Chromosome instability induced by a single defined sister chromatid fusion

Abstract

Chromosome fusion is a frequent intermediate in oncogenic chromosome rearrangements and has been proposed to cause multiple tumor-driving abnormalities. In conventional experimental systems, however, these abnormalities were often induced by randomly induced chromosome fusions involving multiple different chromosomes. It was therefore not well understood whether a single defined type of chromosome fusion, which is reminiscent of a sporadic fusion in tumor cells, has the potential to cause chromosome instabilities. Here, we developed a human cell-based sister chromatid fusion visualization system (FuVis), in which a single defined sister chromatid fusion is induced by CRISPR/Cas9 concomitantly with mCitrine expression. The fused chromosome subsequently developed extra-acentric chromosomes, including chromosome scattering, indicative of chromothripsis. Live-cell imaging and statistical modeling indicated that sister chromatid fusion generated micronuclei (MN) in the first few cell cycles and that cells with MN tend to display cell cycle abnormalities. The powerful FuVis system thus demonstrates that even a single sporadic sister chromatid fusion can induce chromosome instability and destabilize the cell cycle through MN formation.

Target residence of Cas9-sgRNA influences DNA double-strand break repair pathway choices in CRISPR/Cas9 genome editing

“…S8b). Lack of palindromic sequences in these PCR products is consistent with previous studies [ 47 , 52 – 54 ]. It is likely that the collision between DNA replication and Cas9-sgRNA could generate long ssDNA at the lagging strand end and little or no ssDNA overhang at the leading strand end.…”

“…In addition, repair of the three-ended DSBs provides an opportunity for the DNA ends of two sister chromatids to rejoin, not only creating a palindromic chromosome with two centromeres or no centromere [48,49], but also leaving the third end for potential translocation. Both dicentric and acentric palindromic chromosomes are unstable and serves as a potential source for chromothripsis and complex chromosomal rearrangements including large deletions and insertions at the target site [46][47][48][49]64]. Therefore, this study identifies a potential mechanism underlying on-target chromosomal rearrangements previously detected in CRISPR/Cas9 genome editing [65][66][67][68][69] and suggests that Cas9-sgRNA variants with shorter post-cleavage target residence might be a strategy to reduce the occurrence of dangerous chromosomal rearrangements such as palindromic SCF.…”

“…7e). These two ends each can rejoin with the other blunt end of the DSB, or have a potential to directly ligate with each other, the latter generating a palindromic chromosome from sister chromatid fusion (SCF) and potentially promoting chromatid breakage-fusion-bridge (BFB) cycles [46][47][48][49] (Fig. 7e).…”

Micronucleus Is Not a Potent Inducer of cGAS-STING Pathway

“…Consequently, it serves as a reliable biomarker for chromosome instability (Krupina et al, 2021). MNs can form as a result of chromosome missegregation due to lagging chromosomes, acentric chromosome fragments (Fenech et al, 2011; Thompson & Compton, 2011) and breakage of anaphase chromatin bridges (Kagaya et al, 2020; Umbreit et al, 2020). Genetic material in MNs undergoes dysregulated DNA replication and DNA damage repair (Crasta et al, 2012), potentially leading to chromothripsis events (Zhang et al, 2015; Ly et al, 2016, 2019; Kneissig et al, 2019; Umbreit et al, 2020).…”

Cas9-sgRNA target residence alters DNA double strand break repair pathway choices in CRISPR/Cas9 genome editing

“…In addition, repair of the three-ended DSBs provides an opportunity for the DNA ends of two sister chromatids to rejoin, not only creating a palindromic chromosome with two centromeres or no centromere [42,43], but also leaving the third end for potential translocation. Both dicentric and acentric palindromic chromosomes are unstable and serves as a potential source for chromothripsis and complex chromosomal rearrangements including large deletions and insertions at the target site [40][41][42][43]54]. Therefore, this study identi es a potential mechanism underlying on-target chromosomal rearrangements previously detected in CRISPR/Cas9 genome editing [55][56][57][58][59].…”