CRISPR-Cas9 screen upon Olaparib+CldU treatment

DNA single-strand breaks (SSBs) disrupt DNA replication and induce chromosome breakage. However, whether SSBs induce chromosome breakage when present in nascent strands behind replication forks or template strands ahead of replication forks is unclear. To address this question, we exploited an exquisite sensitivity of SSB repair-defective human cells lacking PARP activity or XRCC1 to the thymidine analog 5-chloro-2'-deoxyuridine (CldU). We show that incubation with CldU in these cells results in chromosome breakage, sister chromatid exchange, and cytotoxicity by a mechanism that depends on the S phase activity of uracil DNA glycosylase (UNG). Importantly, we show that CldU incorporation in one cell cycle is cytotoxic only during the following cell cycle when it is present in template DNA. In agreement with this, while UNG induces SSBs both in nascent strands behind replication forks and in template strands ahead of replication forks, only the latter triggers fork collapse and chromosome..., Two independent clones of P53-deficient RPE-1 cells expressing Cas9 nuclease were infected with the Brunello library containing gRNAs targeting the whole genome (coverage 500x). After selection for infected cells and population sampling 9 days after infection (samples \"T6clon4\" or \"T6clon7\"), the starting cell populations were divided into three groups. One group was an untreated control (sample name from the end of treatment: \"T21clon4UN\" or \"T21clon7UN\"), a second group was treated continuously with a sub-lethal concentration of olaparib (500 nM) (sample name from the end of the treatment: \"T21clon4OLA\" or \"T21clon7OLA\"), and a third population was continuously treated with both 500 nM olaparib and 0.2 uM CldU; a combination that kills ~20% of cells (IC20) (samples name from the end of the treatment: \"T21clon4OLACldU20\" or \"T21clon7OLACldU20\"). Infected cell populations were sampled every 3 days from days 9-24, genomic DNA was isolated, and integrated gRNA abundances were measured rel..., , # CRISPR-Cas9 screen upon Olaparib+CldU treatment

## Description of the data and file structure

TSV files from the raw counts of sequencing.

## Sharing/Access information

These data are discussed in Serrano-Benitez et al. 2023, if you require more data from the sequencing please email: [[e-mail]](mailto:[e-mail])