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 ...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.展开更多
文摘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.
