ABSTRACT Telomeres consist of TTAGGG repeats bound by protein complexes that serve to protect the natural ends of linear chromosomes and ensure genome stability. Due to the end replication problem, telomeres progressively shorten with each cellular division until they become critically short, resulting in cellular senescence or apoptosis. A universal feature of human cancer cells is the ability to bypass this proliferation barrier by engaging a telomere elongation mechanism, which is most often the expression of the reverse transcriptase telomerase. However, a significant portion of cancer cells (approx 10-15%) utilize a telomerase-independent pathway named the Alternative Lengthening of Telomeres (ALT) pathway that relies on homologous recombination between telomeric sequences to extend telomeres and evade replicative arrest. Cancer cells that utilize ALT are characterized by heterogeneous telomere lengths, extrachromosomal telomeric DNA and the presence of promyelocytic leukemia (PML) bodies at telomeres. Although the ALT pathway has been the subject of intense investigation, the mechanism by which it occurs is still incompletely understood. In order to shed light on the ALT pathway, we will employ CRISPR/Cas9 to generate genetic knockouts of the known ALT-associated factors PML and SUMO2 to assess their essentiality in ALT activity. Additionally, we will use CRISPR/Cas9 to perform a genome-wide gRNA knockout screen to identify novel factors required for ALT. In this way, we will identify factors essential for ALT to expand our understanding of this telomere maintenance mechanism and reveal potential therapeutic targets for the treatment of ALT cancers.