{
  "abstract": "Background Identification of genes modulating the intrinsic immunogenicity of tumor cells may reveal biology underlying resistance to immunotherapy. Targeting these genes pharmacologically may potentiate durable anti-tumor immunity when combined with immune checkpoint blockade. Utilizing high throughput CRISPR-Cas9 screening assays, our group and others have identified Setdb1 as a negative regulator of antigen processing and presentation. While Setdb1 as a target has been validated, a structural understanding of the protein is necessary for rational drug design. Here we present the results of a CRISPR base editor screen tiling across the protein-coding sequence of Setdb1 to identify domains regulating antigen processing and presentation in tumor cells.Methods We transduced B16F10-ova cells with two base editor libraries (pRDA_478 BE3.9 C>T; pRDA_479 ABE8e A>G) encompassing ~2600 sgRNAs targeting the protein coding regions of the Setdb1 gene, and including appropriate positive and negative controls. Stably BE-expressing cells were cultured +/- IFN-β for 48hr and then positively selected for surface expression of SIINFEKL peptide presented on H2-kb. sgRNA abundance in positive- and negative-sorted fractions was deconvoluted by NGS and predicted missense mutations were mapped onto a 3D projection of the Setdb1 protein.Results Our initial screen identified multiple missense mutations, splice site alterations, and introduction of premature termination codons that phenocopied the full Setdb1 knockout. We validated 14 hits from the initial screen through standard CRISPR-Cas9 follow-up: single sgRNA cloning, transduction, establishment of cell lines, functional validation by FACS assay of modulation of H2-Kb-SIINFEKL surface staining +/- IFN-β, and confirmation by Sanger sequencing of correct edit. Top hit inducing a missense Cys759Arg mutation reproduced from initial screen, phenocopied the immunogenicity of full Setdb1 knockout in response to IFN-β exposure, and maps to a region of Setdb1 homologous between mouse and human.Conclusions We performed a CRISPR base editor screen tiling across the Setdb1 gene to identify function domains of the protein that, when mutated, would enhance susceptibility to immune-mediated killing. When overlaid onto 3D structure, hits provide target domains for medicinal chemistry efforts.",
  "authors": [
    {
      "affiliations": [
        "Belfer Center for Applied Cancer Science, Boston, MA, USA",
        "Dana-Farber Cancer Institute, Boston, MA, USA"
      ],
      "name": "Patrick H Lizotte"
    },
    {
      "affiliations": [
        "Belfer Center for Applied Cancer Science, Boston, MA, USA",
        "Dana-Farber Cancer Institute, Boston, MA, USA"
      ],
      "name": "Lily M Blanchard"
    },
    {
      "affiliations": [
        "Belfer Center for Applied Cancer Science, Boston, MA, USA",
        "Dana-Farber Cancer Institute, Boston, MA, USA"
      ],
      "name": "Kelsey Hagen"
    },
    {
      "affiliations": [
        "Belfer Center for Applied Cancer Science, Boston, MA, USA",
        "Dana-Farber Cancer Institute, Boston, MA, USA"
      ],
      "name": "Nat Spicer"
    },
    {
      "affiliations": [
        "Servier Bio-Innovation LLC, Boston, MA, USA"
      ],
      "name": "Cristovao Sousa"
    },
    {
      "affiliations": [
        "Servier Bio-Innovation LLC, Boston, MA, USA"
      ],
      "name": "Donald Simons"
    },
    {
      "affiliations": [
        "Servier Bio-Innovation LLC, Boston, MA, USA"
      ],
      "name": "Jiqin Yang"
    },
    {
      "affiliations": [
        "Belfer Center for Applied Cancer Science, Boston, MA, USA",
        "Dana-Farber Cancer Institute, Boston, MA, USA"
      ],
      "name": "Cloud Paweletz"
    }
  ],
  "title": "1234 CRISPR base editor screen reveals functional domains of Setdb1 modulating tumor immunogenicity",
  "uid": "cec2a3db-298a-5268-a80c-c764f45855e2"
}