Arrayed CRISPR/Cas9 Screening for the Functional Validation of Cancer Genetic Dependencies

Area of Science:

• Biomedical Research
• Functional Genomics
• Cancer Biology

Background:

• CRISPR/Cas9 screening is crucial for identifying genetic dependencies in cancer.
• Existing protocols can be time-consuming and less versatile.

Purpose of the Study:

• To develop an efficient and versatile protocol for CRISPR/Cas9 screening.
• To enable high-throughput assessment of gene knockout effects on cellular fitness and other parameters.

Main Methods:

• Lentiviral vector-based cloning of guide RNAs (gRNA) in a 96-well format.
• Production of lentiviral supernatants and transduction of target cells.
• Competition-based cell proliferation assay using flow cytometry for assessing gene knockout effects.

Main Results:

• The protocol allows for efficient cloning, lentiviral production, and cell transduction.
• Flow cytometry-based assay enables rapid and reproducible screening of multiple gene knockouts.
• The method can assess various cellular parameters beyond proliferation, including protein expression and cell death.

Conclusions:

• This protocol provides a fast and reproducible method for functional genomics screening.
• It enables the assessment of 50-300 gene knockouts on diverse cellular parameters within eight weeks.
• The validated protocol is applicable to leukemia research and other areas of biomedical investigation.