CRISPR-mediated Deletion of Regulatory Elements in Mouse Leukemia Cells

Mouse leukemia cells carrying Npm1c/Flt3-ITD/Cas9 (termed DM-Cas9 cells)^{42 (link)} were cultured in X-Vivo medium (Lonza) plus 10% fetal bovine serum (Gibco) supplemented with cytokines as described above. In the presence of Cas9, using dual guide RNAs (gRNAs) to specifically target two genomic loci has been demonstrated previously^{52 (link)}. Paired dual gRNAs targeting specific cis-regulatory elements or scramble control dual gRNAs were designed with IDT web tools (https://eu.idtdna.com/site/order/designtool/index/CRISPR_CUSTOM) and their sequences were available in Supplementary Table 12. Oligos of dual gRNAs were cloned into lentivirual vector pKLV2.2-h7SKgRNA5(SapI)-hU6gRNA5(BbsI)-PGKpuroBFP-W (#72666, Addgene). Production of gRNA particles and transduction of DM-Cas9 cells were carried out same as for shRNA. To confirm the target deletion in the bulk transduced cells, PCR primers were designed to amplify the wildtype or modified allele (Supplementary Table 12). Size of PCR products was checked by agarose gel electrophoresis. Furthermore, PCR products were subject to TA cloning (Promega). Plasmid DNA was extracted from individual colonies and was confirmed for deletion by Sanger sequencing.