CRISPR/Cas9-based gene activation and base editing in Populus

Abstract

The genus Populus has long been used for environmental, agroforestry and industrial applications worldwide. Today Populus is also recognized as a desirable crop for biofuel production and a model tree for physiological and ecological research. As such, various modern biotechnologies, including CRISPR/Cas9-based techniques, have been actively applied to Populus for genetic and genomic improvements for traits such as increased growth rate and tailored lignin composition. However, CRISPR/Cas9 has been primarily used as the active Cas9 form to create knockouts in the hybrid poplar clone “717-1B4” (P. tremula x P. alba clone INRA 717-1B4). Alternative CRISPR/Cas9-based technologies, e.g. those involving modified Cas9 for gene activation and base editing, have not been evaluated in most Populus species for their efficacy. Here we employed a deactivated Cas9 (dCas9)-based CRISPR activation (CRISPRa) technique to fine-tune the expression of two target genes, TPX2 and LecRLK-G which play important roles in plant growth and defense response, in hybrid poplar clone “717-1B4” and poplar clone “WV94” (P. deltoides “WV94”), respectively. We observed that CRISPRa resulted in 1.2-fold to 7.0-fold increase in target gene expression through transient expression in protoplasts and Agrobacterium-mediated stable transformation, demonstrating the effectiveness of dCas9-based CRISPRa system in Populus. In addition, we applied Cas9 nickase (nCas9)-based cytosine base editor (CBE) to precisely introduce premature stop codons via C-to-T conversion, with an efficiency of 13%–14%, in the target gene PLATZ which encodes a transcription factor involved in plant fungal pathogen response in hybrid poplar clone “717-1B4”. Overall, we showcase the successful application of CRISPR/Cas-based technologies in gene expression regulation and precise gene engineering in two Populus species, facilitating the adoption of emerging genome editing tools in woody species.

Acknowledgements

We thank Dr. Yiping Qi from University of Maryland for providing the CRIPSR-Act3.0 and A3A/Y130F-BE3 related vectors. We thank Miranda Clark, David McLennan, and Jamie McBrien for growing and maintaining plants in ORNL greenhouses. This work was supported by the Center for Bioenergy Innovation, a U.S. Department of Energy (DOE) Bioenergy Research Center supported by the Biological and Environmental Research program and the DOE Genomic Science Program, as part of the Secure Ecosystem Engineering and Design Scientific (SEED) Focus Area and the Plant-Microbe Interfaces (PMI) Scientific Focus Area. Oak Ridge National Laboratory is managed by UT-Battelle, LLC for the U.S. Department of Energy under Contract Number DE-AC05-00OR22725.

Author contributions

G.A.T., J-G.C. and X.Y. conceived the research. T.Y., G.Y., H.L. executed and analyzed the experiments. T.Y., G.Y., H.L., and Y.L. drafted the manuscript. J.Z., W.M., G.A.T., J-G.C., and X.Y. revised the manuscript. W.M., J-G.C. and X.Y. supervised the research. All authors have read and agreed to the published version of the manuscript.

Data availability

The authors confirm that all data from this study are available and can be found in this article and in supplementary information. The plasmids will be available at Addgene.

Conflict of interest

The authors declare no competing interests.

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