A gene synthesis method that promises to help researchers understand how mutations give rise to disease has been dramatically increased in power in work co-led by a scientist in the University of Oregon’s Phil and Penny Knight Campus for Accelerating Scientific Impact.
With a series of tweaks, researchers can now synthesize four times the number of genes with five times the fidelity — the amount of completely correct molecules — than was possible when the method, DropSynth, was unveiled in 2018 in the journal Science by scientists at UCLA.
The improvements were detailed by a five-member team, which includes UO synthetic biologist Calin Plesa, in the journal Nucleic Acids Research. Plesa, who joined the Knight Campus in August 2019, was a member of the UCLA group as a postdoctoral researcher. He is now working to improve the method even further in his UO lab.
“Our improvements reduce the cost of using this process by 43 percent, or 90 times cheaper than current commercially available processes,” Plesa said. “We have increased the number of genes we can work with from 384 to 1,536 in a single tube reaction and can accurately reassemble the gene fragments we produce with over 20 percent fidelity.”
When initially announced, DropSynth’s fidelity was just under 4 percent, too low for use in many applications.
“Biological systems are incredibly complex and diverse,” he said. “These improvements will allow researchers to study these systems at large scales. The gains in fidelity makes these gene libraries easier to use in downstream processes and will allow us to assemble even longer length genes.”
The improvement in scale reduces the cost per gene and makes it realistic to synthesize tens of thousands of genes. Numerous other researchers have been trained to use the method, added Plesa, who also is an affiliate member of the UO’s Department of Chemistry and Biochemistry and the Institute of Molecular Biology.
Synthesizing genes is important in basic medical research, especially for developing new drugs and finding disease mechanisms, but the process is expensive. Before DropSynth, the process involved sewing small strands of oligonucleotides, short nucleic acid polymers that can combine with DNA or RNA sequences, into sequences one at a time after their creation.
DropSynth simplifies the process by assembling oligonucleotides together inside tiny droplets. The strands, in effect, become the equivalent of bar-coded library materials, providing genetic components that can be reconstructed to, for instance, design proteins needed to understand the trajectory of disease based on mutations introduced into genetic materials or tissue types.
“Natural populations do not contain sufficient genetic variation in a well-controlled manner to allow a thorough understanding of how different mutations lead to various diseases,” Plesa said. “Each one of us has mutations that are completely unique to us. Understanding the clinical impact of these mutations will require large-scale, sequence-function mapping efforts that can be carried out in the lab using methods like DropSynth to systematically characterize large parts of the corresponding sequence space.”
Because of their complexity, he said, engineering biological systems for practical purposes often requires testing many different designs and improving models through feedback by characterizing model-driven predictions. Such applications can benefit from synthesizing large libraries of gene variants at low costs, he said.
When DropSynth was introduced, the cost for such synthesis was estimated at $2 per gene. Researchers previously could only obtain gene sequences from commercial vendors for $50 to $100 per gene.
Co-authors with Plesa on the paper are UCLA scientists Angus M. Sidore, Joyce A. Samson, Nathan B. Lubock and the paper’s corresponding author Sriram Kosuri.
Plesa’s part of the research was supported by a grant from the Human Frontier Science Program, part of an organization based in Strasbourg, France; a Rubicon fellowship from the Netherlands Organization for Scientific Research; and an award from the Burroughs-Wellcome Fund’s Career Awards at the Scientific Interface program.
Other team members were supported by grants from the National Science Foundation, National Institutes of Health, Department of Energy and Searle Scholars Program.
—By Jim Barlow, University Communications