CRISPR Screening to Identify DNA Repair Pathway Targets That Sensitize Tumor Cells to Chemotherapy
Creators: name: Guo, Alyssa
Publisher: name: The University of North Carolina at Chapel Hill University Libraries
Year: 2019

description: Background: DNA Damage Response (DDR) is crucial for maintaining genomic stability by detecting and repairing damage to cellular DNA, and by eliminating cells with irreparable DNA. Disruption of the DDR is observed in many cancers and is hypothesized to contribute to the mutagenic processes driving tumorigenesis, influence tumor development, and determine the drug treatment resistance. We implemented a custom Cluster Regularly Interspaced Short Palindromic Repeats (CRISPR) library targeting DDR genes to identify those genes that when expression is lost result in synthetic lethality (sensitization) or synthetic viability (resistance) in conjunction with chemotherapeutic treatments.  Furthermore, because different tumor types lead to different treatment responses, we utilized two cancer mouse cell line models, C3Tag2 (mammary cancer) and KPC4662 (P53 knock-out pancreatic cancer), to identify one or more pathways that are in common with multiple cancer types. Methods: 1) Doxorubicin (Adriamycin), 5-flurouracil (5-FU), and Camptothecin (CPT) are chemotherapeutic drugs that were selected based on their distinct mechanisms of action. 2) The DDR CRISPR library was cloned into a DD-CAS9 lentiviral vector (DD-Cas9 with filler sequence and Venus (EDCPV) was a gift from Raffaella Sordella (Addgene plasmid #90085; http://n2t.net/addgene:90085; RRID: Addgene_90085)), containing mVenus as a marker and a destabilized Cas9 allowing for timed activation of Cas9 activity.  Following low multiplicity of infection, cell populations were sorted to give a uniformly transduced population based on mVenus expression.  3) During the experiment, Cas9 was stabilized for 24 hours in the respective cell populations followed by 48 hours of drug exposure.  The cells were then allowed to recover, with passages every 2 days, for 10 population doublings in C3Tag2, and 10 and 15 in KPC4662. 4) Genomic DNA was then harvested.  The sgRNA sequence was PCR amplified from the integrated virus.  The 217 base pair product was then subjected to 8 PCR cycles to add Illumina sequencing adapters and indices, resulting in a 326 base pair product. Results & Conclusion: Drug concentrations that resulted in no more than a 50% reduction in cell number as compared to no drug treatment and allowed cells to recover equally were (for C3Tag2, KPC4662 cell lines): Adriamycin (5 nM, 5nM), 5-FU (100 nM, 200 nM), CPT (10 nM, 10 nM). Each cell line/drug combination was done in triplicate. The resulting 72 libraries plus 3 additional controls were pooled into a single multiplexed library and sequenced on a NexSeq500.  Following sequencing data analysis in the FASTQ files using our custom pipeline Völundr, we expect to have a list of synthetic lethal and synthetic viable genes. Future Direction: We will utilize CRISPR knockout as well as pharmacological inhibitors to validate synthetic lethal and viable DDR gene targets. We will utilize similar approaches to include additional cancer cell lines and drug-screening data for commonly used chemotherapeutics. By understanding and exploiting these gene-drug interactions, we expect to generate a database to personalize the implementation of chemotherapeutics by selecting synthetic lethal or avoiding synthetic viable combination therapies based on a patient’s tumor-specific mutations.