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 source codes used to generate the library are available in GitHub (https://github.com/anatshafir1/sgRNA_filtering_procedure).
Alonso, J. M. et al. Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301, 653–657 (2003).
Hauser, F. et al. A genomic-scale artificial microRNA library as a tool to investigate the functionally redundant gene space in Arabidopsis. Plant Cell 25, 2848–2863 (2013).
Henry, I. M. et al. Efficient genome-wide detection and cataloging of EMS-induced mutations using exome capture and next-generation sequencing. Plant Cell 26, 1382–1397 (2014).
Tal, I. et al. The Arabidopsis NPF3 protein is a GA transporter. Nat. Commun. 7, 11486 (2016).
Tang, X. et al. A single transcript CRISPR-Cas9 system for efficient genome editing in plants. Mol. Plant 9, 1088–1091 (2016).
Wang, L. et al. Construction of a genomewide RNAi mutant library in rice. Plant Biotechnol. J. 11, 997–1005 (2013).
Zhang, Y. et al. A transportome-scale amiRNA-based screen identifies redundant roles of Arabidopsis ABCB6 and ABCB20 in auxin transport. Nat. Commun. 9, 4204 (2018).
Panchy, N., Lehti-Shiu, M. & Shiu, S. H. Evolution of gene duplication in plants. Plant Physiol. 171, 2294–2316 (2016).
Li, Z. et al. Gene duplicability of core genes is highly consistent across all angiosperms. Plant Cell 28, 326–344 (2015).
Rensing, S. A. Gene duplication as a driver of plant morphogenetic evolution. Curr. Opin. Plant Biol. 17, 43–48 (2014).
Lu, X. et al. Gene-indexed mutations in maize. Mol. Plant 11, 496–504 (2018).
Gaillochet, C., Develtere, W. & Jacobs, T. B. CRISPR screens in plants: approaches, guidelines, and future prospects. Plant Cell 33, 794–813 (2021).
Chen, K., Wang, Y., Zhang, R., Zhang, H. & Gao, C. CRISPR/Cas genome editing and precision plant breeding in agriculture. Annu. Rev. Plant Biol. 70, 667–697 (2019).
Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).
Jacobs, T. B., Zhang, N., Patel, D. & Martin, G. B. Generation of a collection of mutant tomato lines using pooled CRISPR libraries. Plant Physiol. 174, 2023–2037 (2017).
Chen, K. et al. A FLASH pipeline for arrayed CRISPR library construction and the gene function discovery of rice receptor-like kinases. Mol. Plant 15, 243–257 (2021).
Liu, H. J. et al. High-throughput CRISPR/Cas9 mutagenesis streamlines trait gene identification in maize. Plant Cell 32, 1397–1413 (2020).
Lu, Y. et al. Genome-wide targeted mutagenesis in rice using the CRISPR/Cas9 system. Mol. Plant 10, 1242–1245 (2017).
Meng, X. et al. Construction of a genome-wide mutant library in rice using CRISPR/Cas9. Mol. Plant 10, 1238–1241 (2017).
Bai, M. et al. Generation of a multiplex mutagenesis population via pooled CRISPR–Cas9 in soya bean. Plant Biotechnol. J. 18, 721–731 (2020).
Ramadan, M. et al. Efficient CRISPR/Cas9 mediated pooled-sgRNAs assembly accelerates targeting multiple genes related to male sterility in cotton. Plant Methods 17, 1–13 (2021).
Lorenzo, C. D. et al. BREEDIT: a multiplex genome editing strategy to improve complex quantitative traits in maize. Plant Cell 35, 218–238 (2023).
Grützner, R. et al. High-efficiency genome editing in plants mediated by a Cas9 gene containing multiple introns. Plant Commun. 2, 1–15 (2021).
Proost, S. et al. PLAZA 3.0: an access point for plant comparative genomics. Nucleic Acids Res. 43, D974–D981 (2015).
Hyams, G. et al. CRISPys: optimal sgRNA design for editing multiple members of a gene family using the CRISPR system. J. Mol. Biol. 430, 2184–2195 (2018).
Joung, J. et al. Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening. Nat. Protoc. 12, 828–863 (2017).
Kang, J. et al. Plant ABC transporters. Arabidopsis Book 9, e0153 (2011).
Tsutsui, H. & Higashiyama, T. PKAMA-ITACHI vectors for highly efficient CRISPR/Cas9-mediated gene knockout in Arabidopsis thaliana. Plant Cell Physiol. 58, 46–56 (2017).
