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’ (Populus 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 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.

Editing Metabolism, Sex, and Microbiome: How Can We Help Poplar Resist Pathogens?

“…To date, the following CRISPR/Cas9 system delivery approaches have been used in plant cells: (1) agrotransformation, (2) biolytic bombardment, (3) electroporation, (4) nanoparticle application, (5) polyethylene glycol (PEG), ( 6) viral delivery, and (7) meristem transplantation for editing into a Cas9 overexpressing plant [318]. Each of these methods has disadvantages in obtaining genetically homogeneous edited poplar lines, which can be overcome by obtaining protoplasts, and CRISPR/Cas-editing followed by whole-plant regeneration [312]. The development of CRISPR/Cas9-based genome editing technologies using plant cell cultures may completely change the concept of poplar breeding.…”

“…Another way to avoid harmful Cas9 off-targets is through the use of base editors. In recent work, a Cas9-based cytosine base editor was used to knockout PLATZ (plant AT-rich protein and zinc-binding protein), encoding a transcription factor involved in plant response to fungal pathogens, in the poplar hybrid P. tremula × P. alba clone INRA 717-1B4 [312]. The knockout was accomplished by introducing premature stop codons into the PLATZ coding region via C-to-T transformations with an efficiency of 13-14%.…”

“…CRISPR-based artificial transcription factors provide another way to effectively and reversibly influence gene activity without changing their sequence. For example, CRISPR activator was applied to increase the expression of two target endogenous genes, TPX2 and LecRLK-G, which play an important role in plant growth and the defense response of poplar trees [312]. The application of CRISPRa resulted in a 1.2-to 7-fold increase in expression of the target genes through temporal expression in poplar protoplasts and stable Agrobacterium-mediated transformation.…”

Male-Specific Sequence in Populus simonii Provides Insights into Gender Determination of Poplar

“…Populus deltoids (section Aigeiros) and P. simonii (section Tacamahaca) are important parents for poplar hybrid breeding. It has been suggested that the F 1 hybrid population of P. deltoids and P. simonii has obvious heterosis, showing fast growth, straight trunks, disease resistance, and stress tolerance [14][15][16]. The development of a molecular marker for the sex identification of P. deltoids, P. simonii, and their hybrid population at the seedling stage could significantly improve breeding efficiency.…”

CRISPR/Cas9-mediated mutation of FERULATE 5-HYDROXYLASE enhances adsorption capacity of lignocellulose-based porous carbon from paper mulberry

“…In summary, employing the recently developed CRISPR/Cas9-mediated gene editing technology [ 9 , 10 ] we generated a BpF5H mutant and altered the biosynthesis of S and G lignins in paper mulberry, leading to increased adsorption capacity of adsorbate molecules in their corresponding porous carbon. These findings underscore the potential of F5H as a promising target for developing high-value germplasm through lignin bioengineering not only in paper mulberry and but also in other wood species.…”