Fig. 2

A pipeline for in vivo CRISPR-Cas9 dropout screens in ALL PDX models. A Workflow of CRISPR-Cas9 screening experiments. Primary leukemia cells from patients were transplanted into mice, re-isolated, and lentivirally transduced with split-Cas9 and a lenti-CRISPR library. Cells were cultured for 10 days in vitro, and MACS-enriched Cas9/sgRNA double-positive cells were transplanted into mice (n = 5 for ALL-199, n = 8 for ALL-265). PDX cells were re-isolated from mice with advanced leukemia and sgRNA distribution was analyzed by next generation sequencing (NGS) in comparison to the input control. B Distribution of all sgRNAs present in the library was analyzed by NGS in the plasmid pool and in re-isolated cells from split-Cas9-negative PDX samples (n = 2) after 6 weeks of in vivo growth. C Depletion score calculated using the MAGeCK robust ranking algorithm (RRA) for dropout genes in both samples or exclusively in ALL-199 or ALL-265. Dotted line represents cut-off at < 0.01. D Workflow of in vivo competitive validation assay. Split-Cas9-GFP-transgenic PDX cells were either used as control (CTRL, GFP-positive) or lentivirally transduced with sgRNAs targeting the gene of interest (GOI, mTagBFP-positive). Cells were cultured for 10 days, before sgRNA-positive cells were enriched by FACS. KO cells and CTRL cells were mixed in a 1:1 ratio and injected into three mice, one mouse per sgRNA. Animals were sacrificed at advanced leukemia and the distribution of the two re-isolated cell populations was evaluated by flow cytometry. E Representative flow cytometry plots of in vivo competitive validation assay for CXCR4 and ITGB1 in ALL-199 and ALL-265. Distribution of mTagBFP-positive KO cells and mTagBFP-negative CTRL cells in the injection mixture (1:1 ratio, Input, upper panel) and in re-isolated PDX cells after 6 weeks of in vivo growth (Output, lower panel) is shown. F Quantification of in vivo competitive validation assay for CXCR4 and ITGB1. Percentage of the KO populations in the injection mixture and in corresponding re-isolated PDX cells of ALL-199 (n = 3 each GOI, each 3 animals with 3 BM measurements) and ALL-265 (n = 3 for CXCR4 and n = 3 for ITGB1 with 3 animals with the same ITGB1 sgRNA; for each GOI 3 animals with 3 BM measurements) are depicted. *** p < 0.001, ** p < 0.01 by paired t-test