Western Blot Analysis of Cas9 Expression

Cells were collected and lysed in 2 × reducing sample buffer, and then incubated at 98°C for 15 min. All samples collected from an individual experiment were run on the same gel to compare protein expression levels as accurately as possible. Samples were separated by a Criterion 4–20% gel (No. 5671095; Bio-Rad), and then transferred to PVDF membranes. Antibody incubation was conducted as previously reported.^{42 (link)} Primary antibodies used include rabbit anti-Cas9 (No. ab189380, 1:5000; Abcam), mouse anti-tubulin (No. T5168, 1:10,000; Sigma-Aldrich), and rabbit anti-FLAG (No. F7425, 1:5000; Sigma-Aldrich). Secondary antibodies used were goat anti-rabbit IRdye800 (No. 925-32211, 1:10,000; LI-COR) and goat anti-mouse IRdye680 (No. 926-69020, 1:10,000; LI-COR).