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Updated: Sep 28, 2025
Pooled CRISPR-Based Genetic Screens in Mammalian Cells
Published on: September 4, 2019
CRISPR Guide RNA Library Screens in Human Induced Pluripotent Stem Cells
Yan Zhou1,2, Qiang Fu3, Huijun Shi3
1Department of Medical Cell Biology and Genetics, Guangdong Key Laboratory of Genomic Stability and Disease Prevention, Shenzhen Key Laboratory of Anti-aging and Regenerative Medicine, and Shenzhen Engineering Laboratory of Regenerative Technologies for Orthopaedic Diseases, Health Science Center, Shenzhen University, Shenzhen, China. yanzhou_2017@163.com.
This study details a method for high-throughput CRISPR screens to identify gene functions. It optimizes puromycin and lentiviral CRISPR guide RNA (gRNA) titration for unbiased gene discovery in human iPSC models.
Area of Science:
- * CRISPR/Cas9 gene editing technology
- * Functional genomics and high-throughput screening
Background:
- * Pooled CRISPR/Cas9 screens are essential for unbiased identification of gene functions.
- * These screens link genes to cellular phenotypes through guide RNA (gRNA) depletion or enrichment.
Purpose of the Study:
- * To describe a detailed method for optimizing CRISPR screens in Cas9-expressing human induced pluripotent stem cells (Cas9+MNhiPSC).
- * To enable reliable and unbiased gene function discovery using CRISPR/Cas9 technology.
Main Methods:
- * Optimization of puromycin (PURO) concentration and lentiviral CRISPR gRNA library titration.
- * Performing pooled CRISPR gRNA library screens in Cas9+MNhiPSC.
- * Genomic DNA extraction, sequencing library preparation, and next-generation sequencing (NGS) for gRNA read counts.
Main Results:
- * Established protocols for titration of critical screening components (PURO and gRNA library).
- * Demonstrated the application of these methods in Cas9+MNhiPSC for pooled CRISPR screens.
- * Achieved a target transduction efficiency of 30% (MOI=0.3) for single gRNA delivery.
Conclusions:
- * The described method provides a robust framework for conducting CRISPR screens.
- * This approach is adaptable for various CRISPR perturbations (knockout, activation, repression, base editing) across diverse cell models and species.
