CHK2 and PARP inhibitor response

A CRISPR/Cas9 screen was conducted in murine Eu-Myc lymphoma cells to investigate PARP inhibitor related toxicity. We focused on the DNA damage response and generated a custom CRISPR/Cas9 library targeting 174 established DNA repair genes, 10 essential genes (e.g. Rpa3, Rpl11, Psmb2) and Hprt1, each with 7 guides per gene, together with 150 non-targeting controls. Cas9 expressing Eu-Myc lymphoma cells were transduced with the sgRNA pools, a subset of the cells were collected and frozen down for a baseline control (day 3). The remaining cells were either treated with olaparib (40nM or 2uM), or left untreated, followed by collection at day 21. Genomic DNA was extracted from the viable cells, sgRNA sequences were amplified and subjected to amplicon sequencing to determine enrichment.To determine whether blocking p53 phosphorylation would impair PARPi induced cell killing, a CRISPR/Cas9 base editor was used to target a key site phosphorylated by CHK2. Nucleofection was used to deliver a base editor plasmid pCAGIG-hA3A-BE3 into murine Eu-Myc lymphoma cells expressing an sgRNA targeting Trp53 Ser23. GFP positive cells were collected by FACS sorting, then they were treated with DMSO, S63845 (500nM), RG-7388 (1uM) or olaparib (2uM) for 7 days. After treatment, cells were harvested for genomic DNA extraction. The region surrounding Ser23 in Trp53 was amplified by PCR, followed by Illumina sequencing to determine the enrichment of each type of base edit.