A genotype–phenotype mapping system has been developed that can allow researchers to fine-tune the activity of multiple genes simultaneously. The system, which is called the MAGIC, represents a powerful synthetic biology tool. It may be used to investigate fundamental biological questions as well as engineer complex phenotypes for biotechnological applications.
MAGIC, which stands for multifunctional genome-wide CRISPR, has been shown to work in yeast, but it could also be adopted for use in higher eukaryotic organisms, enabling the engineering of complex phenotypes for biotechnological applications.
MAGIC was developed by an interdisciplinary research group at the University of Illinois’ Carl R. Woese Institute for Genomic Biology (IGB), which develops synthetic biology tools to support biological systems engineering. The group is led by Huimin Zhao, PhD, who declares that researchers can use MAGIC to modulate almost all the 6000 genes or so in the yeast genome “individually or in combination to various expression levels.”
Details about the MAGIC system appeared December 19 in Nature Communications, in an article titled, “Multi-functional genome-wide CRISPR system for high throughput genotype–phenotype mapping.” The article describes how MAGIC improves on existing methods for genotype–phenotype mapping, which are limited to a single mode of genomic alteration, that is, overexpression, repression, or deletion. MAGIC, the article maintains, can control the expression level of defined genes to desired levels throughout the whole genome.
“By combining the trifunctional CRISPR system and array-synthesized oligo pools, MAGIC is used to create, to the best of our knowledge, one of the most comprehensive and diversified genomic libraries in yeast ever reported,” the article’s authors wrote. “The power of MAGIC is demonstrated by the identification of previously uncharacterized genetic determinants of complex phenotypes, particularly those having synergistic interactions when perturbed to different expression levels.”
Genomic research has unlocked the capability to edit the genomes of living cells; yet so far, the effects of such changes must be examined in isolation. In contrast, the complex traits that are of interest in both fundamental and applied research involve many genes acting in concert.
Researchers design their own DNA sequences that work within CRISPR systems to precisely edit the genomes of living things. The molecules originally borrowed from bacteria have been tweaked so that they can have one of several effects on the gene toward which they are targeted, either increasing, decreasing, or completely eliminating gene activity, according to the way that cuts in the genome are made and repaired.
Until now, though, there has been no easy way to use more than one of these editing modes simultaneously. Researchers could explore the effects of different changes but could not easily combine them, as if playing improv in a jazz trio in which only one instrument could be playing at any given time.
“We have developed the trifunctional CRISPR system which can be used to engineer the expression of specific genes to various expression levels,” Zhao said. In other words, MAGIC allows researchers to bring two or all three instruments into the music session at once. When combined with the comprehensive “library” of custom DNA sequences created in Zhao’s lab, his group can explore the effects of turning up, turning down, and turning off any combination of genes in the yeast genome simultaneously.
Zhao and colleagues used MAGIC to look for combinations of edits that helped their yeast strain tolerate the presence of furfural, a byproduct of cellulosic hydrolysates that can limit the survival and activity of yeast cells used for cellulosic biofuels production. The resulting engineered furfural tolerant yeast strain could produce more biofuels than the parent yeast strain in fermentation.
The researchers introduced sequences from their MAGIC library into yeast and looked for yeast cells that could withstand high levels of furfural. They found that some of surviving cells had taken in MAGIC sequences that altered the activity of genes known to be involved in tolerating furfural; the involvement of other genes was discovered for the first time by this experiment. The team was able to integrate one of these effective MAGIC sequences into the yeast genomic DNA and then test how further sequences might enhance tolerance.
“We were most excited about the ability of MAGIC to identify novel genetic determinants and their synergistic interactions in improving a complex phenotype [like furfural tolerance], particularly when these targets must be regulated to different expression levels,” Zhao noted. Because MAGIC allows researchers to examine how different genetic changes might work in combination to produce an effect, the new system can lead to clearer analyses of how different biological processes are involved in a trait.
Zhao added that among several technical challenges of the work was the development of a screening method that could be carried out efficiently at a large scale, a capability he hopes to expand to other scientific questions and other organisms.
“These challenges,” he emphasized, “should be addressed in order to apply MAGIC to other eukaryotic systems such as industrial yeast strains and mammalian cells.”