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Multi-Knock—a multi-targeted genome-scale CRISPR toolbox to overcome functional redundancy in plants

Nature Plants volume 9, pages 572–587 (2023)Cite this article

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Abstract

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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Fig. 1: Overview of Multi-Knock, the genome-scale, multi-targeted CRISPR platform.
Fig. 2: An overview of sgRNA design strategy for gene families.
Fig. 3: Multi-targeted genome-scale sgRNA design and construction.
Fig. 4: Transportome-specific Multi-Knock screen.
Fig. 5: PUP7, PUP8 and PUP21 redundantly regulate shoot growth and phyllotaxis.
Fig. 6: PUP7, PUP8 and PUP21 regulate cytokinin transport and shoot meristem.

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Data availability

All the data supporting the findings of this study are available within the article and Supplementary Materials. Source data are provided with this paper.

Code availability

All source codes used to generate the library are available in GitHub (https://github.com/anatshafir1/sgRNA_filtering_procedure).

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Acknowledgements

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.).

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  1. These authors contributed equally: Priyanka Patra, Odelia Pisanty.

Authors and Affiliations

  1. School of Plant Sciences and Food Security, Tel Aviv University, Tel Aviv, Israel

    Yangjie Hu, Priyanka Patra, Odelia Pisanty, Anat Shafir, Jenia Binenbaum, Shir Ben Yaakov, Gal Hyams, Yuqin Zhang, Maor Trabulsky, Omer Caldararu, Daniela Weiss, Adi Avni, Itay Mayrose & Eilon Shani

  2. Laboratoire Reproduction et Développement des Plantes, Université de Lyon, ENS de Lyon, CNRS, INRAE, Lyon, France

    Priyanka Patra, Bihai Shi & Teva Vernoux

  3. DynaMo Center, Department of Plant and Environmental Sciences, University of Copenhagen, Frederiksberg, Denmark

    Zeinu Mussa Belew, Christoph Crocoll & Hussam Hassan Nour-Eldin

  4. Department of Biology, University of Fribourg, Fribourg, Switzerland

    Laurence Charrier & Markus Geisler

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Contributions

Y.H. and E.S. conceived and designed the study and wrote the manuscript. Y.H. performed the research. P.P. assisted in cloning the Multi-Knock libraries and with PUP genetics. O.P. cloned and characterized the PUP amiRNA and PUP reporter lines. A.S., G.H. and O.C. carried out the Multi-Knock sgRNA library bioinformatics design and analysis. Z.M.B. performed transport assays in the Xenopus oocytes. J.B. carried out the Multi-Knock sgRNA library deep-sequencing analysis. S.B.Y. cloned the OLE:CITRINE Cas9 vector. B.S. carried out the TCS:VENUS assays. L.C. performed the tobacco transport assays. C.C. performed the LC–MS analysis. Y.Z. assisted in the PUP gene discovery. M.T. assisted in the library screen. D.W. assisted in the Multi-Knock sgRNA library design. A.A. provided the UBI:Cas9 vector. H.H.N.-E., M.G., T.V. and I.M. designed and supervised the work and edited the manuscript. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Itay Mayrose or Eilon Shani.

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Competing interests

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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Peer review information

Nature Plants thanks Kevin Zhao, Thomas Jacobs and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–10.

Reporting Summary

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

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

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