Chromothripsis as an on-target consequence of CRISPR-Cas9 genome editing.

This study deals with unintended on-target changes in the genome of human cell lines after a DNA double-strand break (DSB) was generated at the target region using the gene scissors CRISPR/Cas.
It is important to consider the structure and function of chromosomes to properly understand the effects found here: Humans have two complete sets of chromosomes, one from the father and the other from the mother; one speaks of homologous chromosomes in this sense. A chromosome consists of two copies, also called sister chromatids, which are held together by a centromere. During cell division, the centromere is used to ensure that both daughter cells receive one copy (one sister chromatid each) of the chromosomes. In the course of the cell cycle, the sister chromatids are then doubled again.
Here is what was found in the study: When the gene scissors CRISPR/Cas was used, the DNA was cut into two parts, one part that remained connected to the centromere of the chromosome, and another part that was no longer connected to the centromere. These “free” pieces of DNA can be enclosed in so-called micronuclei, which are then present in the cell in addition to the cell nucleus. Micronuclei can arise in cells through various effects, for example as a result of an unrepaired DNA double-strand break. Micronuclei are an indication of genetic instability.
The scientists investigated human cell lines that were equipped with the gene scissors CRISPR/Cas in form of already preassembled ribonucleoproteins (RNPs) to change different target regions on four different chromosomes in independent experiments. It was found that the frequency of the formation of micronuclei is between 2.1-6.6%. The use of CRISPR/Cas in the human cells appears to increase the formation of micronuclei in comparison to the controls. The experiments, in which the DNA for the gene scissors components are stably integrated in the genome, but have yet to be expressed to form the gene scissors, gave similar results.
The DNA fragments in the micronuclei of the genome-edited human cell lines were examined more closely: the DNA fragments have been massively restructured and some are present in different copies. An effect called “chromothripsis” has occurred. Chromothripsis describes a mutation process that can occur in a cell as a result of a one-time “catastrophic” event: in this case, when the DNA DSB is generated at the target region of the CRISPR/Cas experiments. Structural rearrangements occur and the number of copies of the DNA sections fluctuates greatly due to deletions and duplications of these areas. Chromothripsis is common in human cancer cells and in certain hereditary diseases. This effect is particularly problematic when CRISPR/Cas is used therapeutically. The authors of the study argue that the of Cas variants must be further developed, which do not cut both DNA double strands and thus make this effect less likely.
In plants, this link between the use of CRISPR/Cas and chromothripsis has not yet been described. Chromothripsis, however, has already been observed during the production of double haploid plants .

Leibowitz ML, Papathanasiou S, Doerfler PA, Blaine LJ, Yao Y, Zhang C-Z, Weiss MJ, Pellman D (2020) Chromothripsis as an on-target consequence of CRISPR-Cas9 genome editing. bioRxiv:2020.2007.2013.200998. doi:10.1101/2020.07.13.200998