Figure 4.

CRISPR/Cas9 editing of MEN1 decreases the fitness of MM and B-ALL cells harboring edited alleles. (A–C) NALM6 and L363 cells were transfected with RNP complexes containing tandem crRNAs targeting the 1st exon of MEN1 or control tandem crRNAs targeting the 3rd intron of TNNI3. (A) The target sequences in the first exon of MEN1 are shown; the pictures were generated with GenePalette (Version 2.1.1, University of California). (B) MEN1 protein expression one week after KO was measured by immunoblot. (C) The proportion of alleles harboring inactivating mutations (frameshift or indel exceeding 21 bp) is shown as a knockout coefficient (KO coefficient) normalized to the starting KO values. The statistical significance of the continuous decrease in the proportion of the MEN1 KO alleles was analyzed by linear regression analysis (L363: slope = −3.697; R² = 0.6161; p = 0.0122; NALM6: slope = −2.412; R² = 0.9406; p < 0.0001). The proportion of TNNI3 KO alleles slightly increased (L363: slope = 0.2004; R² = 0.0020; p = 0.9089; NALM6: slope = 1.487; R² = 0.2941; p = 0.1314). Data obtained from 3–4 independent experiments are shown.