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UC Berkeley Researchers Improve CRISPR/Cas9 Technology for Short DNA Regions

NEW YORK (GenomeWeb) – Researchers at the University of California, Berkeley, have developed a technique that improves on existing CRISPR/Cas9 technology. As they reported today in Nature Biotechnology, their technique achieves a 60 percent success rate when replacing DNA regions no longer than 30 base pairs.

Targeted manipulation of the genome using Cas9 is most effective via non-homologous end joining, but is much less efficient at replacing DNA sequences by homology-directed repair, the team wrote. Their findings show that Cas9 slowly dissociates from double-stranded DNA substrates, remaining attached to the chromosome for up to six hours, but asymmetrically releases the 3′ end of the cleaved DNA strand that is not complementary to the single-guide RNA before it completely dissociates. These findings led postdoc Christopher Richardson to invent the new approach.

"By rationally designing single-stranded DNA donors of the optimal length complementary to the strand that is released first, we increase the rate of [homology-directed repair] in human cells when using Cas9 or nickase variants to up to 60 percent," the researchers wrote. "We also demonstrate HDR rates of up to 0.7 percent using a catalytically inactive Cas9 mutant, which binds DNA without cleaving it."

This technique may be especially useful when repairing genetic mutations that cause hereditary diseases, as those are typically caused by problems in short regions of DNA, including single base-pair mutations, according to the researchers.

"The exciting thing about CRISPR-Cas9 is the promise of fixing genes in place in our genome, but the efficiency for that can be very low," senior author Jacob Corn, scientific director of the Innovative Genomics Initiative at UC Berkeley, said in a statement.

"Our data indicate that Cas9 breaks could be different at a molecular level from breaks generated by other targeted nucleases, such as TALENS and zinc-finger nucleases, which suggests that strategies like the ones we are using can give you more efficient repair of Cas9 breaks," Richardson added.

The researchers also found that Cas9 variants that bind to DNA without cutting it can also insert new DNA sequences at certain sites in the genome, forming a bubble structure on the target DNA that also attracts the repair template, the team said. This kind of technique would have the advantage of being safer than typical gene editing by removing the danger of off-target cutting, Corn added.