DOI: 10.5061/dryad.2v6wwpzm9
title: CRISPR/Cas9 genome-wide screens for sensitisers and suppressors of AZD6738 efficacy in Atm WT and Atm KO mESCs

description: The protein kinase ATR plays pivotal roles in DNA repair, cell cycle
 checkpoint engagement and DNA replication. Consequently, ATR inhibitors
 (ATRi) are in clinical development for the treatment of cancers, including
 tumours harbouring mutations in the related kinase ATM. However, it still
 remains unclear which functions and pathways dominate long-term ATRi
 efficacy, and how these vary between clinically relevant genetic
 backgrounds. Elucidating common and genetic-background specific mechanisms
 of ATRi efficacy could therefore assist patient stratification and
 pre-empting drug resistance. Here, we use CRISPR-Cas9 genome-wide
 screening in ATM-deficient and proficient mouse embryonic stem cells to
 interrogate cell fitness following treatment with the ATRi, ceralasertib
 (AZD6738). We identify factors that enhance or suppress ATRi efficacy,
 with a subset of these requiring intact ATM signalling. Overall,
 our work identifies novel biomarkers of ATRi efficacy in ATM-proficient
 and ATM-deficient cells, and suggests new ATM-dependent pathways that
 compensate in the absence of functional ATR.
description: This dataset contains the results of CRISPR/Cas9 whole-genome
 screens. Atm WT and Atm KO mouse embryonic stem cells (mESCs) were
 infected with pooled plasmid libraries targeting every gene in the mus
 musculus genome (Kosuke Yusa V2 library,
 https://doi.org/10.1038/nbt.2800), selected in puromycin for 14 days, and
 then subjected to either DMSO (control), low dose IC10 ATRi AZD6738 (100
 nM Atm KO, 350 nM Atm WT) or high dose IC90 ATRi AZD6738 (350 nM Atm KO,
 900 nM Atm WT) treatment for 6 days followed by a 2 day recovery in
 drug-free medium. Cells were partitioned into three technical replicates
 following 6 days puromycin treatment. Samples were harvested and DNA
 extracted at the following time points for each genotype and technical
 replicate: 48 h (indicative of the inital library represeation prior to
 puromycin selection), Day 14 (indicative of the pre-treatment library
 representation following puromycin selection) and post DMSO/IC10/IC90
 treatment and recovery. PCR was used to isolate and amplify the
 sgRNA-containing lentiviral casettes that were contained on plasmids in
 the CRISPR library, and were subsequently integrated into the host genome.
 The sgRNA abundances are included here. MAGeCK (V0.5.5,
 https://sourceforge.net/p/mageck) was used to obtained significance values
 for gene enrichment/depletion from these abundances by either summing the
 counts across technical replicates, or treating them as independent
 replicates using the inbuilt MAGeCK replicates function. The input
 and output files are included here, along with a summary file containing
 detailed descriptions of each sample, and the MAGeCK commands
 used.
description: Due to noise in low read-count essential genes sometimes
 confounding drug-treatment results, sgRNAs with low read-counts in the
 DMSO control were excluded prior to analysis. No sgRNAs were excluded for
 essential-gene comparisons between 48 h and Day 14 (pre-treatment)
 samples. For summed analyses, sgRNAs with DMSO read-counts &lt;30
 after combining the replicates were excluded (SUM_30). For replicates
 analyses, sgRNAs with DMSO read-counts of 0 in any 2 replicates and
 &lt;10 in the third were excluded (REP_10). Sample
 B33 (IC10 techncial replicate 1 in WT mESCs) was excluded from downstream
 analyses after PCA and other exploratory analyses revealed close
 resemblance of this samples to DMSO-treated WT mESCs.
 Files: README.rtf - information on
 abbreviations/acronymns, column/row headers and files included to
 facilitate usage of our data summary file.xlsx -
 details about samples in our screens and analyses performed using
 MAGeCK. readcounts.tsv - guide abundances from our
 screens. .csv files in the format cell
 line_method_dose_exclusion threshold - MAGeCK input files (descriptions of
 data contained within each file are provided in the summary
 file). MAGeCK output files.xlsx - files output by
 MAGeCK (each tab contains a seperate analysis, named in accordance with
 the respective input file.)