Novel Cas9-nickase technique repairs mutation without off-target modifications

While the CRISPR-Cas system has shown potential for stem cell-based therapies, the answer for how to make interventions that are safe enough for human use remains elusive. One such approach, called Cas9-nickase, looks to provide a way to use the system without causing off-target mutations. When using this technique, however, the targeted site has been prone to additional unwanted on-target modifications. Recent work holds clues for how to make such a system work better.

Alateeq et al. have demonstrated a technique that combines Cas9-double nickase with a piggyBac transposon-modified donor vector for targeted editing of the DNA mutation of induced pluripotent stem cells of patients with beta-thalassemia, a common and sometimes severe inherited blood disorder. Using a synonymous protospacer adjacent motif-blocking mutation for one guide and destroying the binding site for the other guide, the authors were able to achieve a repair of the disease-causing mutation with no detectable off-target modifications.

The authors have said that unexpected on-target mutagenic activity of monomeric Cas9-nickase in iPSCs highlights continued safety concerns with the system.

“There is much emphasis on the versatility and ease of CRISPR editing, but it is a very human thing to do to ignore the details,” said Ernst Wolvetang, an author on the paper. “Our paper is one of only a few that make its unintended effects a feature rather than burying it in the supplementary data.”

To verify the system’s effectiveness, the group delivered a CRISPR-mediated transcriptional activator, which allowed for accurate splice junction modeling in the cell lines.

Wolvetang said he hopes the group’s method for rapidly and easily assessing the system’s ability to restore gene function will see wider use in the research community.

Source: “Identification of on-target mutagenesis during correction of a beta-thalassemia splice mutation in iPS cells with optimised CRISPR/Cas9-double nickase reveals potential safety concerns,” by Suad Alateeq, Dmitry Ovchinnikov, Timothy Tracey, Deanne Whitworth, Abdullah Al-Rubaish, Amein Al-Ali, and Ernst J. Wolvetang, APL Bioengineering (2018). The article can be accessed at https://doi.org/10.1063/1.5048625 .