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Updated: Dec 8, 2025
Pooled CRISPR-Based Genetic Screens in Mammalian Cells
Published on: September 4, 2019
Systematic screening for potential therapeutic targets in osteosarcoma through a kinome-wide CRISPR-Cas9 library
Yuanzhong Wu1, Liwen Zhou1, Zifeng Wang1
1Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Guangzhou 510060, China.
Abstract:
Objective: Osteosarcoma is the most common primary malignant bone tumor. However, the survival of patients with osteosarcoma has remained unchanged during the past 30 years, owing to a lack of efficient therapeutic targets. Methods: We constructed a kinome-targeting CRISPR-Cas9 library containing 507 kinases and 100 nontargeting controls and screened the potential kinase targets in osteosarcoma. The CRISPR screening sequencing data were analyzed with the Model-based Analysis of Genome-wide CRISPR/Cas9 Knockout (MAGeCK) Python package. The functional data were applied in the 143B cell line through lenti-CRISPR-mediated gene knockout. The clinical significance of kinases in the survival of patients with osteosarcoma was analyzed in the R2: Genomics Analysis and Visualization Platform. Results: We identified 53 potential kinase targets in osteosarcoma. Among these targets, we analyzed 3 kinases, TRRAP, PKMYT1, and TP53RK, to validate their oncogenic functions in osteosarcoma. PKMYT1 and TP53RK showed higher expression in osteosarcoma than in normal bone tissue, whereas TRRAP showed no significant difference. 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.
Insights
This study identified 53 potential kinase targets for osteosarcoma treatment using CRISPR screening. Three kinases, TRRAP, PKMYT1, and TP53RK, show promise as therapeutic targets for improving patient survival in osteosarcoma.
Area of Science:
- Oncology
- Genetics
- Molecular Biology
Background:
- Osteosarcoma is the most common primary bone cancer.
- Patient survival rates for osteosarcoma have stagnated due to a lack of effective therapeutic targets.
- Identifying novel therapeutic targets is crucial for improving osteosarcoma treatment outcomes.
Purpose of the Study:
- To identify potential kinase targets for osteosarcoma through a kinome-wide CRISPR-Cas9 screen.
- To validate the oncogenic function and clinical significance of identified kinase targets in osteosarcoma.
- To establish a functional genetic screening paradigm for osteosarcoma drug discovery.
Main Methods:
- Construction of a kinome-targeting CRISPR-Cas9 library (507 kinases).
- CRISPR screening and data analysis using the MAGeCK Python package.
- Functional validation of kinase targets (TRRAP, PKMYT1, TP53RK) in osteosarcoma cell lines (143B) and clinical data analysis.
Main Results:
- Identification of 53 potential kinase targets in osteosarcoma.
- PKMYT1 and TP53RK exhibited higher expression in osteosarcoma compared to normal bone tissue.
- High expression of TRRAP, PKMYT1, and TP53RK correlated with poorer patient prognosis.
Conclusions:
- The study identified TRRAP, PKMYT1, and TP53RK as potential therapeutic targets for osteosarcoma.
- A CRISPR-Cas9 screening approach provides a valuable paradigm for osteosarcoma functional genetic screening and drug discovery.
- The methodology may accelerate drug discovery for other cancer types by identifying novel therapeutic targets.
