Adapting CRISPR Cas9 dropout screens to in vivo PDX models of acute leukemias

As acute leukemias require improved treatment, we aimed at identifying therapeutic targets on a patient-individual level by establishing CRISPR Cas9 dropout screens in PDX models of acute leukemia in vivo. Genetic barcoding in Cas9 negative samples was performed in order to ensure sufficient library coverage. Larger libraries can be screened in ALL compared to AML PDX models, which might reflect lower intra-sample heterogeneity and higher leukemia stem cell frequency in ALL. A customized library of 146 target genes was designed with 5 sgRNAs per gene using the CLUE platform. Either a puromycin-resistance cassette or an H-2Kk surface marker, each combined with a fluorescent marker was used for enrichment of CRISPR Cas9 sgRNA library positive cells to >90%. Cells were injected into NSG mice and re-isolated at advanced disease stage. Next generation sequencing (NGS) data was analyzed using MAGeCK to detect gene dropouts. Commonly depleted genes might represent interesting future therapeutic options. In summary, we established a CRISPR Cas9 screening pipeline to investigate therapeutic targets on a patient-individual level in ALL and, for the first time, in AML PDX models in vivo.

Publication History

Article published online:
17 May 2022