Arrayed CRISPR libraries for the genome-wide activation, deletion and silencing of human protein-coding genes
Jiang‐An Yin, Lukas Frick, Manuel C. Scheidmann, Tingting Liu, Chiara Trevisan, Ashutosh Dhingra, Anna Spinelli, Yancheng Wu, Longping Yao, Dalila Laura Vena, Britta Knapp, Jingjing Guo, Elena De Cecco, Kathi Ging, Andrea Armani, Edward J. Oakeley, Florian Nigsch, Joel Jenzer, Jasmin Haegele, Michal Pikusa, Joachim Täger, Salvador Rodrı́guez-Nieto, Vangelis Bouris, Rafaela Vanin Pinto Ribeiro, Federico Baroni, Manmeet Sakshi Bedi, Scott M. Berry, Marco Losa, Simone Hornemann, Martin Kampmann, Lucas Pelkmans, Dominic Hoepfner, Peter Heutink, Adriano Aguzzi
Abstract
Abstract Arrayed CRISPR libraries extend the scope of gene-perturbation screens to non-selectable cell phenotypes. However, library generation requires assembling thousands of vectors expressing single-guide RNAs (sgRNAs). Here, by leveraging massively parallel plasmid-cloning methodology, we show that arrayed libraries can be constructed for the genome-wide ablation (19,936 plasmids) of human protein-coding genes and for their activation and epigenetic silencing (22,442 plasmids), with each plasmid encoding an array of four non-overlapping sgRNAs designed to tolerate most human DNA polymorphisms. The quadruple-sgRNA libraries yielded high perturbation efficacies in deletion (75–99%) and silencing (76–92%) experiments and substantial fold changes in activation experiments. Moreover, an arrayed activation screen of 1,634 human transcription factors uncovered 11 novel regulators of the cellular prion protein PrP C , screening with a pooled version of the ablation library led to the identification of 5 novel modifiers of autophagy that otherwise went undetected, and ‘post-pooling’ individually produced lentiviruses eliminated template-switching artefacts and enhanced the performance of pooled screens for epigenetic silencing. Quadruple-sgRNA arrayed libraries are a powerful and versatile resource for targeted genome-wide perturbations.