which was then "planted" by M. capricolum bacteria with a removed chromosome. As a result, M. capricolum was transformed into M. mycoides, or rather reprogrammed [2]. These studies have raised a whole wave of ethical discussions – does a person have the right to interfere so much in the work of nature? However, from the point of view of synthetic biology, the answer is simple: life is a molecular process that has no moral restrictions at the cellular level [3]. Of course, one can disagree with this, but instead of philosophical reasoning, scientists have gone further and have already taken up the eukaryotic genome.

A few years ago, a sensational article was published in the journal Science. As part of a project called Synthetic Yeast Genome Project 2.0, or Sc2.0, a group of American scientists led by Jeff Boeke synthesized the first artificial chromosome of a representative of eukaryotes – Saccharomyces cerevisiae, that is, baker's yeast [4, 5]. Then, in 2014, it was a slightly modified chromosome 3 – synIII. The global goal of this and subsequent work of the project was to gradually, step by step, create an artificial eukaryotic genome. And so, in March 2017, the participants of this project announced their new success: as many as five new S chromosomes were fully modeled and synthesized. cerevisiae: synII, synV, synVI, synX and synXII [6]! Yeast has 16 chromosomes in total, so five out of sixteen are almost a third of the yeast genome. Impressive, isn't it?

In fact, scientists have not just recreated an existing yeast genome – they have added improvements to it. The eukaryotic genome is in constant motion: nucleotides are removed and added, deletions and duplications occur. This is the fault of special inhabitants of the genome – the so-called mobile genetic elements (mainly transposons), which can move through yeast DNA, changing its structure [7, 8]. All these innovations and novelties of genetic structures in S. cerevisiae are nothing but a whim of evolution [9]. We used to think that nature creates perfect creatures, but this is not quite true.

When creating the first synthetic chromosome, biologists significantly corrected it: they threw out many introns and elements responsible for genome instability (transport RNA genes that were relocated to a separate "chromosome", and transposons), and also improved the genetic code by replacing the TAG stop codons with TAA. In general, they worked hard (see the inset) and brought cleanliness and order. What is left after the "general cleaning"?

Assembling a synthetic genome is not an easy task (Fig. 1). The genome is constructed by sequential assembly. First, 3-6 DNA fragments with a length of about 10 thousand nucleotide pairs are synthesized, which are interconnected in vitro using restriction enzymes and ligase into megafragments: each fragment at the ends contains binding sites of restriction endonucleases, after cutting the fragment with these enzymes, non-palindromic protrusions ("sticky ends") remain for crosslinking with the previous and subsequent fragments. Next, the genome assembly continues in vivo – sequential replacement with synthetic megafragments of native host DNA. Megafragments of DNA (blue, green, purple, orange and gray lines in the figure) are embedded in the host genome (black line) from left to right due to the mechanism of homologous recombination (black X). The right end of each megafragment, except the last one, contains the marker gene Ura3 or Leu2 (red and blue triangles). This makes it possible to select successfully transformed cells – those in whose genome a new megafragment has been accurately embedded (positive selection): without the marker, the cells would not grow in an environment without uracil or leucine. During the assembly process, the markers alternate: with homologous recombination, the previous marker is "rewritten" – it is replaced by a section of a new megafragment containing another marker. The last megafragment, "unmarked", should erase the Ura3 label from the penultimate fragment, and then negative selection is already carried out: cells containing the Ura3 gene, when 5-fluoroorotic acid (5-fluoroorotic acid, 5-FOA) are added, die, because in them this acid turns into a toxic substance 5-fluorouracil.

The artificial genome perfectly ensured the synthesis of all those proteins for which it was initially responsible [10]. This means that all the removed elements were not vital, and without them the cell could function safely. It turns out that in the updated form, the chromosomes not only continue to work properly, but also have an improved design. And it is also much easier for scientists to work with such a chromosome [4]. It is worth considering how many questions a person will be able to answer when he learns to control the eukaryotic genome. Why do we need transposons? What is the "living wage" of genes in the genome? Is it possible to create synthetic biological fuel [11]?

However, like any joyful event in the scientific field, the successful synthesis of yeast chromosomes raised a large number of very different questions. And one of them is about the ethics of such experiments. At the moment, only S chromosomes are being created. cerevisiae, and there is no danger from the point of view of bioethics in this. But synthetic biology as a whole is at risk. What happens if an artificial living organism is inadvertently created? Is it ethical towards him? Is a person ready to take on such responsibility? Raises these questions Presidential Commission on Bioethics of the United States of America. Of course, it is unlikely that scientists in the excitement of their research will not notice how they will create a Frankenstein monster, but it is still necessary to act extremely carefully. The Commission recommends that scientists actively interact with the press, explaining to people what they are doing and why. Jeff Boeke's research group does just that – their research is widely discussed by journalists. For example, in an online publication The Christian Science Monitor published an article about the artificial chromosome immediately after the scientific publication of the data [12].

Where do these kinds of discoveries lead us? What will happen when we can manage the genome and improve our own DNA? The answers to these questions will appear only when we live up to these events. But whatever happens, it's worth thinking about the good, because, as you know, thought is material.

Literature

1. biomolecule: "Hand-assembled genome";
2. biomolecule: "Life in the era of synthetic life";
3. biomolecule: "The meanings of "life"";
4. Annaluru N., Muller H., Mitchell L.A., Ramalingam S., Stracquadanio G., Richardson S.M. et al. (2014). Total synthesis of a functional designer eukaryotic chromosome. Science. 344 (6179), 55–58;
5. biomolecule: "Synthetic chromosome";
6. Zahn L.M. and Riddihough G. (2017). Building on nature’s design. Science. 355 (6329), 1038–1039;
7. biomolecule: "Mobile genetic elements of prokaryotes: stratification of the "society of "vagrants and stay-at-home";
8. biomolecule: "There is no such thing as a lot of diversity: what do the mobile elements of the genome in the brain do";
9. biomolecule: "The human genome: a useful book, or a glossy magazine?";
10. Richardson S.M., Mitchell L.A., Stracquadanio G., Yang K., Dymond J.S., DiCarlo J.E. et al. Design of a synthetic yeast genome. Science. 355 (6329), 1040–1044;
11. Synthetic Yeast 2.0. FAQ;
12. Chowdhury S. (2014). Artificial yeast chromosome brings science one step closer synthetic life. The Christian Science Monitor.

Portal "Eternal youth" http://vechnayamolodost.ru  22.03.2017