TY - JOUR
T1 - Double-strand break toxicity is chromatin context independent
AU - Friskes, Anoek
AU - Koob, Lisa
AU - Krenning, Lenno
AU - Severson, Tesa M.
AU - Koeleman, Emma S.
AU - Vergara, Xabier
AU - Schubert, Michael
AU - van den Berg, Jeroen
AU - Evers, Bastiaan
AU - Manjón, Anna G.
AU - Joosten, Stacey
AU - Kim, Yongsoo
AU - Zwart, Wilbert
AU - Medema, René H.
N1 - Publisher Copyright: © The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.
PY - 2022/9/23
Y1 - 2022/9/23
N2 - Cells respond to double-strand breaks (DSBs) by activating DNA damage response pathways, including cell cycle arrest. We have previously shown that a single double-strand break generated via CRISPR/Cas9 is sufficient to delay cell cycle progression and compromise cell viability. However, we also found that the cellular response to DSBs can vary, independent of the number of lesions. This implies that not all DSBs are equally toxic, and raises the question if the location of a single double-strand break could influence its toxicity. To systematically investigate if DSB-location is a determinant of toxicity we performed a CRISPR/Cas9 screen targeting 6237 single sites in the human genome. Next, we developed a data-driven framework to design CRISPR/Cas9 sgRNA (crRNA) pools targeting specific chromatin features. The chromatin context was defined using ChromHMM states, Lamin-B1 DAM-iD, DNAseI hypersensitivity, and RNA-sequencing data. We computationally designed 6 distinct crRNA pools, each containing 10 crRNAs targeting the same chromatin state. We show that the toxicity of a DSB is highly similar across the different ChromHMM states. Rather, we find that the major determinants of toxicity of a sgRNA are cutting efficiency and off-target effects. Thus, chromatin features have little to no effect on the toxicity of a single CRISPR/Cas9-induced DSB.
AB - Cells respond to double-strand breaks (DSBs) by activating DNA damage response pathways, including cell cycle arrest. We have previously shown that a single double-strand break generated via CRISPR/Cas9 is sufficient to delay cell cycle progression and compromise cell viability. However, we also found that the cellular response to DSBs can vary, independent of the number of lesions. This implies that not all DSBs are equally toxic, and raises the question if the location of a single double-strand break could influence its toxicity. To systematically investigate if DSB-location is a determinant of toxicity we performed a CRISPR/Cas9 screen targeting 6237 single sites in the human genome. Next, we developed a data-driven framework to design CRISPR/Cas9 sgRNA (crRNA) pools targeting specific chromatin features. The chromatin context was defined using ChromHMM states, Lamin-B1 DAM-iD, DNAseI hypersensitivity, and RNA-sequencing data. We computationally designed 6 distinct crRNA pools, each containing 10 crRNAs targeting the same chromatin state. We show that the toxicity of a DSB is highly similar across the different ChromHMM states. Rather, we find that the major determinants of toxicity of a sgRNA are cutting efficiency and off-target effects. Thus, chromatin features have little to no effect on the toxicity of a single CRISPR/Cas9-induced DSB.
UR - http://www.scopus.com/inward/record.url?scp=85138478649&partnerID=8YFLogxK
U2 - https://doi.org/10.1093/nar/gkac758
DO - https://doi.org/10.1093/nar/gkac758
M3 - Article
C2 - 36107780
SN - 0305-1048
VL - 50
SP - 9930
EP - 9947
JO - Nucleic acids research
JF - Nucleic acids research
IS - 17
ER -