Arrayed CRISPR libraries for the genome-wide activation, deletion and silencing of human protein-coding genes.

Autor: Yin JA; Institute of Neuropathology, University of Zurich, Zurich, Switzerland. jiang-an.yin@uzh.ch., Frick L; Institute of Neuropathology, University of Zurich, Zurich, Switzerland., Scheidmann MC; Novartis Institutes for Biomedical Research, Novartis Campus, Basel, Switzerland., Liu T; Institute of Neuropathology, University of Zurich, Zurich, Switzerland., Trevisan C; Institute of Neuropathology, University of Zurich, Zurich, Switzerland., Dhingra A; German Center for Neurodegenerative Diseases (DZNE), Tübingen, Germany., Spinelli A; Institute of Neuropathology, University of Zurich, Zurich, Switzerland., Wu Y; Institute of Neuropathology, University of Zurich, Zurich, Switzerland., Yao L; Institute of Neuropathology, University of Zurich, Zurich, Switzerland., Vena DL; Institute of Neuropathology, University of Zurich, Zurich, Switzerland., Knapp B; Novartis Institutes for Biomedical Research, Novartis Campus, Basel, Switzerland., Guo J; Institute of Neuropathology, University of Zurich, Zurich, Switzerland., De Cecco E; Institute of Neuropathology, University of Zurich, Zurich, Switzerland., Ging K; Institute of Neuropathology, University of Zurich, Zurich, Switzerland., Armani A; Institute of Neuropathology, University of Zurich, Zurich, Switzerland.; Department of Biomedical Sciences, University of Padua, Padova, Italy., Oakeley EJ; Novartis Institutes for Biomedical Research, Novartis Campus, Basel, Switzerland., Nigsch F; Novartis Institutes for Biomedical Research, Novartis Campus, Basel, Switzerland., Jenzer J; Novartis Institutes for Biomedical Research, Novartis Campus, Basel, Switzerland., Haegele J; Novartis Institutes for Biomedical Research, Novartis Campus, Basel, Switzerland., Pikusa M; Novartis Institutes for Biomedical Research, Novartis Campus, Basel, Switzerland., Täger J; German Center for Neurodegenerative Diseases (DZNE), Tübingen, Germany., Rodriguez-Nieto S; German Center for Neurodegenerative Diseases (DZNE), Tübingen, Germany., Bouris V; Institute of Neuropathology, University of Zurich, Zurich, Switzerland., Ribeiro R; Institute of Neuropathology, University of Zurich, Zurich, Switzerland., Baroni F; Institute of Neuropathology, University of Zurich, Zurich, Switzerland., Bedi MS; Novartis Institutes for Biomedical Research, Novartis Campus, Basel, Switzerland., Berry S; Department of Molecular Life Sciences, University of Zurich, Zurich, Switzerland.; EMBL Australia Node in Single Molecule Science, School of Medical Sciences, University of New South Wales, Sydney, Australia., Losa M; Institute of Neuropathology, University of Zurich, Zurich, Switzerland., Hornemann S; Institute of Neuropathology, University of Zurich, Zurich, Switzerland., Kampmann M; Institute for Neurodegenerative Diseases, Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, CA, USA., Pelkmans L; Department of Molecular Life Sciences, University of Zurich, Zurich, Switzerland., Hoepfner D; Novartis Institutes for Biomedical Research, Novartis Campus, Basel, Switzerland., Heutink P; German Center for Neurodegenerative Diseases (DZNE), Tübingen, Germany., Aguzzi A; Institute of Neuropathology, University of Zurich, Zurich, Switzerland. adriano.aguzzi@uzh.ch.
Jazyk: angličtina
Zdroj: Nature biomedical engineering [Nat Biomed Eng] 2024 Dec 04. Date of Electronic Publication: 2024 Dec 04.
DOI: 10.1038/s41551-024-01278-4
Abstrakt: 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.
Competing Interests: Competing interests: J.-A.Y., L.F. and A. Aguzzi are listed as inventors on a patent (Molecular Cloning Method and Vector Therefore, WO/2023/089153) owned by the University of Zurich, whose claims are supported by the present study. L.P. has ownership interest in Sagimet Biosciences, Apricot Therapeutics and Element Biosciences. M.K. is an inventor on US Patent 11,254,933 related to CRISPRi and CRISPRa screening, serves on the Scientific Advisory Boards of Engine Biosciences, Casma Therapeutics, Cajal Neuroscience, Alector and Montara Therapeutics, and is an advisor to Modulo Bio and Recursion Therapeutics. All authors with the affiliation ‘Novartis Institutes for Biomedical Research’ are employees of Novartis Pharma AG and may own stock in the company. The other authors declare no competing interests.
(© 2024. The Author(s).)
Databáze: MEDLINE
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