Rewriting the Genetic Code
Synthetic Chromosome Construction from Natural Components in Yeast
Artificial chromosome synthesis is a powerful technique that enables scientists to elucidate the mechanisms underlying cellular life and its diversity. From mapping the genetic basis of trait differences to discerning the structural requirements of genomes, synthesizing chromosomes makes it possible to explore fundamental biological questions that have yet to be answered.1 However, de novo chromosome synthesis is expensive and time-consuming, which hinders its deployment to the wider scientific community.
To overcome these challenges and break down the barrier of high costs, molecular biologists at the University of Southern California invented a new method of building synthetic chromosomes from natural components in the budding yeast Saccharomyces cerevisiae. Their technique, called CReATiNG (Cloning, Reprogramming, and Assembling Tiled Natural Genomic DNA), consisted of first cloning natural chromosome segments with unique adapter sequences specifying how the segments would recombine during assembly, and then transforming cloned segments into cells and assembling them via homologous recombination.1 Using this system, the scientists were able to analyze phenotypic effects tied to specific chromosome segments.
First, target-specific cloning vectors were produced and harvested with the ZymoPURE II Plasmid Midiprep Kit. S. cerevisiae cells were then transformed with the linearized vector, repair template, and multiple guide RNAs. Cloned segments were extracted from the yeast cells and removed from the cloning vector using restriction enzyme digestion. These segments of interest were purified using the Zymoclean Large Fragment DNA Recovery Kit following gel electrophoresis.
To assess the impact of chromosome restructuring on gene expression, RNA was extracted from S. cerevisiae cells using Zymo Research’s YeaStar RNA Kit. Several phenotypically relevant genes were then analyzed by RT-qPCR to confirm that structural changes in synthetic chromosomes can cause gene overexpression, resulting in phenotypic outcomes such as growth defects. To mitigate unintended phenotypic outcomes on donor yeast cells, cloning reactions were designed to avoid disruption of annotated functional elements.1
With the help of Zymo Research’s cutting-edge extraction technology, the researchers successfully developed a cost-effective chromosome synthesis method called CReATiNG, which leverages natural components within yeast cells. This technology may also be adapted for use in other biological systems such as bacterial and mammalian cells, highlighting its diverse applications in the fields of genetics, genomics, and evolution.
