CRISPR/Cas9-based gene activation and base editing in Populus
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- Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States). Center for Bioenergy Innovation (CBI)
- Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States). Center for Bioenergy Innovation (CBI); Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)
- Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States); Central Community College, Hastings, NE (United States)
- Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)
- Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States); Zhejiang A & F University, Hangzhou (China)
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.
- Research Organization:
- Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States). Center for Bioenergy Innovation (CBI)
- Sponsoring Organization:
- USDOE Office of Science (SC), Biological and Environmental Research (BER)
- Grant/Contract Number:
- AC05-00OR22725
- OSTI ID:
- 1986236
- Journal Information:
- Horticulture Research (online), Vol. 10, Issue 6; ISSN 2052-7276
- Publisher:
- Springer Nature - Nanjing Agricultural UniversityCopyright Statement
- Country of Publication:
- United States
- Language:
- English
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