Figure 3. CRISPR/Cas9 knockout of REV7 recapitulates the FA phenotype.

(A) Western blot of whole cell lysates shows no detectable REV7 protein in the REV7^–/– line. FANCD2 is still efficiently monoubiquitylated in response to 100 ng/ml MMC. (B) Western blot showing reconstitution of WT REV7 expression in REV7^–/– cells. (C–D) Increase in chromosome aberrations (left graph) and radials (right) after 48-hour treatment with 20 ng/ml MMC as compared with cells complemented with WT REV7 cDNA. Original magnification, ×1,000. (E) Increase in G₂/M arrest with and without MMC treatment (20 ng/ml). (F) Impaired clonogenic capacity of REV7^–/– cells over 10-day treatment with MMC. (G) REV7 is required for normal hematopoiesis; CFU scoring of bone marrow Lin^– cells after lentiviral silencing using REV7 or scramble (scr) shRNAs. Fancg^-/- cells are used as FA cell control. CFUs were numbered after 7 days on methylcellulose; 2 passages (P1 and P2) were performed. (H) Cell population percentages in CFUs after 7 days of culture.