Imagine a word processor that allowed you to change letters or words but balked when you tried to cut or rearrange whole paragraphs. Biologists have faced such constraints for decades. They could add or disable genes in a cell or even—with the genome-editing technology CRISPR—make precise changes within genes. Those capabilities have led to recombinant DNA technology, genetically modified organisms, and gene therapies. But a long-sought goal remained out of reach: manipulating much larger chunks of chromosomes in Escherichia coli, the workhorse bacterium. Now, researchers report they've adapted CRISPR and combined it with other tools to cut and splice large genome fragments with ease.
"This new paper is incredibly exciting and a huge step forward for synthetic biology," says Anne Meyer, a synthetic biologist at the University of Rochester in New York who was not involved in the paper published in this week's issue of Science. The technique will enable synthetic biologists to take on "grand challenges," she says, such as "writing of information to DNA and storing it in a bacterial genome or creating new hybrid bacterial species that can carry out novel [metabolic reactions] for biochemistry or materials production."
The tried and true tools of genetic engineering simply can't handle long stretches of DNA. Restriction enzymes, the standard tool for cutting DNA, can snip chunks of genetic material and join the ends to form small circular segments that can be moved out of one cell and into another. (Stretches of linear DNA don't survive long before other enzymes, called endonucleases, destroy them.) But the circles can accommodate at most a couple of hundred thousand bases, and synthetic biologists often want to move large segments of chromosomes containing multiple genes, which can be millions of bases long or more. "You can't get very large pieces of DNA in and out of cells," says Jason Chin, a synthetic biologist at the Medical Research Council (MRC) Laboratory of Molecular Biology in Cambridge, U.K.
What's more, those cutting and pasting tools can't be targeted precisely, and they leave unwanted DNA at the splicing sites—the equivalent of genetic scars. The errors build up as more changes are made. Another problem is that traditional editing tools can't faithfully glue large segments together. These issues can be a deal-breaker when biologists want to make hundreds or thousands of changes to an organism's genome, says Chang Liu, a synthetic biologist at the University of California, Irvine.
Now, Chin and his MRC colleagues report they have solved these problems. First, the team adapted CRISPR to precisely excise long stretches of DNA without leaving scars. They then altered another well-known tool, an enzyme called lambda red recombinase, so it could glue the ends of the original chromosome—minus the removed portion—back together, as well as fuse the ends of the removed portion. Both circular strands of DNA are protected from endonucleases. The technique can create different circular chromosome pairs in other cells, and researchers can then swap chromosomes at will, eventually inserting whatever chunk they choose into the original genome. "Now, I can make a series of changes in one segment and then another and combine them together. That's a big deal," Liu says.
The new tools will bolster industrial biotechnology by making it easier to vary the levels of proteins that microbes make, Liu and others say. They also promise an easy way to rewrite bacterial genomes wholesale, Meyer adds. One such project aims to alter genomes so they can code not just for proteins' normal 20 amino acids, but also for large numbers of nonnatural amino acids throughout the genome. That could lead to synthetic life forms capable of producing molecules far beyond the reach of natural organisms.