Murine CLL cells with disrupted MyD88 are not negatively selected in vivo. (A) Schematic representation of the procedure to investigate the impact of CRISPR/Cas9-mediated disruption of the MyD88 gene on the growth of adoptively transferred Eμ-TCL1 CLL cells. The scheme illustrates expected outcome in case TLR signals are required for leukemia growth in vivo. (B) Indel analysis by amplicon capillary electrophoresis of the targeted region of MyD88 in TCL1-355– and TCL1-333–injected and recovered leukemia cells isolated from peritoneal cavity (PC) and spleen. The wild type allele is indicated by a red arrow, and mutant alleles are indicated by black arrows. (C) Immunoblotting analysis of wild type (WT) and MyD88-edited TCL1-355 and TCL1-333 leukemia cells. (D) Amplicon capillary electrophoresis of the targeted region of MyD88 in TCL1-699–injected and recovered leukemia cells isolated from PC and spleen. Experiment was performed with the second MyD88 guide RNA. (E) MyD88 mutant allele frequency (MAF) in injected leukemia cells and cells isolated from the PC and spleen of mice 21 to 35 days after adoptive transfer. Seventeen independent experiments were performed with the 8 TCL1-derived CLL and the 2 TCL1-derived RS lines. MAF was calculated by dividing the area of the mutated alleles with the total area of all amplified alleles (mutant + WT) detected in the amplicon capillary electrophoresis. Statistical analysis was done using 1-way repeated measures ANOVA.