Coronavirus Whole Genome Cloning Using Yeast Based Method
According to a paper published in Nature on May 4, researchers have generated a full-length clone of the novel coronavirus genome using artificial chromosomes in brewers’ yeast. Many laboratories have generated clones of the novel coronavirus by other methods, but the yeast system is better in terms of speed and stability.
Microbiologist and coronavirus expert, Susan Weiss of the University of Pennsylvania, said the exciting thing about the yeast is that it’s fast. Other methods are difficult and tedious.
The starting point of many research endeavors in virology is to reconstruct and modify the genomes of disease-causing viruses. To study the virus’s method of infection, it’s replication, and drugs and vaccines that might work against it, these genetic manipulations are essential.
Darwyn Kobasa, a virologist at the University of Manitoba, said speed is the essence for cloning during this pandemic, especially if new variants emerge. The new technique is really quite impressive in terms of speed, he added.
Volker Thiel at the University of Bern, co-author of the paper, said the idea behind the research is to learn about the virus and its weaknesses.
The most commonly used method for cloning viral
genomes is by introducing DNA fragments into Escherichia coli bacteria for replication. But this approach could be problematic for some viruses, including coronaviruses. The bacteria may find it difficult to cope with their extraordinarily large genomes. Moreover, some parts of the genome could be toxic to the bacteria for some unknown reasons.
Yeast cells can handle bigger pieces of DNA as they are larger than bacteria. Joerg Jores, the co-author of the study, said there is another big advantage that yeast cells have the inherent ability to assemble the fragments of DNA into one big molecule.
It means there is no need to reconstruct the viral DNA before introducing into cells as in the case of bacteria; yeast can magically put all the fragments together. The heart of the cloning method used by researchers is this automatic fragment assembly. The method called transformation associated recombination (TAR) was used to introduce the 14 fragments of Viral DNA to the yeast. The short region of an overlapping sequence of each fragment was shared with the next so that yeast cells can identify the matching ends.
Both the end fragments of the genome shared overlapping sequences with a plasmid vector so that it can form a yeast artificial chromosome (YAC) that contains the viral genome. The gluing process is called homologous recombination and involves trimming away nucleotides at the end of one strand of DNA and annealing the remaining complementary sequences (the overlapping section) to another fragment.
Saccharomyces cerevisiae cells were introduced with the DNA fragments. After two days, they picked and tested the colonies for the presence of the full-length genome. The DNA extracted from such clones were then subjected to in vitro transcription, and the obtained RNA was used to infect cultured mammalian cells. This whole process took one week to complete. For high-throughput drug screens, they also cloned a version of SARS-CoV-2 encoding a fluorescent reporter.
Previously, few researchers had reported the construction of a full-length SARS-CoV-2 genome using in vitro ligation in E.Coli. This method is also quite fast, but the fragments had to be individually cloned before ligation. The yeast system for cloning coronavirus genome has the benefit of stably cloned YAC that can be reused. So there is no need to reassemble by in vitro ligation each time infectious virus particles are needed.
However, one advantage of the bacterial system is that anyone can do it. Most molecular biology laboratories around the world are used to genetic manipulations in E.Coli, but using yeast in this way is less prevalent.
The team also cloned other viruses like Zika and MERS using the TAR technique. But currently, they are focused on SARS-CoV-2.
