While CRISPR/Cas9 is used for editing individual genes, CRISPR screening scales genome engineering to the entire genome in a single experiment, identifying genes that confer resistance, sensitivity, or a specific phenotype.

Principle

A pooled CRISPR screen uses a library of single-guide RNA (sgRNA) sequences targeting every gene in the genome (or a focused subset). Each sgRNA directs Cas9 to create a double-strand break at a specific genomic locus, producing a knockout. By tracking which sgRNAs become enriched or depleted in a population under selection, the screen identifies genes that are functionally relevant.

Library Design

Genome-wide CRISPR libraries (Brunello, TKOv3, GeCKO) contain 4–6 sgRNAs per gene, plus 100–1000 non-targeting controls. Each sgRNA is a 20 nt sequence targeting a protospacer adjacent motif (PAM)-adjacent site in the genome. The library is synthesized as an oligonucleotide pool, cloned into a lentiviral vector, and packaged into lentivirus.

Screen Workflow

1. Transduction: infect Cas9-expressing cells with the lentiviral sgRNA library at a low multiplicity of infection (MOI ~0.3) so most cells receive a single sgRNA. Use enough cells to maintain ~500× representation of each sgRNA.
2. Selection: 48 hours after transduction, add puromycin (or another selection antibiotic) to select for cells that have integrated the library.
3. Phenotypic selection: after 7–14 days of propagation, apply the experimental condition: drug treatment, toxin exposure, starvation, or sort cells based on a reporter (e.g., a signaling pathway reporter). A parallel control population is cultured without selection.
4. Sequencing: extract genomic DNA from both populations, PCR-amplify the sgRNA-encoding region, and sequence on an Illumina platform.
5. Analysis: align reads to the library reference, count sgRNAs per gene, and calculate fold-change between selected and control populations. Genes with significantly depleted sgRNAs are essential for survival under the condition; enriched sgRNAs indicate resistance genes.

Key Considerations

• Representation: maintain at least 500 cells per sgRNA throughout the screen to avoid dropout artifacts.
• Cas9 expression: cells must express Cas9 at high levels. Stable Cas9-expressing lines or Cas9 knock-in lines are preferred.
• Controls: include a toxicity control (e.g., a high dose of the drug) to confirm that depletion is specific, and a no-selection control to account for growth effects of the sgRNAs themselves.

Applications

CRISPR screens have been used to identify resistance mechanisms to chemotherapeutics, targeted therapies, and immunotherapies; discover host factors required for viral infection; map synthetic lethal interactions in cancer; and identify genes regulating cell differentiation, migration, and metabolism.

Comparison with shRNA Screens

CRISPR screens create complete knockouts (frameshift mutations) rather than knockdowns (mRNA degradation), giving more consistent and complete loss of function. They have fewer off-target effects than shRNA because of the longer targeting sequence (20 nt vs. 19 nt) and the requirement for a PAM sequence. However, CRISPR screens are limited to protein-coding genes, whereas shRNA screens can target non-coding RNAs.