Genome-scale CRISPR-mediated control of gene repression and activation
CRISPR-Cas9 cut irreversibly, limiting essential-gene study, and RNAi had strong off-target effects. This paper defined dCas9 CRISPRi/CRISPRa targeting rules and built genome-scale sgRNA libraries. CRISPRi typically achieved 90-99% knockdown with minimal off-target effects.
Key findings
- CRISPRi using dCas9-KRAB achieved 90–99% knockdown of endogenous genes. A tiling screen across 54,810 sgRNAs targeting 49 genes defined optimal positioning from −50 to +300 bp relative to the TSS, with protospacers of 18–21 bp showing maximal activity. CRISPRi outperformed shRNA with higher specificity and minimal off-target effects in head-to-head comparisons.
- Gilbert et al. developed genome-scale CRISPRi and CRISPRa libraries each targeting every annotated human gene with 10 sgRNAs, enabling expression modulation over a ~1,000-fold dynamic range. The platform was validated in two independent pooled screens for growth phenotypes and cholera-diphtheria toxin sensitivity. Growth screens reproducibly identified essential genes and tumor suppressors with high concordance across sgRNAs.
- Pooled screens with a cholera-diphtheria fusion toxin (CTx-DTA) revealed broad mechanisms of pathogen entry, retrotranslocation, and toxicity at genome scale. CRISPRi/a provided complementary and non-overlapping functional information relative to CRISPR knockout approaches. The system enabled reversible, inducible perturbation of essential genes and noncoding loci inaccessible to cutting or RNAi.