Chromosomal translocation was detected in the TIGIT-CD96-CD226 triple-KO cells. (A) Schematics of the TIGIT, CD96, and CD226 loci show the PCR primers, sgRNA target positions, and the expected sizes of PCR fragments. (B) We used a PCR-based assay and 15 primer sets to probe various patterns of chromosomal translocation. PCR was performed for 28 and also 35 cycles to increase the detection sensitivity. All DNA gels were loaded with the same amount of DNA ladder (marked as M) for parallel comparison and visualized by SYBR Gold staining.