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

Abstract

Genome editing has promising therapeutic potential for genetic diseases and cancer (1, 2). However, the most practicable current approaches rely on the generation of DNA double-strand breaks (DSBs), which can give rise to a poorly characterized spectrum of structural chromosomal abnormalities. Here, we show that a catastrophic mutational process called chromothripsis is a previously unappreciated consequence of CRISPR-Cas9-mediated DSBs. Chromothripsis is extensive chromosome rearrangement restricted to one or a few chromosomes that can cause human congenital disease and cancer (3–6). Using model cell systems and a genome editing protocol similar to ones in clinical trials (7) (NCT03655678, NCT03745287) we show that CRISPR-Cas9-mediated DNA breaks generate abnormal nuclear structures—micronuclei and chromosome bridges—that trigger chromothripsis. Chromothripsis is an on-target toxicity that may be minimized by cell manipulation protocols or screening but cannot be completely avoided in many genome editing applications.

Small but mighty: the causes and consequences of micronucleus rupture

“…3a, b). Together with other recent work using micronuclei whose contents were known a priori, it is now clear that micronuclei generate chromothripsis at remarkably high rates, regardless of whether the bulk of the micronucleus is reincorporated into the primary nucleus [48][49][50][51] .…”

“…1b and 3a) [44][45][46][47] . The suggestion that micronuclei might lead to chromothripsis was later affirmed, as discussed below [48][49][50][51] (Fig. 3b).…”

“…The lack of requirement of bulk reincorporation is surprising, as it was thought that fragments needed to be reincorporated into a repair-competent primary nucleus for rearrangement to occur 48,50,62 . However, in these cases, it is possible that some fragments not visible by microscopy are reincorporated and repaired.…”

“…Notably, micronuclei and bridge formation frequently follow one another during tumor development 64,69,70 . One possible explanation for this sequence is that micronuclei arising from DNA double-strand breaks leave behind the uncapped centromere-containing part of the chromosome, which is then susceptible to improper repair and chromosome bridge formation 50 . Chromosome bridges can then undergo cycles of instability, including the formation of new micronuclei 64,65,69,71 .…”

Small but strong: Mutational and functional landscapes of micronuclei in cancer genomes

“…Micronucleus is the most accepted hypothesis of chromothripsis origin because it is spatially separated from the main nucleus and is subject to extensive mutagenesis or Zhang et al demonstrated that micronucleated chromosomes undergo fragmentation and subsequent reassembly, providing direct evidence that chromothripsis occurs in micronucleus 82. Recently, SCS analysis revealed that chromothripsis occurs in 10% intact micronuclei in G2 daughter cells32 and in 66.7% of micronuclei that are persisted for one cell cycle,83 indicating chromothripsis is an immediate Although PCC is thought to be the leading cause of micronucleus shattering, whether the PCC phenotype indeed reflects a fragmented micronucleus remains unclear. Directly testing this hypothesis is technically challenging because it requires the combination of SCS after creation and detection of PCC by imaging.…”