NEW YORK – A new method to create synthetic chromosomes out of natural DNA offers an efficient way to engineer structural genomic variation.
Developed by researchers at the University of Southern California, CReATiNG (Cloning, Reprogramming, and Assembling Tiled Natural Genomic DNA) uses yeast to manipulate large chunks of genomic material. In a paper published last month in Nature Communications, the authors demonstrated the ability to make yeast chromosomes approximately 200 kb in length. "But we're pretty confident we could be making molecules quite a bit larger," possibly even on the megabase scale, said senior author Ian Ehrenreich.
This approach can help engineer genomic features that even CRISPR-Cas9-based genome editing can't do easily, he said. "This opens up a world of possibilities in synthetic biology, enhancing our fundamental understanding of life and paving the way for groundbreaking applications."
"There are parts of this workflow that have been employed somewhat routinely," said Sudarshan Pinglay, a synthetic biology researcher at the University of Washington, who was not involved in the study. "What this paper shows very neatly is, it packages it nicely into a workflow other people can use."
The current state of the art for synthesizing large molecules of DNA starts with synthetic DNA, making it expensive, Ehrenreich said, noting that it also takes many steps. Recently, firms like Elegen, Moligo Technologies, and Ribbon Biolabs have advertised their ability to create DNA in the range of 5 kb to 20 kb in that way.
This can be useful when trying to change the base-by-base sequence of DNA, "but sometimes you just want to change the structure in a complex way or generate a chromosome containing DNA from multiple individuals," he said.
That's the type of molecule that CReATiNG is best suited for. Ehrenreich's team has developed "a whole strategy of using CRISPR and specialized vectors where we could clone segments inside yeast and add on new sequences at the ends," he said. "These adapters make it possible to utilize them for new purposes."
The method clones DNA inside yeast and transfers the molecules to Escherichia coli for amplification. Then the molecules are stitched back together in yeast to produce a continuous chromosome.
Ehrenreich estimated that it cost around $500 to build one chromosome, the smallest in the yeast genome. "You can go through it all in a week and a half or two weeks if everything goes right on the first try," he said. For comparison, GenScript says it can produce custom sequences up to 200 kb long for $.45 per base in about a month.
"Our lab now mostly gets this stuff right," he added. The biggest challenge is ineffective CRISPR guides; if they don't work, the cloning won't happen, but it's not always possible to predict how they'll work.
Ehrenreich has applied for a patent on the method and said that licensing the intellectual property "seems to be USC's preferred strategy." However, he did not say whether anyone had licensed the IP yet.
In proof-of-principle experiments, the study authors replaced an entire chromosome inside of a yeast cell, taking one from another species of yeast.
This is one application of the method. "If you want to study how evolution works, but you can't easily make the organisms, or they don't go through meiosis well, it shows you can synthesize the chromosome to bypass that," he said.
CReATiNG could also help to delete many regions in a cell's genome, something that gets harder to do with CRISPR as multiplicity rises. Or it could remove large stretches of a chromosome all at once. "You can eliminate almost 40 percent of a chromosome just by cloning the parts essential to the organism and programming them to recombine with each other," Ehrenreich said.
Pinglay said he's interested in trying out CReATiNG, but not necessarily to study yeast. The method should be applicable to mammalian DNA, as well, he suggested. A 100 kb stretch of DNA would allow him to study different sequence elements associated with a disease by mixing and matching them to pinpoint which ones actually underlie the disease.
He may be working with CReATiNG-produced DNA sooner than most. Pinglay's lab will be associated with the recently announced Seattle Hub for Synthetic Biology, and Alessandro Coradini, first author of the new paper describing the method, will be joining the Seattle Hub's DNA foundry this year.
Using CReATiNG to look closer at genomic loci associated with disease is in line with Ehrenreich's broader, more fundamental goal of understanding how genetic changes produce phenotypic effects.
Ehrenreich said it's likely that CReATiNG can work with DNA from other organisms, but that yeast will remain involved. "The best system we have right now for building big pieces of DNA is actually yeast," he said, due to its tendency to recombine DNA.
"Any workflow for synthetic genomics is going to have to go through yeast. This is all compatible with synthetic genomics in other systems," he said.