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.

CRISPR and Chromothripsis: Proceed with Caution

“…1B). 15 In the paper, the authors observed high overall editing efficiency and a significant increase in micronucleus formation compared to cells transfected without guide RNA. Micronucleation frequency ranged between 4% and 7.5% of edited cells.…”

“…Writing in Nature Genetics, Leibowitz et al put large genomic rearrangements under the microscope once more, revealing a diversity of genomic alterations that have not previously been appreciated. 15 Importantly, the authors identify evidence for chromothripsis-a process where a single catastrophic event shatters the genome, and the resulting reassembly leads to mass genomic reorganization (Fig. 1A).…”

“…1A). 15 The mechanism of shattering and repair is not well understood. However, chromothripsis is frequently accompanied by micronuclei (small nuclei that encapsulate acentric chromosomal fragments).…”

“…Those cells with micronuclei showed functional defects as a result of DNA damage, including defective replication and rupture of the nuclear envelope. 15 The authors also examined the specific genetic consequences of micronucleus formation using the Look-Seq method. This method, developed by the Pellman lab in 2015, combines long-term live-cell imaging with singlecell whole-genome sequencing.…”

“…The authors observed a wide variety of deleterious structural variations, including copy number alterations, copy-neutral loss of heterozygosity, and deleted sequence arising from dicentric bridge formation. 15 The authors attribute many of these alterations to chromothripsis and exclude the possibility of these variants occurring through simple mechanisms such as resection (as this is typically limited to a few kilobases).…”