Chromothripsis as an On-Target Consequence of CRISPR–Cas9 Genome Editing

Abstract

Genome editing has therapeutic potential for treating genetic diseases and cancer. However, the currently most practicable approaches rely on the generation of DNA double-strand breaks (DSBs), which can give rise to a poorly characterized spectrum of chromosome structural abnormalities. Here, using model cells and single-cell whole genome sequencing, as well as by editing at a clinically relevant locus in clinically relevant cells, we show that CRISPR-Cas9 editing generates structural defects of the nucleus—micronuclei from missegregation of acentric chromosome fragments during mitosis, and chromosome bridges from fusion of cut centric sister chromatids—that initiate a mutational process called chromothripsis. Chromothripsis is extensive chromosome rearrangement restricted to one or a few chromosomes that can cause human congenital disease and cancer. These results demonstrate that chromothripsis is a previously unappreciated on-target consequence of CRISPR-Cas9-generated DSBs. As genome editing is implemented in the clinic, the potential for extensive chromosomal rearrangements should be monitored, and methods for genome editing that minimize DSB formation and chromosome missegregation into micronuclei should be considered. Genome editing produces acentric micronuclei, however micronuclei from whole chromosomes also often arise in cancer and embryogenesis. Despite the presence of centromeric DNA on these chromosomes, they are often unable to properly segregate into primary nuclei. The reason for persistent missegregation of micronuclei is unknown, although early work suggested that micronuclei might completely fail to assemble a kinetochore. In this thesis, we show that kinetochores can be functional in micronuclei, however they exhibit an assembly defect. This defect manifests as reduced levels of inner kinetochore proteins, and reduced levels or a complete lack of outer kinetochore proteins. Because micronuclei are known to have reduced import, we suggest that defects in kinetochore assembly in micronuclei are caused by a failure to import kinetochore proteins into micronuclei.