Optimized libraries for CRISPR-Cas9 genetic screens with multiple modalities

• Published in: Nature communications Vol. 9; no. 1; pp. 5416 - 15
• Main Authors: Sanson, Kendall R., Hanna, Ruth E., Hegde, Mudra, Donovan, Katherine F., Strand, Christine, Sullender, Meagan E., Vaimberg, Emma W., Goodale, Amy, Root, David E., Piccioni, Federica, Doench, John G.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 21.12.2018; Nature Publishing Group; Nature Portfolio
• Subjects: 42; 42/47; 42/89; 45; 45/44; 49; 49/40; 631/337/4041; 631/553/2490; 631/61/212; CRISPR; CRISPR-Associated Protein 9; CRISPR-Cas Systems; Geckos; Genes; Genetic screening; Genomes; Genomic Library; Humanities and Social Sciences; Interference; Interrogation; Libraries; multidisciplinary; Negative selection; Open reading frames; Perturbation; Positive selection; RNA-mediated interference; Science; Science (multidisciplinary); Streptococcus pyogenes
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-018-07901-8

The creation of genome-wide libraries for CRISPR knockout (CRISPRko), interference (CRISPRi), and activation (CRISPRa) has enabled the systematic interrogation of gene function. Here, we show that our recently-described CRISPRko library (Brunello) is more effective than previously published libraries at distinguishing essential and non-essential genes, providing approximately the same perturbation-level performance improvement over GeCKO libraries as GeCKO provided over RNAi. Additionally, we present genome-wide libraries for CRISPRi (Dolcetto) and CRISPRa (Calabrese), and show in negative selection screens that Dolcetto, with fewer sgRNAs per gene, outperforms existing CRISPRi libraries and achieves comparable performance to CRISPRko in detecting essential genes. We also perform positive selection CRISPRa screens and demonstrate that Calabrese outperforms the SAM approach at identifying vemurafenib resistance genes. We further compare CRISPRa to genome-scale libraries of open reading frames (ORFs). Together, these libraries represent a suite of genome-wide tools to efficiently interrogate gene function with multiple modalities. Genome-wide libraries for CRISPR knockout, interference, and activation have allowed the systemic interrogation of gene function. Here, the authors evaluate the Brunello CRISPRko library and introduce Dolcetto and Calabrese for CRISPRi and CRISPRa, respectively.