Exploring_NKG2DL_cell_surface_regulation_using_CRISPR_CAS9

Patients with acute myeloid leukemia (AML) often achieve remission after initial therapy, but subsequently die of relapse arising from persistent leukemic stem cells (LSCs). In addition to their enhanced chemoresistance, LSCs also selectively escape natural killer (NK) cell immune surveillance by downregulating the surface expression of ligands for activating NKG2D receptors on NK and T cells (Paczulla et al., Nature 2019). In this project, we aim to leverage published RNA-seq data of NKG2DLneg vs NKG2DLpos populations from five primary AML patients (Paczulla et al., Nature 2019) to explore methods of upregulating NKG2D ligands on LSCs augmenting their immunogenicity. Specifically, we will use an unbiased CRISPR/Cas9 screening approach and transduce AML cell lines with a customized sgRNA library targeting the most significantly up- or downregulated genes to explore their role in regulating NKG2DL cell surface presentation as determined by flow cytometry. We will use Kosuke Yusa´s two-plasmid system (Tzelepis et al., Cell Rep 2016) to deliver sgRNAs and introduce the desired gene knockouts.