An In Vivo CRISPR Screening Platform to Evaluate the Therapeutic Impact of DNA Damage Response Gene Mutations

Abstract

<h3>Purpose/Objective(s)</h3> Alterations in DNA damage response (DDR) genes are prevalent in human cancers. How these genetic changes modify responses to DNA-directed therapeutics (DDTs) is poorly understood. Here we describe an <i>in vivo</i> CRISPR screening platform to systemically identify DDR gene mutations that confer sensitivity or resistance to different classes of DDTs in a transgenic breast cancer model. <h3>Materials/Methods</h3> Murine breast tumors were induced by mammary intraductal injection of <i>Rosa26^{LSL-Myc;LSL-Cas9};Trp53^flox/flox</i> 6-10 week old female mice with 5 × 10⁵ transduction units of lentivirus expressing Cre recombinase and a library of small guide RNAs targeting 310 DDR genes ("DDR-CRISPR" library). Mammary tumors were analyzed for sgRNA representation by amplicon-based next generation sequencing. A pool of 39 DDR-CRISPR mammary tumor lines was orthotopically injected into a cohort of female <i>NOD/RAG1^−/-</i> mice. When tumors reached 8-10mm in maximal dimension, cohorts of six tumors each were treated with the following DDTs: doxorubicin/hydroxydaunorubicin, Paclitaxel, Capecitabine, Olaparib, AZD6738, 2Gyx4, and 8Gyx1. Tumors were harvested when they reached 15mm in maximal dimension. Statistically significant changes in sgRNA abundance in drug treated tumors versus mock treated controls were identified using two-tailed t-tests. <i>FANCA-</i>knockout clones of MDA-MB-231 cells were generated by dual Cas9-sgRNA targeting. Sensitivity to Olaparib and AZD6738 were assessed by colony forming assay and <i>in vivo</i> by tumor growth analyses in female <i>NOD/RAG1^−/−</i> mice. <h3>Results</h3> Mock-treated mammary tumor pools contained approximately 500 sgRNAs targeting 230 distinct DDR genes. Each type of DDT resulted in distinct yet reproducible fluctuations in sgRNA representation, likely reflecting the variable biological effects of DDR gene mutations on therapeutic sensitivity. Olaparib and AZD6738 treatments resulted in depletion of sgRNAs targeting genes in the canonical homologous recombination (HR) repair pathway (e.g., <i>Brca2, Mre11, Nbn</i>, and <i>Rad54b</i>). However, non-HR genes such as <i>FANCA</i> were also significantly depleted by PARP and ATR inhibitor treatments. Using an isogenic MDA-MB-231 cell line model, we show that <i>FANCA</i> deficiency does not impair HR but results in significant accumulation of single-stranded gaps upon PARP and ATR inhibitor treatment. Single and dual treatment with PARP and/or ATR inhibitors resulted in substantial effects on tumor growth delay and mouse survival. <h3>Conclusion</h3> Our findings illustrate that genetic mixtures of DDR-deficient breast cancers generated through an <i>in vivo</i> CRISPR screen can be used to efficiently quantify the impact of DDR gene mutations on therapeutic sensitivity. Using this platform, we discovered <i>FANCA</i> deficiency as a determinant of sensitivity to PARP and ATR inhibitors due to an accumulation of single-stranded DNA gaps, despite being proficient in HR repair.