Results from previous publications
Previous experiments on human cell lines showed that cuts, also called double-strand breaks (DSB), caused by CRIPSR/Cas gene scissors in the genome can lead to large, unwanted DNA rearrangements (see, for example, Leibowitz et al. (2021), Zuccaro et al. (2020), Weisheit et al. (2020)):
The genome of most target organisms is organised in chromosomes. A chromosome consists of two chromosome arms that are held together at a centromere. During cell division, the centromere serves to ensure that both daughter cells each receive a copy (a so-called sister chromatid) of the chromosomes. Most of the commonly used gene scissor approaches cut the DNA of the target chromosome into two pieces: one piece remains linked to the centromere of the chromosome and the other piece is no longer linked 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 nucleus. Various factors can lead to the presence of micronuclei in cells, e.g. as a result of an unrepaired DNA double strand break, and they are an indication of genetic instability.
Examination of the DNA-fragments in the micronuclei showed that some were extensively restructured and existed in different copies, an effect called “chromothripsis”. Chromothripsis is a mutational process triggered by the generation of the DSB at the target region. In this process, the copy number of DNA segments can vary greatly due to deletions as well as duplication of the regions. Chromothripsis often occurs in human cancer cells and in certain hereditary diseases.
Results of the current study by Papathanasiou et al.
The Papathanasiou et al. study focused on unintended changes that can be induced by CRISPR/Cas during the early embryonic development of mouse embryos. The gene scissors were introduced as an enzyme complex into mouse zygotes, each with two different gRNAs (recognition component of the gene scissors), through which the gene scissors were brought to the respective target sites (i.e. the second and seventeenth chromosome, respectively). The scientists performed the experiments on the mouse embryos to investigate whether DNA fragments are also enclosed in micronuclei and whether, as in other studies with human cell lines, there was a loss of whole chromosomes. This involved examining the 8-cell stage of the injected mouse embryos in more detail: using microscopic analyses, the scientists were able to prove that micronuclei formation does occur. For a more precise analysis of the concrete chromosomal changes, they subsequently isolated the individual cells in the mouse embryos (a total of 24 embryos and 202 individual cells) and used a technique called single cell sequencing.
In some cells they observed micronuclei, so-called chromosome bridges and the loss of entire chromosomes. Chromosome bridges connect the shortened sister chromatids with each other, which are missing the piece of DNA enclosed in the micronuclei. The sister chromatids connected by chromosome bridges are jointly inherited by a daughter cell during cell division, in which case the other daughter cell does not receive a copy of this chromosome. This also explains the loss of whole chromosomes in other cells of the respective embryo. Chromothripsis was not detected in the experiments. This is most likely due to the relatively small number of cells that were examined.
This study is distinguished by the precise single cell analysis of the genome of the mouse embryos. Single cell sequencing makes it possible to trace how chromosomal aberrations arise during embryonic development and are passed on in subsequent cell divisions.
Papathanasiou, S., Markoulaki, S., Blaine, L.J. et al. Whole chromosome loss and genomic instability in mouse embryos after CRISPR-Cas9 genome editing. Nat Commun 12, 5855 (2021). https://doi.org/10.1038/s41467-021-26097-y
Leibowitz ML, Papathanasiou S, Doerfler PA, Blaine LJ, Sun L, Yao Y, Zhang C-Z, Weiss MJ, Pellman D (2021) Chromothripsis as an on-target consequence of CRISPR–Cas9 genome editing. Nature Genetics. Doi: 10.1038/s41588-021-00838-7
Zuccaro MV, Xu J, Mitchell C, Marin D, Zimmerman R, Rana B, Weinstein E, King RT, Palmerola KL, Smith ME, Tsang SH, Goland R, Jasin M, Lobo R, Treff N, Egli D (2020) Allele-Specific Chromosome Removal after Cas9 Cleavage in Human Embryos. Cell. doi: https://doi.org/10.1016/j.cell.2020.10.025
Weisheit I, Kroeger JA, Malik R, Klimmt J, Crusius D, Dannert A, Dichgans M, Paquet D (2020) Detection of Deleterious On-Target Effects after HDR-Mediated CRISPR Editing. Cell Rep 31 (8):107689. doi: https://doi.org/10.1016/j.celrep.2020.107689