A Dual-gRNA CRISPR/Cas9 System for Efficient Generation of Large Fragment Deletions in Poplar

CRISPR/Cas9-based genome editing is a powerful approach for functional genomics and bioenergy research in woody plants. However, conventional single guide RNA (gRNA) strategies predominantly generate small insertions or deletions that may not fully disrupt gene function and often require extensive sequencing for mutation identification. Here, we present an optimized protocol for the efficient generation of large-fragment deletion mutants in Populus tremula × P. alba clone INRA 717-1B4 using a dual-gRNA CRISPR/Cas9 system. Co-expression of two gRNAs flanking the target region induces double-strand breaks at both sites, enabling the deletion of the intervening genomic fragment, typically larger than 50 bp. This protocol describes step-by-step procedures for gRNA design, vector construction, Agrobacterium-mediated transformation, plant regeneration, and molecular validation. Using the PtFBX230 gene as a representative target, large deletions are readily identified by conventional PCR and agarose gel electrophoresis, enabling rapid and cost-effective genotyping. This protocol can be readily adopted to other loci in poplar and related woody species and provides a robust framework for generating null alleles to support functional genomics and bioenergy-related trait engineering in woody plants. Key features • Enables efficient deletion of large genomic fragments (>50 bp) using a dual-gRNA CRISPR/Cas9 strategy. • Optimized for the hybrid poplar (Populus tremula × P. alba clone INRA 717-1B4) using the pORE303N vector system. • Compatible with Agrobacterium-mediated transformation and tissue culture-based regeneration. • Simplifies mutant screening through PCR-based genotyping, minimizing reliance on sequencing. • Facilitates simultaneous editing of homologous genes in bioenergy-relevant poplar lines.