Simple, efficient and open-source CRISPR/Cas9 strategy for multi-site genome editing in Populus tremula × alba

Area of Science:

• Plant Biotechnology
• Molecular Biology
• Forestry Science

Background:

• CRISPR/Cas9 gene editing is valuable for crop improvement but underutilized in forest trees.
• Efficient genetic tools are needed to study gene function in woody plants like poplar.

Purpose of the Study:

• To establish and validate an efficient CRISPR/Cas9 gene editing strategy for hybrid poplar (Populus tremula × alba).
• To assess the system's efficacy for generating both single and simultaneous double gene mutants.
• To investigate the role of the SHORT ROOT (SHR) gene in poplar root development.

Main Methods:

• Utilized Golden Gate MoClo cloning for constructing CRISPR/Cas9 vectors with single guide RNAs (sgRNAs).
• Employed Agrobacterium rhizogenes-mediated hairy root transformation in hybrid poplar.
• Applied Sanger sequencing to confirm mutations and analyze phenotypic effects.

Main Results:

• Successfully generated single (PtaSHR) and double (yuc4/plt1, lbd12/lbd4) gene mutants with high efficiency.
• Confirmed biallelic and homozygous knockout mutations, with deletions and indels being common.
• Observed loss of endodermal cell layer in SHR mutants, indicating conserved function.

Conclusions:

• The developed CRISPR/Cas9-Golden Gate MoClo system is a rapid, simple, and standardized tool for hybrid poplar gene editing.
• This method facilitates the study of essential developmental processes, such as radial cell differentiation, in forest trees.
• The findings provide a foundation for leveraging advanced gene editing in poplar functional genomics and breeding.