Lentiviral transduction and CRISPR/Cas9-mediated gene deletion

For sgRNA experiments, Ocy454 cells were stably transduced with a blasticidin-resistant Cas9 expressing lentivirus which caused no discernable effects on sclerostin production, PTH responsiveness, or FFSS-responsiveness. sgRNA sequences were subcloned into pLentiGuide-Puro (Addgene; plasmid# 52963) plasmid (see Supplementary Dataset ² for sgRNA sequences used). To design sgRNA target sequences, we used “Design sgRNA for CRISPRko” web tool (http://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design) and selected top two guide sequences for cloning. To produce lentiviruses, Ocy454 cells transfected with pLentiGuide, psPAX2 (Addgene; plasmid #12260), and MD2.G (Addgene; plasmid #12259) using Fugene HD (Promega, WI, US). Medium was changed the next day, and then collected 48 h later. Cells were exposed to lentiviral particles overnight at 33 °C in the presence of polybrene (2.5 µg mL⁻¹). Media was then changed with puromycin (4 µg mL⁻¹) and blasticidin (5 µg mL⁻¹). Cells were maintained in selection medium throughout the duration of the experiment. Some bulk cell populations were seeded in 96-well plates at 0.2 cell/well. Media were changed twice a week, and 3 weeks later single-cell colonies were identified by microscopy. Single-cell-derived colonies were expanded and analyzed for loss of target protein expression by immunoblotting. For FAK CRISPR-KO experiments, at least six independent clones from each bulk sgRNA cell line were tested by immunoblotting, and clones with 100% protein deletion were used for experiments. Consistent results with respect to reduced SOST expression were seen in all single-cell clones where FAK protein was absent by immunoblotting. FAK-KO clone 1–4 (FAK-KO c1–4), derived from the bulk population of cells transduced with sgRNA-1, was used for subsequent experiments. Control cells were transduced with an empty sgRNA-expressing lentivirus.

HDAC5-deficient cells as described²³ were infected with FLAG-tagged HDAC5 variants in a pLX_311 backbone (Addgene plasmid 118018) followed by blasticidin selection. HDAC5 S259/498A mutant cDNA was obtained from Addgene (plasmid 32216), HDAC5 Y642F construct was synthesized de novo (VectorBuilder).