Putative LEUTX enhancer regions 1 and 2 were predicted from Tet-On DUX4 hESC NET-CAGE dataset.9 (link) Putative CRX enhancer and promoter regions were predicted from NET-CAGE data introduced in this study. The guide RNAs targeting the each of the putative enhancers or promoters were designed using the Benchling CRISPR tool (https://benchling.com ), targeting them to the proximal promoters (−400 to −50 base pairs from transcription start site) or +/−200 base pairs of the putative enhancer midpoint. Guide sequences were selected according to their on- and off-target score and position. Guide RNA transcriptional units (gRNA-PCR) were prepared by PCR amplification with Phusion polymerase (Thermo Fisher), using as template U6 promoter and terminator PCR products amplified from pX335 together with a guide RNA sequence-containing oligo to bridge the gap. The oligos for guide RNA transcriptional units are as in (Balboa et al., 2015).67 (link) PCR reaction contained 50 pmol forward and reverse primers, 2 pmol guide oligo, 5 ng U6 promoter and 5 ng terminator PCR products in a total reaction volume of 100μL. The PCR reaction program was 98°C/10 sec, 56°C/30 sec, 72°C/12 sec for 35 cycles. Amplified gRNA-PCRs were purified and transfected to HEK293 cells.
HEK 293 cells were seeded on tissue culture treated 24-well plates one day prior to transfection (5 × 104 cells/well). Cells were transfected using FuGENE HD transfection reagent (Promega) in fibroblast culture medium with 500 ng of dCas9VP192 transactivator encoding plasmid and 200 ng of guide RNA-PCR product or TdTomato guide RNA plasmid. Cells were cultured for 72 h post-transfection, after which samples were collected for qRT-PCR. Successful activation of LEUTX and CRX was confirmed by qPCR.
In order to introduce LEUTX guides to DD-dCas9 activator cell line, guide cassettes containing either four guide oligos targeting LEUTX promoter or five guide oligos targeting enhancers 1 or 2 were assembled in a GoldenGate reaction using the four different LEUTX promoter guide oligos and 5 different guide oligos targeting enhancers 1 and 2 as described in (Balboa et al., 2015).67 (link) Guide cassettes containing both promoter and enhancer guides was further cloned together. Finally, the guide cassettes were cloned to piggyBac vector. Primer sequences for promoter and enhancer guide oligos are provided inTable S12 . See Figure S5 for LEUTX enhancer validation.
HEK 293 cells were seeded on tissue culture treated 24-well plates one day prior to transfection (5 × 104 cells/well). Cells were transfected using FuGENE HD transfection reagent (Promega) in fibroblast culture medium with 500 ng of dCas9VP192 transactivator encoding plasmid and 200 ng of guide RNA-PCR product or TdTomato guide RNA plasmid. Cells were cultured for 72 h post-transfection, after which samples were collected for qRT-PCR. Successful activation of LEUTX and CRX was confirmed by qPCR.
In order to introduce LEUTX guides to DD-dCas9 activator cell line, guide cassettes containing either four guide oligos targeting LEUTX promoter or five guide oligos targeting enhancers 1 or 2 were assembled in a GoldenGate reaction using the four different LEUTX promoter guide oligos and 5 different guide oligos targeting enhancers 1 and 2 as described in (Balboa et al., 2015).67 (link) Guide cassettes containing both promoter and enhancer guides was further cloned together. Finally, the guide cassettes were cloned to piggyBac vector. Primer sequences for promoter and enhancer guide oligos are provided in
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