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Scientists Improve CRISPR/Cas9 Knock-in Efficiency by Disrupting NHEJ

NEW YORK (GenomeWeb) – Disrupting key molecules in a quick and dirty DNA repair mechanism led to an eightfold increase in gene knock-in efficiency with CRISPR/Cas9 genome editing, according to a study published this week in Nature Biotechnology.

Knocking out genes with CRISPR/Cas9 has been much easier than knocking them in. That's because the double-stranded breaks induced by the Cas9 nuclease are more quickly repaired by non-homologous end-joining (NHEJ), a DNA repair mechanism that often drops nucleotides from the ends being joined. This works well for inducing gene knockouts because it leads to frameshift, but is not ideal for knocking in genes, which requires greater precision.

Gene knock-ins are best facilitated by homology-directed repair (HDR), a more precise but less efficient DNA repair mechanism that relies on a template to perform the repair.

To promote HDR and therefore get the precision needed to knock in genes with greater efficiency, scientists, led by Van Trung Chu and Ralf Kühn of the Max-Delbrück-Center for Molecular Medicine in Berlin, were able to turn off key molecules in the NHEJ pathway – particularly DNA ligase IV but also KU70 and KU80 – using gene silencing and other methods to suppress the molecules.

Gene silencing directed at KU70 and DNA ligase IV upped the efficiency of HDR four to five times; however, the most effective way of inhibiting NHEJ turned out to be by degrading the molecules with adenovirus 4 proteins.

By adding in adenovirus 4 proteins E1B55K and E4orf6, the researchers improved the efficiency of HDR up to eightfold and "essentially abolished NHEJ activity in both human and mouse cell lines," they wrote.

The researchers said they were able to reach gene knock-in frequencies of 50 to 66 percent in mouse cells transfected with a combination of CRISPR/Cas9 and adenovirus 4 protein plasmids. The process could be enhanced even further in human induced pluripotent stem cells by delivering Cas9 and sgRNAs as synthetic RNAs, the authors wrote.

The findings provide other scientists with several tools to increase the frequency of precision gene knock-ins using the CRISPR/Cas9 system in mammalian cells, the researchers said, and could allow them to insert gene corrections into embryos of other model organisms and mammalian cell lines.