C-terminal RUNX1 mutations are frequently frameshift and nonsense, resulting in transcripts that are exempt from nonsense-mediated decay. (A) Lollipop plot of hematopoietic mutations in RUNX1 (isoform 1c NP_001754.2) in the COSMIC database with accompanying transcript exons displayed (top). Truncating mutations include nonsense, nonstop, frameshift deletion, frameshift insertion, and splice site. In-frame deletions and in-frame insertions are considered in-frame mutations, and all other nonmissense mutations are labeled as “Other.” Enlarged region of exons 7 and 8 of RUNX1 denoting NMD exempt mutations (bottom). Mutations that result in a premature stop codon in the final exon (exon 8) or within 50 nucleotides upstream of the last exon-exon junction (exon 7-8) are predicted to be exempt from NMD. (B) Analysis of C-terminal RUNX1 mutations beyond the RHD. Frameshift and nonsense mutations represented 304 of 387 mutations (78.55%), whereas all other in frame mutations consisting of missense, in-frame insertions and deletions, coding silent substitutions, and compound substitution combined account for 83 of 387 mutations (21.45%). NMD analysis was performed on 304 frameshift and nonsense mutations, examining premature stop codons within the region defined in panel (A). A total of 76.3% (232/304) of C-terminal frameshift and nonsense mutations were predicted to be exempt from NMD. (C) Schematic of RUNX1 protein domains and knock-in R320^∗ mutation using CRISPR-Cas9. (D) Sanger sequencing of the RUNX1^R320∗ homozygous knock-in mutation compared with the wild-type RUNX1 sequence. K562 cells were nucleofected with Cas9, RUNX1 targeting gRNA, and R320^∗ donor template. The gRNA (black underline) targeted exon 7 (isoform 1c NM_001754.5) and the donor oligo template results in a TAA codon from TCG at R320. Single-cell clones were screened for homozygous mutations, confirmed by sequencing the targeted region, and analyzed using the ICE tool by Synthego. (E) Western blot of wild-type (WT) RUNX1 and RUNX1^R320∗ K562 cells along with β-actin loading control. Both lines were subjected to the same nucleofection process as the +/− CRISPR-Cas9 editing components. Whole cell lysate was extracted and used to confirm the presence of both wild-type and RUNX1^R320∗ proteins. Densitometry calculations were performed using β-actin normalization. Arrow indicates a possible nonspecific signal. (F) RUNX1 transcript levels in RUNX1 wild-type and RUNX1^R320∗ cells and RUNX family members as measured using the DESeq2 analysis software package. Each line was subjected to RNA-seq and sampled in triplicate (n = 3). Student t test was used, significance: ∗ P ≤ .05; ∗∗ P ≤ .01; ∗∗∗ P ≤ .001; ∗∗∗∗ P ≤ .0001.