ANGI-01. INDUCIBLE CAS9 IN PATIENT-DERIVED CULTURES OF IDH WILDTYPE GLIOBLASTOMA WITH DISTINCT GROWTH CHARACTERISTICS

Abstract

Abstract To identify new treatment opportunities for IDH wildtype glioblastoma, it is essential to establish robust methods to evaluate candidate genes in patient-derived cells. Here, we report initiative to establish large-scale candidate gene evaluation by CRISPR-Cas9 using well characterized cells from the Uppsala Human Glioma Cell Culture biobank. Previously, we have characterized fully the HGCC cultures (n = 100) in terms of multi-omics, and by systematic injection as patient-derived orthotopic xenografts. Here, we report a procedure to introduce doxycycline-inducible Cas9 into cultures from our biobank. This allows for temporally controlled target gene ablation in patient-derived cells with known growth characteristics. As proof-of-concept, we describe the derivation of clonal Cas9-inducible cells in the cell lines U3013MG (proneural, bulk forming) and U3180MG (classical, diffuse-growing). The Cas9 induction and editing efficiency was evaluated by ablation of the gene encoding the cell surface marker CD44, for which we transduced the Cas9-inducible clones with CD44 dual sgRNA vector and treated then with doxycycline over time, evaluating CD44 knockout by longitudinal flow cytometry. From a library of 106 clones, we identified the best performing clones, as evaluated by CD44 loss at day 21. Ten out of 64 clones assessed, showed potent Cas9 activity, estimating a drop in CD44 levels by 40-60% after 21 days of doxycycline treatment. The amount of editing by inducible Cas9 was higher with CD44 sgRNA administered by lentivirus than by electroporation. To ensure that the Cas9-inducible patient-derived cell lines have reproducible tumor phenotypes and did not divert from their parental growth pattern, they were transplanted orthotopically into athymic nude mice. We also validated the activity of Cas9ind in vivo by checking the editing of CD44 expression in one of our clones. The established procedure provides a basis for large-scale single-gene and pooled in vivo CRISPR studies of IDH wildtype glioblastomas with distinct invasive characteristics.