Tweaking DNA fragments before inserting yields highest efficiency rates: study

CHICAGO, Dec. 23 (Xinhua) -- University of Illinois (UI) researchers achieved the highest reported rates of inserting genes into human cells with the CRISPR-Cas9 gene-editing system, a necessary step for harnessing CRISPR for clinical gene-therapy applications.

According to a news release posted on UI's website on Monday, the researchers looked at 13 different ways to modify the inserted DNA. They found that small changes to the very end of the DNA increased both the speed and efficiency of insertion.

They then tested inserting end-modified DNA fragments of varying sizes at multiple points in the genome, using CRISPR-Cas9 to precisely target specific sites for insertion.

The researchers found that by chemically tweaking the ends of the DNA to be inserted, the new technique is two to five times more efficient than current approaches. They saw improvements at various genetic locations tested in a human kidney cell line, even seeing 65 percent insertion at one site where the previous high had been 15 percent.

"We speculate that the efficiency improved so much because the chemical modification to the end stabilizes the DNA we are inserting," said UI chemical and biomolecular engineering professor Huimin Zhao. "Normally, when you try to transfer DNA into the cell, it gets degraded by enzymes that eat away at it from the ends. We think our chemical addition protects the ends. More DNA is getting into the nucleus, and that DNA is more stable."

The researchers already are using the method to tag essential genes in gene function studies. They purposely used off-the-shelf chemicals to modify the DNA fragments so that other research teams could use the same method for their own genetic studies.

"We've developed quite a few knock-in methods in the past, but we never thought about just using chemicals to increase the stability of the DNA we want to insert," Zhao said. "It's a simple strategy, but it works."

The study has been published in the journal Nature Chemical Biology.


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