AZD1775 (IC25 and IC95) CRISPRi screen using DDR and cell cycle focused library in RPE TP53-/- dCas9 KRAB cell line

RPE-1-hTERT dCas9-KRAB TP53-/- cells were transduced with a lentiviral CRISPRi library (DDR CRISPRi allelic series library targeting 2294 genes with pLG1 (pCRISPRia- v2) backbone provided by the Corn Lab, ETH Zurich) at an MOI of 0.15 via spinfection. Two separate biological replicates of the screen were performed i.e., two separate library transductions. The next day following transduction, fresh medium containing puromycin (2 µg/ml) was added. Cells were negatively selected with puromycin for a total of 9 days, at which point each replicate was divided into different treatments and subcultured every 3 days for a total of 9 days. Cell pellets were frozen at the end of the day 9 treatment (day 19 post-transduction) and as well as day 3 post-transduction for gDNA isolation. Library coverage of at least x750 cells per sgRNA was maintained at every step for the DMSO and the IC25 arm, and at least x300 cells per sgRNA for the IC95 arm. gDNA from cell pellets were isolated using the QIAamp Blood Midi Kit (Qiagen, Cat# 51185) and genome-integrated sgRNA sequences were amplified by PCR using NEBNext Ultra II Q5 Mastermix (New England Biolabs, Cat# M5044L). Sample preparations were performed by amplifying the sgRNA with forward and reverse primers containing single TruSeq indexes. The final magnetic bead-purified products (purified using SPRI MBSpure beads obtained from the Vienna Biocenter, Austria) were sequenced on an Illumina HiSeq3000 system to determine sgRNA representation in each sample. A 15% PhiX spike-in was used.