Plant genomes are characterized by large and complex gene families that often result in similar and partially overlapping functions. This genetic redundancy severely hampers current efforts to uncover novel phenotypes, delaying basic genetic research and breeding programmes. Here we describe the development and validation of Multi-Knock, a genome-scale clustered regularly interspaced short palindromic repeat toolbox that overcomes functional redundancy in Arabidopsis by simultaneously targeting multiple gene-family members, thus identifying genetically hidden components. We computationally designed 59,129 optimal single-guide RNAs that each target two to ten genes within a family at once. Furthermore, partitioning the library into ten sublibraries directed towards a different functional group allows flexible and targeted genetic screens. From the 5,635 single-guide RNAs targeting the plant transportome, we generated over 3,500 independent Arabidopsis lines that allowed us to identify and characterize the first known cytokinin tonoplast-localized transporters in plants. With the ability to overcome functional redundancy in plants at the genome-scale level, the developed strategy can be readily deployed by scientists and breeders for basic research and to expedite breeding efforts.
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All the data supporting the findings of this study are available within the article and Supplementary Materials. Source data are provided with this paper.
All source codes used to generate the library are available in GitHub (https://github.com/anatshafir1/sgRNA_filtering_procedure).
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We thank B. Müller (University of Zurich, Switzerland) for sharing TCS:VENUS seeds. Funding: this work was supported by grants from the Israel Science Foundation (2378/19 and 3419/20 to E.S.), the Human Frontier Science Program (HFSP—RGY0075/2015 and HFSP—LIY000540/2020 to E.S., H.H.N.-E. and Z.M.B.), Danmarks Grundforskningsfond (DNRF99 to H.H.N.-E.), the European Research Council (757683-RobustHormoneTrans to E.S.), the PBC postdoctoral fellowship (to Y.H.), PhD fellowship from the Edmond J. Safra Center for Bioinformatics at Tel Aviv University (to A.S.) and by the Swiss National Funds (31003A-165877/1 to M.G.).
A US Provisional Patent Application (no. 63/329,506) on the Multi-Knock system described in this study has been filed. The authors declare that they have no competing interests.
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Supplementary Figs. 1–10.
Supplementary Data 1
sgRNA library all families.
Supplementary Data 2
Source data for supplementary figures.
Supplementary Data 3
Unprocessed gel image of Supplementary Fig. 7a.
Supplementary Tables 1–8
Supplementary Tables 1–8.
Source Data Fig. 3
NGS source data for Fig. 3e.
Source Data Fig. 4
NGS source data for Fig. 4a.
Source Data Fig. 5
Statistical source data.
Source Data Fig. 6
Statistical source data.
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Hu, Y., Patra, P., Pisanty, O. et al. Multi-Knock—a multi-targeted genome-scale CRISPR toolbox to overcome functional redundancy in plants. Nat. Plants 9, 572–587 (2023). https://doi.org/10.1038/s41477-023-01374-4