Table 1.

Key Considerations When Performing a CRISPR-ko Screen

Screen Phase	Considerations	Advantages/Disadvantages
I. Targets  What genes will be studied?	Choice of library	• Genome-scale libraries have the benefit of being comprehensive but can be resource-intensive. Predesigned genome-wide CRISPR libraries are available • Targeted libraries focus on class of elements (kinases, transcription factors, etc.) or can be custom-designed and generated by the investigator
II. Model  What cells should be used?	Cas9 expression	• Primary cells require delivery of both Cas9 and sgRNA, which may be technically challenging • Stably expressing Cas9 cell lines provide uniform high expression of Cas9
III. Assay  How are cells screened?	Phenotype	• A classical screen results in positive or negative selection from a selective pressure (e.g., exposure to a drug) • Combining CRISPR screens with other genetic tools such as reporter cell lines can facilitate screening for diverse phenotypes
IV. Analysis  How are screen results evaluated?	Measuring screen outputs	• Changes in sgRNA abundance, measured by next-generation sequencing, are a classical output of CRISPR-ko screens • A variety of validated CRISPR screen analysis pipelines are publicly available

Abbreviations: CRISPR-ko, CRISPR knockout; sgRNA, single guide RNA.