Arrayed CRISPR libraries for the genome-wide activation, deletion and silencing of human protein-coding genes
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<sup>C</sup>, 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.
Authors
- Britta Knapp
- Kathi Ging (ORCID: https://orcid.org/0000-0001-7765-9821)
- Joachim Täger
- Jiang‐An Yin (ORCID: https://orcid.org/0000-0002-2134-8693)
- Jingjing Guo (ORCID: https://orcid.org/0000-0001-6139-4777)
- Tingting Liu (ORCID: https://orcid.org/0000-0003-2756-9697)
- Edward J. Oakeley (ORCID: https://orcid.org/0000-0001-6226-7470)
- Michal Pikusa
- Elena De Cecco (ORCID: https://orcid.org/0000-0002-0148-2596)
- Adriano Aguzzi (ORCID: https://orcid.org/0000-0002-0344-6708)
- Lucas Pelkmans (ORCID: https://orcid.org/0000-0002-6754-9730)
- Manuel C. Scheidmann
- Ashutosh Dhingra (ORCID: https://orcid.org/0000-0003-2997-7467)
- Longping Yao (ORCID: https://orcid.org/0000-0002-7239-1392)
- Peter Heutink (ORCID: https://orcid.org/0000-0001-5218-1737)
- Lukas Frick (ORCID: https://orcid.org/0000-0002-2147-5369)
- Florian Nigsch (ORCID: https://orcid.org/0000-0002-2919-8749)
- Anna Spinelli (ORCID: https://orcid.org/0000-0002-8829-7405)
- Vangelis Bouris (ORCID: https://orcid.org/0000-0002-6959-5765)
- Simone Hornemann (ORCID: https://orcid.org/0000-0002-2674-9891)
- Chiara Trevisan (ORCID: https://orcid.org/0000-0002-3997-094X)
- Rafaela Vanin Pinto Ribeiro (ORCID: https://orcid.org/0000-0002-5627-9157)
- Martin Kampmann (ORCID: https://orcid.org/0000-0002-3819-7019)
- Salvador Rodrı́guez-Nieto
- Jasmin Haegele
- Andrea Armani (ORCID: https://orcid.org/0000-0002-4765-9748)
- Dominic Hoepfner (ORCID: https://orcid.org/0000-0001-8450-7543)
- Dalila Laura Vena (ORCID: https://orcid.org/0009-0007-6863-7349)
- Joel Jenzer
- Scott M. Berry
- Yancheng Wu (ORCID: https://orcid.org/0000-0003-2809-4865)
- Marco Losa (ORCID: https://orcid.org/0000-0003-3428-418X)
- Federico Baroni
- Manmeet Sakshi Bedi
Institutions
- Novartis (Switzerland) (CH)
- University of Padua (IT)
- University of California, San Francisco (US)
- University of Zurich (CH)
- German Center for Neurodegenerative Diseases (DE)
- UNSW Sydney (AU)
- EMBL Australia (AU)
- Novartis Institutes for BioMedical Research
Publication Details
- Journal
- Nature Biomedical Engineering
- Published
- 2024-12-04
- DOI
- https://doi.org/10.1038/s41551-024-01278-4
- Citations
- 23
- Primary Topic
- CRISPR and Genetic Engineering
- Type
- article
- Field-Weighted Citation Impact
- 5.38