Journal Article

DZNE-2025-00230

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png Arrayed CRISPR libraries for the genome-wide activation, deletion and silencing of human protein-coding genes.

Yin, J.-A. ; Frick, L. ; Scheidmann, M. C. ; Liu, T. ; Trevisan, C. ; Dhingra, A.DZNE* ; Spinelli, A. ; Wu, Y. ; Yao, L. ; Vena, D. L. ; Knapp, B. ; Guo, J. ; De Cecco, E. ; Ging, K. ; Armani, A. ; Oakeley, E. J. ; Nigsch, F. ; Jenzer, J. ; Haegele, J. ; Pikusa, M. ; Täger, J.DZNE* ; Rodriguez-Nieto, S.DZNE* ; Bouris, V. ; Ribeiro, R. ; Baroni, F. ; Bedi, M. S. ; Berry, S. ; Losa, M. ; Hornemann, S. ; Kampmann, M. ; Pelkmans, L. ; Hoepfner, D. ; Heutink, P.DZNE* ; Aguzzi, A.

2025
Nature Research Tokyo

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Please use a persistent id in citations: doi:10.1038/s41551-024-01278-4

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 PrPC, 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.

Keyword(s): Humans (MeSH) ; Gene Silencing (MeSH) ; CRISPR-Cas Systems: genetics (MeSH) ; Genome, Human: genetics (MeSH) ; Gene Library (MeSH) ; RNA, Guide, CRISPR-Cas Systems: genetics (MeSH) ; Plasmids: genetics (MeSH) ; Gene Deletion (MeSH) ; HEK293 Cells (MeSH) ; Clustered Regularly Interspaced Short Palindromic Repeats: genetics (MeSH) ; Transcription Factors: genetics (MeSH) ; Transcription Factors: metabolism (MeSH) ; RNA, Guide, CRISPR-Cas Systems ; Transcription Factors

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Contributing Institute(s):

1. Genome Biology of Neurodegenerative Diseases (AG Heutink)
2. Cellomics Facility (AG Täger)

Research Program(s):

1. 354 - Disease Prevention and Healthy Aging (POF4-354) (POF4-354)
2. 899 - ohne Topic (POF4-899) (POF4-899)

Appears in the scientific report 2025

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Database coverage:
; ; ; Clarivate Analytics Master Journal List ; DEAL Nature ; Essential Science Indicators ; IF >= 25 ; JCR ; SCOPUS ; Science Citation Index Expanded ; Web of Science Core Collection

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