Cas9-sgRNA RNP targeting of IKZF1 efficiently eliminates protein expression in Nalm-6 cells. (A) sgRNAs targeting the early coding exons of IKZF1 (targeted exons 2 and 3, shown in blue; red indicates noncoding exon 1). Vertical black rectangles denote zinc fingers. (B) Flow cytometry histograms showing CXCR4 cell surface protein using sgRNA against CXCR4 as a control marker for CRISPR efficiency in Nalm-6. (C) IKAROS immunoblotting of single cell–derived Nalm-6 clones with Sanger sequencing–verified IKZF1 frameshift mutations. (D) Reduced-concentration RNP delivery yields a higher frequency of heterozygous clones with varying protein levels. Relative quantification was normalized to ACTIN. Bold font of clones on panels C and D indicates those used in further experiments.