First there was CRISPR, the molecular “scissors” hailed in the scientific world — and beyond — as a faster, cheaper and more precise way to edit the DNA of plants, mice, potentially even humans.
But while CRISPR has proved useful at snipping out bits of DNA, some scientists say it has been less effective when it comes to inserting pieces of genetic material or making changes that can switch genes on and off.
Now a University of Nebraska Medical Center researcher and his team, with collaborators from seven institutions as far away as Japan, have taken that next step, using a new patent-pending twist on CRISPR called Easi-CRISPR to make the kinds of changes needed to create designer animal models for research on human diseases even more quickly and inexpensively than with the original.
In a paper published recently in the journal Genome Biology, Dr. Channabasavaiah Gurumurthy and his collaborators reported that they were able to insert long sequences of DNA at 13 points on CRISPR-cut mouse genomes, with success rates of between 8.5 percent and 100 percent.
Gurumurthy, assistant professor of developmental neuroscience at UNMC’s Munroe-Meyer Institute, noted that the results with three genes were 100 percent and most were more than 40 percent successful.
“The point of this one is, you can insert genes, you can modify genes very easily,” he said.
Being able to create designer animal models allows scientists to identify the basic function of genes, study the role they and their mutations play in human disease and disability and explore the use of gene therapy, new drugs and other treatments, said Gurumurthy, the paper’s lead author. Other members of his team are Rolen Quadros and Donald Harms. Researchers from several other UNMC departments also were involved.
Scientists began using what’s now called genetic engineering to inactivate, or “knock out,” single genes in mouse embryos some 40 years ago.
Using such “knock-out” animals, researchers have systematically determined the function of roughly 5,000 of the approximately 20,000 genes in the human body, which are largely the same as those in mice. But those efforts, funded by organizations such as the National Institutes of Health, have cost about a third of a billion dollars and left about 15,000 genes to go.
As a result, scientists and research organizations have been looking to speed up the process and trim the cost.
CRISPR came on the scene in 2013. Derived from a defense mechanism bacteria use to fend off viruses, it refers to a system with a guide RNA that leads to the correct place in the DNA to cut and an enzyme that does the slicing.
But it still has proved somewhat difficult — and expensive — to make some of the more complicated models that involve adding genes or creating those with switches that, say, turn on only when an animal is given an antibiotic. Such models, the authors wrote, now make up 90 percent of mouse models created.
George Church, the pioneering Harvard geneticist, said CRISPR editing has produced only incrementally better results than previous methods. Making the more complicated models, which is important, also has been slow and difficult.
Easi-CRISPR, he said in an email, shows much greater precision, and the method “looks like it lives up to its acronym ‘Easi.’ ” Church was not involved in the research.
Dr. Karoly Mirnics, the Munroe-Meyer Institute’s director, said Easi-CRISPR helps solve the previous inefficiencies involved in inserting DNA into genomes.
“This technical advance will be immediately useful for developing much-needed complex genetic models used in biomedical research,” he said.
Gurumurthy and Mirnics noted that such technologies can help cure diseases down the road, but they do raise ethical implications. CRISPR has spurred considerable debate, and the NIH has said it will not fund the use of the technology in human embryos.
William C. Skarnes, director of cellular engineering for the Jackson Laboratory for Genomic Medicine in Farmington, Connecticut, said Easi-CRISPR is a significant advance in genome-editing technology.
“With this tool, researchers can move beyond simple, crude knockouts to make much more sophisticated alleles (variant forms of a given gene) with nucleotide precision,” he said in an email. A leading researcher in the field, Skarnes was not involved in the research.
Gurumurthy said the increased efficiency stems from inserting single-stranded DNA rather than the usual double-stranded version. While the researchers don’t yet know precisely why it works, they suspect that it has to do with how DNA repairs itself.
The idea, he said, came to his long-term collaborator, Masato Ohtsuka of Japan’s Tokai University, while the two were scribbling on a napkin at a conference in Scotland in 2014.
Joe Runge, director of business development at UNeMed Corp., said Easi-CRISPR is a good partner for CRISPR, and one that’s likely to lead to additional advances.
“I think you will see an acceleration of these technologies, so it just raised the bar,” he said.
UNeMed is UNMC’s technology transfer arm.
The results of the current study previously were posted on an online preprint archive operated by Cold Spring Harbor Laboratory on Long Island. Because of that early availability, Gurumurthy said, at least a half-dozen laboratories have been able to replicate his method.
Because of the interest in the technique, Gurumurthy’s team also has posted another article with a step-by-step recipe for using Easi-CRISPR. Gurumurthy and his collaborators also are working on new techniques, including working to make Easi- CRISPR even easier.