Sussholz, O., Pizarro, L., Schuster, S. & Avni, A. SlRLK-like is a malectin-like domain protein affecting localization and abundance of LeEIX2 receptor resulting in suppression of EIX-induced immune responses. Plant J. 104, 1369–1381 (2020).
Fauser, F., Schiml, S. & Puchta, H. Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana. Plant J. 79, 348–359 (2014).
Wang, Z. P. et al. Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation. Genome Biol. 16, 1–12 (2015).
LeBlanc, C. et al. Increased efficiency of targeted mutagenesis by CRISPR/Cas9 in plants using heat stress. Plant J. 93, 377–386 (2018).
Kubis, S. et al. Functional specialization amongst the Arabidopsis Toc159 family of chloroplast protein import receptors. Plant Cell 16, 2059–2077 (2004).
Niittylä, T. et al. A previously unknown maltose transporter essential for starch degradation in leaves. Science 303, 87–89 (2004).
Takano, J. et al. Arabidopsis boron transporter for xylem loading. Nature 420, 337–340 (2002).
Kang, J., Lee, Y., Sakakibara, H. & Martinoia, E. Cytokinin transporters: GO and STOP in signaling. Trends Plant Sci. 22, 455–461 (2017).
Zürcher, E., Liu, J., Di Donato, M., Geisler, M. & Müller, B. Plant development regulated by cytokinin sinks. Science 353, 1027–1030 (2016).
Bürkle, L. et al. Transport of cytokinins mediated by purine transporters of the PUP family expressed in phloem, hydathodes, and pollen of Arabidopsis. Plant J. 34, 13–26 (2003).
Gillissen, B. et al. A new family of high-affinity transporters for adenine, cytosine, and purine derivatives in Arabidopsis. Plant Cell 12, 291–300 (2000).
Xiao, Y. et al. Endoplasmic reticulum-localized PURINE PERMEASE1 regulates plant height and grain weight by modulating cytokinin distribution in rice. Front. Plant Sci. 11, 618560 (2020).
Xiao, Y. et al. Big Grain3, encoding a purine permease, regulates grain size via modulating cytokinin transport in rice. J. Integr. Plant Biol. 61, 581–597 (2019).
Perilli, S., Moubayidin, L. & Sabatini, S. The molecular basis of cytokinin function. Curr. Opin. Plant Biol. 13, 21–26 (2010).
Wybouw, B. & De Rybel, B. Cytokinin—a developing story. Trends Plant Sci. 24, 177–185 (2019).
Ha, S., Vankova, R., Yamaguchi-Shinozaki, K., Shinozaki, K. & Tran, L. S. P. Cytokinins: metabolism and function in plant adaptation to environmental stresses. Trends Plant Sci. 17, 172–179 (2012).
Sakakibara, H. Cytokinins: activity, biosynthesis, and translocation. Annu. Rev. Plant Biol. 57, 431–449 (2006).
Besnard, F. et al. Cytokinin signalling inhibitory fields provide robustness to phyllotaxis. Nature 505, 417–421 (2014).
Nelson, B. K., Cai, X. & Nebenführ, A. A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants. Plant J. 51, 1126–1136 (2007).
Bruno, M. & Jen, S. Cytokinin and auxin interplay in root stem-cell specification during early embryogenesis. Nature 4, 1094–1097 (2008).
O’Malley, R. C. & Ecker, J. R. Linking genotype to phenotype using the Arabidopsis unimutant collection. Plant J. 61, 928–940 (2010).
Park, R. J. et al. A genome-wide CRISPR screen identifies a restricted set of HIV host dependency factors. Nat. Genet. 49, 193–203 (2017).
Wang, T., Wei, J. J., Sabatini, D. M. & Lander, E. S. Genetic screens in human cells using the CRISPR-Cas9 system. Science 343, 80–84 (2014).
Ursache, R., Fujita, S., Dénervaud, V. & Geldner, N. Combined fluorescent seed selection and multiplex CRISPR/Cas9 assembly for fast generation of multiple Arabidopsis mutants Robertas. Plant Methods 17, 111 (2021).
Caesar, K. et al. Evidence for the localization of the Arabidopsis cytokinin receptors AHK3 and AHK4 in the endoplasmic reticulum. J. Exp. Bot. 62, 5571–5580 (2011).
