CRISPR Screen Basics

The CRISPR/Cas9 revolution has opened new and unexpected avenues to gene-editing technology, permitting to perform powerful functional genome-wide screens directly in human cells. The endonuclease Cas9 from S. pyogenes can be directed to make double strand breaks (DSBs) in precise DNA locations by a sgRNA (single-guide RNA) molecule via a 20 bp-long base pairing. Cut DNA is then repaired and cut again until error-prone non-homologous end-joining repairs it with either DNA insertions or deletions (indels), creating a frame-shift into the exon of the targeted gene that inactivates the gene product and creates most of the time a functional knockout. The use of pooled KO sgRNA libraries permits to interrogate the whole genome in a single experiment. These libraries contain hundreds of thousands of sgRNAs molecules cloned into lentiviral plasmids, one sgRNA per plasmid or lentivirus, all in the same tube. Cells are infected at a low multiplicity of infection (MOI) to favor delivery of one sgRNA per cell. As cells are grown in the presence of a chemical inhibiting growth, cells carrying sgRNAs providing either resistance or sensitivity to the effect of the drug will respectively be enriched or depleted in the cell population.

The changes in sgRNA frequencies, as assessed by Next-Generation Sequencing (NGS) of sgRNA sequences, between treated vs untreated cells can be used to compute robust enrichment or depletion scores for all genes. All hits genes, rescues (KO creating resistance, sgRNA enriched, positive score) or synthetic lethals (KO creating sensitivity, sgRNA depleted, negative score) are what we call the chemogenomic signature for a compound.