CBX7 gene targeting using CRISPR-Cas9 results in the outgrowth of WT or heterozygous hit but not homozygous hit OCI-AML3 clones. (A) Experimental setup. RNP complexes (Cas9 plus an atto550-labeled sgRNA targeting CBX7 validated in F. Zhang’s knockout library screen) are transfected into OCI-AML3-CBX7^WT or already heterozygous hit OCI-AML3-CBX7^+/− cells, by nucleofection. After 2 days of recovery, Atto550⁺ cells are single-cell sorted into 96 wells. (B) Amount of Atto550⁺ sorted single-cell–derived clones after nucleofection of mock or CBX7-targeting sgRNAs in either CBX7^WT or CBX7^+/− OCI-AML3 cells. Bars indicate the number of single-cell–derived clones, with blue indicating the amount of outgrown WT CBX7 clones and red the outgrown of heterozygous hit CBX7 clones. (C) Percentage of polymerase chain reaction (PCR)–amplified WT CBX7 sequence, or insertion-mutated CBX7 sequence in the 3 heterozygous hit OCI-AML3 clones. (D) Western blot analysis showing CBX7 and H4 protein expression levels in the 3 heterozygous hit OCI-AML3 clones. (E) Absolute cell count after 4 days of in vitro culturing 250 000 CBX7^WT or CBX7^+/− OCI-AML3 cells. Bars represent the mean of ≥3 replicates with standard error of the mean (SEM) as error bar. Student t test was used to calculate the P value between CBX7^WT and CBX7^+/− OCI-AML3 cells, ∗P < .05. DAPI, 4′,6-diamidino-2-phenylindole.