Increased Genome Editing Efficiency in Poplar by Optimizing Sgrna Length and Copy Number
Industrial Crops and Products（2025）
Abstract
The CRISPR/Cas9 system has achieved remarkable success in genome engineering; however, its application in woody plants remains challenging due to their complex genomic architecture and low transformation efficiency. This study established an optimized CRISPR/Cas9-based genome editing strategy for 84 K poplar (Populus alba × Populus tremula var. glandulosa), achieving editing efficiencies of up to 50 % through systematic optimization of transformation conditions. Comprehensive analysis revealed two critical factors determining editing efficiency: sgRNA length and copy number. Among targeting sequences spanning 18–22 nucleotides, 20 nt sgRNA exhibited optimal performance with 30 % editing efficiency following a normal distribution pattern relative to sgRNA length. Integration of triple sgRNA copies enhanced editing outcomes, particularly for allelic and homologous gene editing. Three candidate reporter genes, PDS, HEMA, and RbcS, were evaluated for monitoring editing outcomes. PDS and HEMA proved effective for detecting biallelic null mutations but were unsuitable for identifying monoallelic editing. This optimized protocol establishes a robust foundation for genetic manipulation in forest trees, advancing fundamental research and biotechnology applications in sustainable forestry.
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Key words
CRISPR/Cas9,sgRNA length,sgRNA copy number,PDS,HEMA
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