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CRISPR–Act3.0 for highly efficient multiplexed gene activation in plants

Abstract

RNA-guided CRISPR activation (CRISPRa) systems have been developed in plants. However, the simultaneous activation of multiple genes remains challenging. Here, we develop a highly robust CRISPRa system working in rice, Arabidopsis and tomato, CRISPR–Act3.0, through systematically exploring different effector recruitment strategies and various transcription activators based on deactivated Streptococcus pyogenes Cas9 (dSpCas9). The CRISPR–Act3.0 system results in fourfold to sixfold higher activation than the state-of-the-art CRISPRa systems. We further develop a tRNA–gR2.0 (single guide RNA 2.0) expression system enabling CRISPR–Act3.0-based robust activation of up to seven genes for metabolic engineering in rice. In addition, CRISPR–Act3.0 allows the simultaneous modification of multiple traits in Arabidopsis, which are stably transmitted to the T3 generations. On the basis of CRISPR–Act3.0, we elucidate guide RNA targeting rules for effective transcriptional activation. To target T-rich protospacer adjacent motifs (PAMs), we transfer this activation strategy to CRISPR–dCas12b and further improve the dAaCas12b-based CRISPRa system. Moreover, we develop a potent near-PAM-less CRISPR–Act3.0 system on the basis of the SpRY dCas9 variant, which outperforms the dCas9–NG system in both activation potency and targeting scope. Altogether, our study has substantially improved the CRISPRa technology in plants and provided plant researchers a powerful toolbox for efficient gene activation in foundational and translational research.

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Fig. 1: Development of the CRISPR–Act3.0 system.
Fig. 2: Multiplexed gene activation by CRISPR–Act3.0 in rice.
Fig. 3: Multiplexed gene activation by CRISPR–Act3.0 in dicot plants.
Fig. 4: Effects of sgRNA position and GC composition on CRISPR–Act3.0-mediated activation in rice.
Fig. 5: Expanding the targeting scope of CRISPR–Act3.0.

Data availability

The 25 Golden Gate and Gateway compatible vectors for the CRISPR–Act3.0 systems are available from Addgene: pYPQ131–tRNA2.0 (no. 158393), pYPQ132–tRNA2.0 (no. 158394), pYPQ133–tRNA2.0 (no. 158395), pYPQ134–tRNA2.0 (no. 158396), pYPQ135–tRNA2.0 (no. 158397), pYPQ136–tRNA2.0 (no. 158398), pYPQ142–ZmUbi–tRNA (no. 158578), pYPQ143–ZmUbi–tRNA (no. 158400), pYPQ144–ZmUbi–tRNA (no. 158402), pYPQ145–ZmUbi–tRNA (no. 158403), pYPQ146–ZmUbi–tRNA (no. 158404), pYPQ141–ZmUbi–RZ–Aac.4 (no. 158406), pYPQ141–ZmUbi–RZ–Aa3.8.5 (no. 158407), pYPQ–dpcoCas9–Act3.0 (no. 158408), pYPQ–dpcoCas9–TV (no. 158409), pYPQ–dpcoCas9–SunTag (no. 158410), pYPQ–dpcoCas9–EV2.1 (no. 158411), pYPQ–dAaCas12b–Act3.0 (no. 158413), pYPQ–dzCas9–Act3.0 (no. 158414), pYPQ–dzCas9–NG–Act3.0 (no. 158415), pYPQ–dSpRY–Act3.0 (no. 158416), pYPQ134B2.0 (no. 167158), pYPQ135B2.0 (no. 167159), pYPQ136B2.0 (no. 167160) and pYPQ141D–gRNA2.1 (no. 167161).

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Acknowledgements

This work was supported by University of Maryland start-up funds, the National Science Foundation Plant Genome Research Program grants (award nos. IOS-1758745 and IOS-2029889), Biotechnology Risk Assessment Grant Program competitive grants (award nos. 2018-33522-28789 and 2020-33522-32274) from the US Department of Agriculture, a Foundation for Food and Agriculture Research grant (award no. 593603) and Syngenta. A.A.M. was supported by an NRT-INFEWS: UMD Global STEWARDS (STEM Training at the Nexus of Energy, Water Reuse and Food Systems) award to the University of Maryland School of Public Health from the National Science Foundation National Research Traineeship Program (award no. 1828910). The content of this publication is solely the responsibility of the authors and does not necessarily represent the official views of these funding agencies.

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Y.Q. and C.P. designed the experiments. C.P., X.W., N.K., Y.Z. and S.S. generated all the constructs. C.P., X.W. and Y.C. performed the transient assays in protoplasts. C.P. generated the stable transgenic rice and analysed the plants. C.P. and A.A.M. generated the stable transgenic Arabidopsis plants. C.P. and X.W. conducted the transcriptional activation assays. K.M. and P.M.S. helped with the design of metabolic pathway gene activation. Y.Q. and C.P. wrote the paper with input from the other authors. All authors read and approved the final manuscript.

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Correspondence to Yiping Qi.

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Y.Q. and C.P. are inventors on a US Provisional Patent Application (no. 63066674) that has been filed on the CRISPR–Act3.0 system in this study. All other authors declare no competing interests.

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Peer review information Nature Plants thanks Jian-feng Li and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary Methods, Figs. 1–13 and Table 1.

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Pan, C., Wu, X., Markel, K. et al. CRISPR–Act3.0 for highly efficient multiplexed gene activation in plants. Nat. Plants 7, 942–953 (2021). https://doi.org/10.1038/s41477-021-00953-7

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