Generation of SET7/9 Knock-out Cell Lines Using CRISPR/Cas9 Gene Editing Technology

SET7/9 knockout (KO) HeLa cells were generated using CRISPR/Cas9 system as described previously⁵³. gRNA targeting sequence (5′-TAGCGACGACGAGATGGTGG-3′) was cloned into gRNA cloning vector (Addgene, 41824) and confirmed by sequencing. To screen for SET7/9 KO clones, HeLa cells were transfected with pcDNA3.3-hCas9 and gRNA expression vectors (Addgene 41815), followed by G418 selection (0.5 mg/ml). Single colonies were subjected to immunoblotting (IB) using anti-SET7/9 antibody to select knock-out ones, which were further validated by PCR using genomic DNA as template followed by Sanger sequencing. The sequencing information for primer sets used was as follows: Forward (F) 5′-CTCCTCCTCCTCCAAACTCG-3′; Reverse (R) 5′-ACTCCTTCCGCGCTCCAG-3′.