Summary
This study reports that CRISPR/Cas9 can cause unexpectedly high off-target mutations in Arabidopsis, even when guide RNAs are predicted to be highly specific, with these effects worsening in later generations. To address this, researchers developed strategies to reduce off-target effects, including an mCherry-based system for selecting Cas9-free mutants and the use of high-specificity SpCas9 variants. They found that fusing tRNA with a mutant sgRNA scaffold greatly improved editing efficiency, though high-specificity Cas9 variants required stronger expression to match wild-type efficiency. The study also revealed that T-DNA integrates frequently at CRISPR/Cas9 cleavage sites, underscoring the need for continued monitoring of off-target effects and offering optimized tools for more precise genome editing in plants.