<i>In vivo</i> genome‐editing screen identifies tumor suppressor genes that cooperate with <i>Trp53</i> loss during mammary tumorigenesis

Abstract

Breast cancer is a heterogeneous disease that comprises multiple histological and molecular subtypes. To gain insight into mutations that drive breast tumorigenesis, we describe a pipeline for the identification and validation of tumor suppressor genes. Based on an in vivo genome‐wide CRISPR/Cas9 screen in Trp53 +/– heterozygous mice, we identified tumor suppressor genes that included the scaffold protein Axin1 , the protein kinase A regulatory subunit gene Prkar1a , as well as the proof‐of‐concept genes Pten , Nf1 , and Trp53 itself. Ex vivo editing of primary mammary epithelial organoids was performed to further interrogate the roles of Axin1 and Prkar1a . Increased proliferation and profound changes in mammary organoid morphology were observed for Axin1/Trp53 and Prkar1a/Trp53 double mutants compared to Pten/Trp53 double mutants. Furthermore, direct in vivo genome editing via intraductal injection of lentiviruses engineered to express dual short‐guide RNAs revealed that mutagenesis of Trp53 and either Prkar1a , Axin1 , or Pten markedly accelerated tumor development compared to Trp53 ‐only mutants. This proof‐of‐principle study highlights the application of in vivo CRISPR/Cas9 editing for uncovering cooperativity between defects in tumor suppressor genes that elicit mammary tumorigenesis.