Data availability
The library design and screening analysis data have been deposited in the Zenodo database with the identifier https://doi.org/10.5281/zenodo.1719195144. The raw screen data is available in the Figshare database with the identifier https://doi.org/10.6084/m9.figshare.25533091.v1. The raw sequencing data is available in the EBI European Nucleotide Archive accession number ERP183979 and are available at the following URL https://www.ebi.ac.uk/ena/browser/view/ERP183979. The editing efficiency and validation data generated in this study are provided in the Supplementary Information and Source data file. Source data are provided with this paper.
Code availability
The code used to generate analysis has been deposited in the Zenodo database (https://doi.org/10.5281/zenodo.17191951)44,45 under a Creative Commons Attribution 4.0 International license (CC BY 4.0). Additional information for the following analyses can be found on the links below. Pilot library design: https://github.com/EmanuelGoncalves/crispy/blob/master/notebooks/dualguide/Library2Composition.py Genetic interaction library design: https://github.com/ibarrioh/DualGuide_COLO1/ Screen analysis: https://github.com/ibarrioh/DualGuide_COLO1/.
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Acknowledgements
We would like to thank the flow cytometry facility in Sanger Cellular Operations for analysis support and DNA sequencing facility in Sanger Scientific Operations for library construction and sequencing. pSpCas9(BB)−2A-GFP (PX458) was a gift from Feng Zhang (Addgene plasmid # 48138; http://n2t.net/addgene:48138; RRID:Addgene_48138)46. pKLV2-U6gRNA5(BbsI)-PGKpuro2ABFP-W, pKLV2-U6gRNA5(BbsI)PGKpuro-2A-mCherry-W and pKLV2-U6gRNA5(BbsI)PGKpuro-2A-mAG-W were gifts from Kosuke Yusa (Addgene plasmid # 67974, #67976, #67977; http://n2t.net/addgene:67974http://n2t.net/addgene:67976http://n2t.net/addgene:67977; RRID:Addgene_67974 RRID:Addgene_67976 RRID:Addgene_67977)24. We are grateful to Fiona Behan for advice and contributions to experimental design, Russell Walton for helpful comments on the manuscript and members of the Garnett and Bassett labs for their advice, discussions and support. This research was funded in part by the Wellcome Trust 220540/Z/20/A as institute core funding to M.G. and A.B. and OpenTargets OTAR2062. Funding from FCT (Fundação para a Ciência e Tecnologia), under projects UIDB/50021/2020 (DOI:10.54499/UIDB/50021/2020), https://doi.org/10.54499/2024.07252.IACDC (through RE-C05-i08.M04), and https://doi.org/10.54499/LISBOA2030-FEDER-00868200 supported E.G. For the purpose of Open Access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission.
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Competing interests
A.B. is a founder of and consultant for Ensocell therapeutics. M.G. is a founder of and consultant for Mosaic Therapeutics, receives research funding from GSK and Astex Pharmaceuticals and is a consultant for Bristol-Myers Squibb. The remaining authors declare no competing interests.
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Burgold, T., Karakoc, E., Gonçalves, E. et al. A next-generation dual guide CRISPR system for genetic interaction library screening. Nat Commun (2025). https://doi.org/10.1038/s41467-025-67256-9
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DOI: https://doi.org/10.1038/s41467-025-67256-9
