Pluto Bioinformatics

GSE128342: Inhibition of DOT1L and PRMT5 promote synergistic anti-tumor activity in a human MLL leukemia model induced by CRISPR/Cas9

Bulk RNA sequencing

Previous studies have identified frequent MLL-AF4 chromosomal translocation breakpoints in patients. Using CRISPRscan, we designed different sgRNAs recognizing patient-specific sequences in intron 11 of the MLL and exon 3 of the AF4 genes, respectively. Using plasmid- and virus-free delivery of sgRNAs with Cas9 protein, we nucleofected CD34+ target cells derived from human umbilical cord blood. Following nucleofection, the cells were maintained in liquid culture supplemented with cytokines and chemokines optimized for growth of MLLr cells. On day 30 (MLL-AF4) of liquid culture, MLL translocations were detcted in 100% of cells by FISH analysis allowing for further analysis of pure MLLr cells. MLL-AF4 translocated cells and the respective control cells from the two different donors were maintained in similar culture conditions. After reaching 100% purity of the MLLr cells, they were subjected to RNA-seq. SOURCE: Corina Schneidawind ( - University Hospital Tuebingen

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