CRISPR-Cas9 Activation/Repression in HeLa Cells

HeLa cells were cultured in MEM (HyClone) supplemented with 1% sodium pyruvate, l-glutamate, NEAA and 10% FBS as previously described (94 (link)). 1.5 × 10⁵ HeLa cells were seeded in each T75 flask 16–18 h prior to transfection. Cells were transfected with 100 ng Cas9m4-VP64 (or Cas9m4-KRAB) (95 (link),96 (link)), 100 ng gRNA (either singly or total of a mix), 100 ng of Renilla luciferase- containing plasmid, and 100 ng of plasmid containing the L1 5′ UTR driving Firefly luciferase in the pGL3-basic plasmid (Promega) (83 (link),97 (link)). The Renilla luciferase plasmid contains an HSV-tk promoter, a Renilla luciferase reporter, and a polyA signal (pRL-TK, Promega) as a transfection control. Both plasmids and the gRNAs used are illustrated in Figure 9A. Transfection reaction used 12 uL of plus reagent (Invitrogen) and 5 uL of lipofectamine (Invitrogen). All gRNA pools are designed to transfect the same total amount of gRNA plasmid. The exact gRNA sequences used can be found in Additional File 2. The plasmid containing the gRNA targeting AAV (gRNA_AAVS1-T2) was purchased from Addgene (98 (link)).