gms | German Medical Science

Objectives: The Receptor Activator of NF- κB Ligand (RANKL), and its receptor RANK, is considered as the major driver of pre-osteoclasts to form multinucleated giant cells that actively resorb bone. However, it is known for more than a decade that other cytokines including tumor necrosis factor-α, Interleukin-1 and - to some extent - transforming growth factor β1 can stimulate differentiation of active, mature osteoclasts. RANK negative hosts (e.g. RANK deficient mice) may not be locally available, and gene-silencing methods such as RNAi do not provide sufficient knockdown efficiency for the vast majority of receptors. With this study, we want to depict methodically how to generate a RANK deficient osteoclast-precursor cell line using CRISPR/Cas9.

Methods: The monocyte-macrophage cell line RAW264.7 was purchased from Sigma-Aldrich and cultured according to manufacturers recommendations. Four 19 basepair (bp) polyspacers were designed to target exon 4 of TNFRSF11A (RANK), encoding a transmembrane peptide sequence of the receptor, and cloned into lentiCRISPRv2 (addgene #52961). Production of lentivirus was performed using 293T packaging cells, as well as pDM2 and psPAX2 2nd generation plasmids. RAW264.7 cells were transduced with lentiviral supernatant and grown under Puromycin selection. DNA was harvested after 10 days of selection and on-target efficiency of the four gRNAs was evaluated using a mismatch-repair assay (T7E1). Clones derived from efficient gRNAs were clonally expanded using a serial dilution assay and sub cultured until they reached confluency. Successful RANK deficiency was determined by sequencing exon 4 using a sanger-sequencing service.

Results and Conclusion: With this methodical study, we give a step-by-step guidance how to generate RANK deficient RAW264.7 osteoclast precursor cells by using CRISPR/Cas9. Together, we provide efficient gRNA sequences to generate a model cell-line to study alternative pathways that regulate osteoclast differentiation besides the RANK-RANKL axis. Future studies need to address questions of cell-cell contacts, as well as an interplay between different cytokines to give mechanistic insights into other drivers of osteoclastogenesis. This model cell line could potentially serve as a backbone to guide future understanding of disease that are linked to bone loss, and that bypass the RANKL-RANK axis.