This study investigates the relationship between single sgRNA (sgRNA) activity and gene targeting (GT) efficiency in *Arabidopsis thaliana*. The researchers used a sequential transformation strategy to evaluate the impact of increasing sgRNA copy numbers and using multiple sgRNAs on GT efficiency at two endogenous loci, *Embryo Defective 2410* (EMB2410) and *Repressor of Silencing 1* (ROS1). They found that increasing the number of sgRNAs copy numbers enhances both double-strand break (DSB) frequency and GT efficiency. However, the application of multiple sgRNAs does not always improve GT efficiency. The study also suggests that some inefficient sgRNAs can act as helpers to facilitate the DSB activity of other sgRNAs. The results highlight that DSB efficiency is a key factor in determining GT efficiency and provide insights into the molecular mechanisms of CRISPR/Cas9-mediated GT in plants.This study investigates the relationship between single sgRNA (sgRNA) activity and gene targeting (GT) efficiency in *Arabidopsis thaliana*. The researchers used a sequential transformation strategy to evaluate the impact of increasing sgRNA copy numbers and using multiple sgRNAs on GT efficiency at two endogenous loci, *Embryo Defective 2410* (EMB2410) and *Repressor of Silencing 1* (ROS1). They found that increasing the number of sgRNAs copy numbers enhances both double-strand break (DSB) frequency and GT efficiency. However, the application of multiple sgRNAs does not always improve GT efficiency. The study also suggests that some inefficient sgRNAs can act as helpers to facilitate the DSB activity of other sgRNAs. The results highlight that DSB efficiency is a key factor in determining GT efficiency and provide insights into the molecular mechanisms of CRISPR/Cas9-mediated GT in plants.