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Researchers Develop Genome Editing Technique Without Double-Stranded DNA Breaks

NEW YORK (GenomeWeb) – Researchers at the Leiden University Medical Center in the Netherlands have developed a new genome editing technique that does not involve subjecting donor DNA and chromosomal sequences to double-stranded DNA breaks (DSBs) in order to induce homologous recombination (HR).

Instead, the team reported in Nature Communications on Friday, the coordinated formation of paired single-stranded DNA breaks (SSBs) — or nicks — at donor plasmids and chromosomal target sites triggered seamless homology-directed gene targeting of large genetic payloads in human cells. This approach reduced the mutagenicity of the genome editing process, the researchers noted, and achieved "multiplexed, single-step gene targeting" with "higher frequencies of accurately edited cells when compared to the standard double-stranded DNA break-dependent approach."

Research involving sequence-specific and strand-specific programmable nucleases, or nickases, for generating these so-called nicks is not new, the researchers wrote in their paper. They not only bypass the formation of DSBs, but they also avoid altering regular cell metabolism, unlike small RNAs, drugs, or viral proteins. But studies have so far found that genome editing based solely on nickases is inefficient, the authors added.

For this study, they explored the use of nicking RNA-guided nucleases (RGNs) containing the RuvC Cas9 mutant Asp10Ala (Cas9D10A) or the HNH Cas9 mutant His840Ala (Cas9H840A) to trigger genome editing via the simultaneous formation of SSBs in endogenous and exogenous DNA. "We report that this strategy based on coordinated in trans paired nicking can improve the three main parameters of DNA editing, i.e., efficiency, specificity, and fidelity, and achieves multiplexing homology-directed DNA addition of large genetic payloads," the authors wrote.

They carried out a series of genotyping and PCR experiments to compare the edits induced by Cas9 or Cas9D10A in human cervix carcinoma HeLa cells. They found that unwarranted and potentially adverse genome-modifying events occur more frequently in cells receiving RGNs containing cleaving Cas9 than in those harboring nicking Cas9D10A.

Next, the researchers sought to investigate homology-directed gene targeting based on inducing DSBs compared to SSBs, and chose the DMD gene at Xp21.2 as the target locus. They found that their in trans paired nicking strategy led to significantly higher percentages of genetically modified cells when compared to those obtained through the standard approach.

To further test their approach, the researchers turned to human pluripotent stem cells (PSCs). Despite their importance to biological research, genetic manipulation of these cells remains limited by the typically low efficiency, specificity, and accuracy of homology-directed gene targeting, even when using programmable nucleases, the team noted. However, the researchers showed that in trans paired nicking was superior to standard gene targeting in achieving efficient cell engineering, even in these cells.

Finally, the researchers looked at whether their approach could be used to co-target two donors, each encoding a different reporter, in order to target genes in a multiplexed manner. For these experiments, flow cytometry showed that RGN-induced nicking led to 15-fold and 23-fold higher frequencies of genetically modified cells, respectively, when compared to standard editing.

"Hitherto, multiplexing genome editing has primarily entailed [non-homologous end-joining]-based manipulations such as those involving RGN pairs for knocking-out two genes simultaneously or for creating chromosomal deletions," the researchers wrote. "Such approaches are, however, not applicable for the targeted addition of new genetic information. We thus sought to capitalize on the higher efficiency, specificity, and accuracy of in trans paired nicking over the conventional DSB-dependent strategy at AAVS1, for testing one-step co-targeting of different alleles."

The high specificity and accuracy of this genome editing approach should be particularly useful when precise genetic manipulation of target cell populations is needed, the researchers added.