“On-target toxicity of genome” editing micronucleus induction and chromothripsis

<p>The nature of genome editing is the DNA double-strand break at the target site. Although the "off-target toxicity" due to sequence homology has been the focus of attention as the toxicity of genome editing, from the viewpoint of genotoxicity, the safety of DNA double-strand breaks themselves, i.e., "on-target toxicity" is important.</p><p>As in the case of radiation, DNA double-strand breaks are likely to cause chromosomal aberrations, and it was reported last year that chromosomal aberrations such as micronuclei induced by genome editing with CRISPR-Cas9 can cause Chromothripsis. This has attracted attention as an "on-target toxicity" of genome editing because it causes massive genomic instability by single event and can be a direct cause of cancer and genetic disease. We are attempting to artificially synthesize fusion genes (chromosomal translocations) between the genes that have been cut by genome editing at different locations. The resulted chromosomal aberrations have been examined by micronucleus induction and chromosome painting analysis.</p><p>To create translocation fusion genes between the ALK gene on chromosome 2 and the MET or SMO genes on chromosome 7 in HEK293T cells, we designed sgRNA expression vectors that cut specific sites in each gene and transfected them together with Cas9 expression vectors.</p><p>Chromosome painting analysis of ALK/MET and ALK/SMO fusions immediately after genome editing showed that the target chromosomal translocation was observed in a few percent of the cells, and more frequently, the chromosomal breaks and fragments were observed. On the other hand, we also examined micronucleus induction and observed a large number of micronuclei, which turned out to be an artifact derived from the vector plasmid we used. Therefore, we are now examining micronucleus induction using a method of transfection of Cas9 protein and sgRNA complexes without using a plasmid.</p><p>In the future, we plan to investigate the possibility that these abnormalities result into chromothripsis.</p>