Wulfetange, K. et al. The cytokinin receptors of Arabidopsis are located mainly to the endoplasmic reticulum. Plant Physiol. 156, 1808–1818 (2011).
Lomin, S. N., Yonekura-Sakakibara, K., Romanov, G. A. & Sakakibara, H. Ligand-binding properties and subcellular localization of maize cytokinin receptors. J. Exp. Bot. 62, 5149–5159 (2011).
Ding, W. et al. Isolation, characterization and transcriptome analysis of a cytokinin receptor mutant osckt1 in rice. Front. Plant Sci. 8, 88 (2017).
Antoniadi, I. et al. Cell-surface receptors enable perception of extracellular cytokinins. Nat. Commun. 11, 4284 (2020).
Kubiasová, K. et al. Cytokinin fluoroprobe reveals multiple sites of cytokinin perception at plasma membrane and endoplasmic reticulum. Nat. Commun. 11, 4285 (2020).
Carpaneto, A. et al. Phloem-localized, proton-coupled sucrose carrier ZmSUT1 mediates sucrose efflux under the control of the sucrose gradient and the proton motive force. J. Biol. Chem. 280, 21437–21443 (2005).
Léran, S. et al. Arabidopsis NRT1.1 is a bidirectional transporter involved in root-to-shoot nitrate translocation. Mol. Plant 6, 1984–1987 (2013).
Mussa Belew, Z. et al. Identification and characterization of phlorizin transporter from Arabidopsis thaliana and its application for phlorizin production in Saccharomyces cerevisiae. Preprint at bioRxiv https://doi.org/10.1101/2020.08.14.248047 (2020).
Chen, L. Q. et al. Sugar transporters for intercellular exchange and nutrition of pathogens. Nature 468, 527–532 (2010).
Chen, J. et al. ABCB-mediated shootward auxin transport feeds into the root clock. EMBO Rep. https://doi.org/10.15252/embr.202256271 (2023).
Zhang, Y. et al. ABA homeostasis and long-distance translocation are redundantly regulated by ABCG ABA importers. Sci. Adv. 7, 1–18 (2021).
Develtere, W. et al. SMAP design: a multiplex PCR amplicon and gRNA design tool to screen for natural and CRISPR-induced genetic variation. Nucleic Acids Res. https://doi.org/10.1093/nar/gkad036 (2023).
Ellison, E. E. et al. Multiplexed heritable gene editing using RNA viruses and mobile single guide RNAs. Nat. Plants 6, 620–624 (2020).
Wang, M. et al. Gene targeting by homology-directed repair in rice using a geminivirus-based CRISPR/Cas9 system. Mol. Plant 10, 1007–1010 (2017).
Martin-Ortigosa, S. et al. Mesoporous silica nanoparticle-mediated intracellular cre protein delivery for maize genome editing via loxP site excision. Plant Physiol. 164, 537–547 (2014).
Mitter, N. et al. Clay nanosheets for topical delivery of RNAi for sustained protection against plant viruses. Nat. Plants 3, 16207 (2017).
Clough, S. J. & Bent, A. F. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743 (1998).
Gronau, I. & Moran, S. Optimal implementations of UPGMA and other common clustering algorithms. Inf. Process. Lett. 104, 205–210 (2007).
Doench, J. G. et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat. Biotechnol. 34, 184–191 (2016).
Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760 (2009).
Nour-Eldin, H. H., Hansen, B. G., Nørholm, M. H. H., Jensen, J. K. & Halkier, B. A. Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments. Nucleic Acids Res. 34, e122 (2006).
Jørgensen, M., Crocoll, C., Halkier, B. & Nour-Eldin, H. Uptake assays in Xenopus laevis oocytes using liquid chromatography-mass spectrometry to detect transport activity. Bio Protoc. 7, e2581 (2017).
Ionescu, I. A. et al. Transcriptome and metabolite changes during hydrogen cyanamide-induced floral bud break in sweet cherry. Front. Plant Sci. 8, 1233 (2017).
Jarzyniak, K. et al. Early stages of legume–rhizobia symbiosis are controlled by ABCG-mediated transport of active cytokinins. Nat. Plants 7, 428–436 (2021).
Dereeper, A. et al. Phylogeny.fr: robust phylogenetic analysis for the non-specialist. Nucleic Acids Res. 36, 465–469 (2008).
Prasad, K. et al. Arabidopsis PLETHORA transcription factors control phyllotaxis. Curr. Biol. 21, 1123–1128 (2011).
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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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