Validation of CRISPR targeting for proliferation and cytarabine resistance control genes in the acute myeloid leukemia cell line MOLM-13

Acute myeloid leukemia patients with FMS-like tyrosine kinase 3-internal tandem duplications and mixed lineage leukemia-protein AF9 fusion proteins suffer from poor clinical outcomes. The MOLM-13 acute myeloid leukemia cell line harbors both of these abnormalities and is used in CRISPR experiments to identify disease drivers. However, experimental observations may be biased or inconclusive in the absence of experimentally validated positive control genes. We validated sgRNAs for knockdown of TP53 for cell proliferation and for DCK knockdown and CDA upregulation for cytarabine resistance control genes in MOLM-13 cells. We have provided a detailed CRISPR protocol applicable to both gene knockdown or activation experiments and downstream leukemic phenotype analyses. Inclusion of these controls in CRISPR experiments will enhance the capacity to identify novel myeloid leukemia drivers in MOLM-13 cells. METHOD SUMMARY CRISPR-Cas9 knockdown and upregulation approaches were used for changing the expression of control genes TP53, DCK, and CDA in the acute myeloid leukemia cell line MOLM-13. Lentivirus transduced cells were selected (sorted for green fluorescent protein positive cells or selected by antibiotic treatment), and knockdown or upregulation of genes in the stable cells was confirmed by real-time quantitative PCR and western blot analyses. EdU incorporation assay was used to measure cell proliferation. Colorimetric proliferation/survival assay and flow cytometric cell counting were used to measure cell growth and survival. Results from the experimental and control groups were compared using Student's t-test.