CRISPRthripsis: The Risk of CRISPR/Cas9-induced Chromothripsis in Gene Therapy

Abstract

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 nuclease system has allowed the generation of disease models and the development of therapeutic approaches for many genetic and non-genetic disorders. However, the generation of large genomic rearrangements has raised safety concerns for the clinical application of CRISPR/Cas9 nuclease approaches. Among these events, the formation of micronuclei and chromosome bridges due to chromosomal truncations can lead to massive genomic rearrangements localized to one or few chromosomes. This phenomenon, known as chromothripsis, was originally described in cancer cells, where it is believed to be caused by defective chromosome segregation during mitosis or DNA double-strand breaks. Here, we will discuss the factors influencing CRISPR/Cas9-induced chromothripsis, hereafter termed CRISPRthripsis, and its outcomes, the tools to characterize these events and strategies to minimize them.

Keywords: CRISPR/Cas9; chromosomal instability; chromothripsis; gene therapy; genome editing; genotoxicity; micronuclei.

A DNA double-strand break can lead to the formation of acentric chromosome fragments and micronuclei. After DNA condensation, the chromosome fragment is shattered generating multiple DNA fragments, which are reincorporated in the nuclear genome forming a chromotriptic chromosome. If not reincorporated in the genome, these fragments can be lost (deleted fragments) or form double-minute chromosomes.

Figure 1.

CRISPR/Cas9-induced events. The CRISPR/Cas9 complex is driven to a specific genomic site thanks to the complementarity of the gRNA to the target DNA region. Then, Cas9 induces a DNA double-strand break (DSB, red double arrow) 3-4 nucleotides upstream of the protospacer adjacent motif (PAM). This DSB can be repaired by the homology-directed repair (HDR) in the presence of a donor DNA or by non-homologous end joining (NHEJ) to generate InDels (insertion and deletions). However, if the DSB is not correctly repaired, one of the possible outcomes is CRISPRthripsis, with the formation of micronuclei containing an acentric chromosome fragment (light red box), chromosome bridges, and chromothriptic chromosomes (colored blocks represent shuffled DNA) (see Fig. 2).

Figure 2.

Chromothripsis leads to massive genomic rearrangements. (A) A DNA double-strand break (DSB, red double arrow) can lead to the formation of an acentric chromosome fragment (light red box). After mitosis, this fragment can be enwrapped by a lipid membrane forming a micronucleus (red circle). After DNA condensation, the chromosome fragment is shattered generating multiple DNA fragments, which are eventually reassembled and reincorporated into the nuclear genome forming a chromotriptic chromosome. If not reincorporated in the genome, these fragments can be lost (deleted fragments) or form double-minute chromosomes. (B) A DNA DSB can lead to the formation of sister chromatids with shortened or absent telomeres that form a chromosomal bridge. During the first mitosis, cell division leads to breakage of the chromosomal bridge, which can induce local DNA fragmentation and chromothripsis. During the second mitosis, the broken chromosome missegregates, potentially leading to the formation of micronuclei, which also trigger chromothripsis (see panel A).