Cancer stem cells (CSCs) are a subpopulation of tumor cells with self-renewal ability. Increasing evidence points to the critical roles of CSCs in tumorigenesis, metastasis, therapy resistance, and cancer relapse. As such, the elimination of CSCs improves cancer treatment outcomes. However, challenges remain due to limited understanding of the molecular mechanisms governing self-renewal and survival of CSCs. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 screening has been increasingly used to identify genetic determinants in cancers. In this primer, we discuss the progress made and emerging opportunities of coupling advanced CRISPR screening systems with CSC models to reveal the understudied vulnerabilities of CSCs.

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