Cas9-induced large deletions and small indels are controlled in a convergent fashion

On-target effects are unintended changes in the genome of genome-edited target organisms that can occur when applying the gene scissors CRISPR/Cas. On-target effects include major rearrangements of the genome, such as restructuring large parts of the DNA, large insertions of DNA sequences or the deletion of DNA sections. So far it has not been fully clarified which processes in the cell are involved in the development of these unintended on-target effects.

The gene scissors CRISPR/Cas introduce double-strand breaks in the genome at the target region. In other words: the DNA is cut in two. The two loose ends of the DNA are recognized by the cell as a DNA damage and certain repair mechanisms are activated. These are the NHEJ repair, which is error-prone and often causes small changes at the target sequence (i.e. SDN-1 applications of the gene scissors) and the homology directed repair, which can integrate introduced DNA templates into the target sequence (SDN-2/SDN-3 applications). As an alternative to NHEJ repair, a cellular repair mechanism called microhomology-mediated end joining (MMEJ) can also be activated, which causes short deletions at the target sequence.

Kosicki et al, 2020 investigate in their study which repair mechanisms are involved in the development of on-target effects. The scientists generated embryonic stem cells from mice in which individual repair genes were knocked out. The deactivated repair genes are either part of the NHEJ, MMEJ or homology directed repair. It was found that the NHEJ repair rather prevents larger deletions (on-target effects) and the MMEJ repair rather favors such deletions. Every now and then, scientists block NHEJ repair in CRISPR experiments in order to increase the efficiency of homology directed repair and thus promoting the incorporation of DNA templates. As can now be concluded from this study, large deletions, i.e. on-target effects, are favored if NHEJ is inhibited.

Kosicki M, Allen F, Bradley A (2020) Cas9-induced large deletions and small indels are controlled in a convergent fashion. bioRxiv:2020.2008.2005.216739. doi:10.1101/2020.08.05.216739