Figure 1.

Optimization of the H2B-GFP SRIRACCHA reporter and use with an inducible Cas9^D10A nuclease. A, constituents of the Piggybac target transposon with an H2B-GFP reporter and schematic of the SRIRACCHA process. The target transposon consists of a CMV-driven puromycin resistance gene, the target sequence (cloned into a BstEII site), a polyadenylation (pA) signal, an out-of-frame H2B-GFP open reading frame, an additional SV40 pA signal, and two HSIV core insulator sequences at the 3′ end. The Piggybac transposon sequences, including inverted terminal repeats, are represented by two outward facing arrows that flank the CMV-driven expression cassette. The Let-7 protospacer sequences are highlighted in red, and the PAM is in blue. After transposition into the genome by hyPBase and nuclease cutting, the donor plasmid supplies the template for HDR, which restores expression of H2B-GFP via a T2A cleavage sequence. B, the effects of different ratios of transposon and transposase plasmids (by mass) in HEK293T cells. Cells were transfected with plasmids driving constitutive expression of the transposase, wild type Cas9, gRNAs, and pCMV-tdT along with the target transposon and donor plasmid. The target and gRNA sequences are from a site in mouse Mirlet7g. Puromycin was added 48 h after transfection, and expression of GFP and RFP was analyzed 2 days after puromycin selection. Signals from nonspecific (NS) gRNAs or targeting gRNAs were also compared. C, the effects of different ratios of donor plasmid and gRNA plasmid (by mass) in HEK293T cells. Cells were transfected as in B. D, schematic of plasmids used for Cas9^D10A induction (iCas9N) with dox and 4-OH-T. E, inducible Cas9^D10A SRIRACCHA assay in the presence and absence of the Piggybac transposase. HeLa cells were transfected as above using a 3:1 transposon to transposase ratio and a 3:2 ratio of donor to gRNA but substituting hCas9 with plasmids in D. At 24 h, 500 ng/ml dox and 0.5 μm 4-OH-T was added to the transfected cells to induce Cas9^D10A expression for 48 h. Fluorescence was examined 72 h after withdrawing dox and 4-OH-T. F, inducibility of Cas9^D10A was compared with dox and/or 4-OH-T treatment, as transfected in E, with fluorescence examined 48 h after drug addition. G and H, SRIRACCHA Cas9^D10A assay in HeLa (G) and HEK293T (H) cells. HeLa or HEK293T cells were transfected and induced as in E, with fluorescence examined 24 h (for HeLa) or 48 h (for HEK293T) after drug addition. Error bars represent S.E. of a representative experiment performed in triplicate (and replicated in a total of 2–3 experiments), where Student's t test was performed to determine significance relative to vehicle in E–H, with p < 0.05 (*) or p < 0.01 (**).