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High expression of all 3 kinases was associated with relatively poor prognosis in patients with osteosarcoma. Conclusions Our results not only offer potential therapeutic kinase targets in osteosarcoma but also provide a paradigm for functional genetic screening by using a CRISPR-Cas9 library, including target design, library construction, screening workflow, data analysis, and functional validation. This method may also be useful in potentially accelerating drug discovery for other cancer types.Objective The aim of this study was to in