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

The genus has long been used for environmental, agroforestry and industrial applications worldwide. Today 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 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" ( x 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 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, and which play important roles in plant growth and defense response, in hybrid poplar clone "717-1B4" and poplar clone "WV94" ( "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 -mediated stable transformation, demonstrating the effectiveness of dCas9-based CRISPRa system in . 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 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 species, facilitating the adoption of emerging genome editing tools in woody species.