DNA “nicks” make for safer, more precise genetic analysis

Researchers at Cornell University have developed a safer and more precise way to study how genes function in living tissues by refining a recently developed CRISPR-based genetic technique in fruit flies, enabling researchers to better study how genes contribute to development and disease.

Published May 27 in the Proceedings of the National Academy of Sciences, the work highlights a new method that replaces the harsh DNA cuts used in traditional CRISPR analysis with gentler cuts known as "nicks."

According to, Chun Han, associate professor in the Department of Molecular Biology and Genetics in the College of Agriculture and Life Sciences (CALS) and the Weill Institute for Cell and Molecular Biology, the approach still allows scientists to study how genes function in living tissues, but with far less unintended cellular damage and greater control over the experiment.

CRISPR is a gene-editing technology that allows scientists to precisely cut DNA to study how genes function. One method built around CRISPR, called MAGIC, creates small groups of altered cells inside an otherwise normal organism. Researchers can then observe how specific genes affect development, cell behavior and disease.

The original MAGIC technique relies on creating double-strand breaks, cuts through both strands of DNA, to trigger the genetic changes needed for the analysis.

"We mainly wanted to improve the MAGIC technique by finding a way to avoid the toxicity associated with Cas9, which acts as the "molecular scissors" in the CRISPR gene-editing system." Han said, who led the study with Cornell doctoral student Yifan Shen and undergraduate student Ann Yeung, who is now a doctoral student at Harvard.

The original MAGIC system uses the CRISPR enzyme Cas9 to cut both strands of DNA to induce recombination between homologous chromosomes, chromosome pairs inherited from each parent, in developing cells. The recombination helps create homozygous cells (cells containing two identical copies of a gene derived from a single parent), that researchers need to study gene function, but the DNA breaks can also unintentionally rearrange chromosomes.

"In our original design of MAGIC, we used Cas9 to induce double-strand breaks (DSBs) in developing cells," Han said. "But those DNA breaks can be highly detrimental to chromosomes during cell division, harming or killing cells."

To address the problem, the researchers turned to "nickases," modified versions of Cas9 that cut only one strand of DNA instead of both.

"Nickases are derived from Cas9 but carry mutations that allow them to cut only one strand of DNA," Han said. "Without producing double-strand breaks, nickases do not damage cells in the same way and can still be used with the MAGIC technique."

One of the study's most unexpected findings was that even a single DNA nick could trigger the recombination needed for the MAGIC technique to work, Han said.

The researchers also found that the exact pattern of DNA nicking strongly influenced how often recombination of DNA occurred, offering scientists new ways to tune experiments for different research needs.

The advance could help researchers study genes with greater confidence by reducing the risk that experimental damage itself alters cell behavior. Cleaner, more reliable genetic tools could help researchers better study how genes contribute to development and disease, Han said.

"Our ability to study biology is restrained by the limit of our tools," he said. "Avoiding the unintended DNA damage can make researchers more confident in using this technique and interpreting their results."

Han said the new nickase-based system, combined with a recently developed genome-wide MAGIC toolkit, could expand use of the technique across the fruit fly research community and eventually beyond it.

"Drosophila, the fruit fly, is often the birthplace of new genetic techniques," Han said. "But many genetic techniques invented in Drosophila later found their ways into other organisms."

The research was supported by a grant from the National Institutes of Health with assistance from the Developmental Studies Hybridoma Bank for antibodies, and Bloomington Drosophila Stock Center for fly stocks.

Stephen D'Angelo is the communications manager for biological systems at Cornell Research and Innovation.