This study investigates the impact of multiple sgRNAs on the efficiency of CRISPR/Cas9-mediated gene targeting (GT) in Arabidopsis. The research demonstrates that increasing the number of sgRNAs can enhance DSB frequency and GT efficiency, but this is not always the case. The study shows that some inefficient sgRNAs can act as helpers to facilitate other sgRNAs' DSB activity. The results indicate that DSB efficiency is a critical factor in determining GT efficiency, rather than mutation patterns. The study also reveals that the use of multiple sgRNAs can improve overall GT efficiency, especially when combined with appropriate donor templates. However, the effectiveness of multiple sgRNAs varies depending on the target locus and the specific sgRNAs used. The study highlights the importance of sgRNA design and the relationship between sgRNA expression levels, DSB frequency, and GT efficiency. The findings suggest that optimizing sgRNA combinations and expression levels can enhance the precision and efficiency of CRISPR/Cas9-mediated GT in plants. The study also shows that the use of multiple sgRNAs can lead to higher HDR event frequencies, thereby improving GT efficiency. However, the study also notes that excessive sgRNA expression may not always lead to improved outcomes, and that the optimal number of sgRNAs depends on the specific target and experimental conditions. Overall, the study provides new insights into the relationship between sgRNAs, DSBs, and GTs, and the molecular mechanisms of CRISPR/Cas9-mediated GT in plants.This study investigates the impact of multiple sgRNAs on the efficiency of CRISPR/Cas9-mediated gene targeting (GT) in Arabidopsis. The research demonstrates that increasing the number of sgRNAs can enhance DSB frequency and GT efficiency, but this is not always the case. The study shows that some inefficient sgRNAs can act as helpers to facilitate other sgRNAs' DSB activity. The results indicate that DSB efficiency is a critical factor in determining GT efficiency, rather than mutation patterns. The study also reveals that the use of multiple sgRNAs can improve overall GT efficiency, especially when combined with appropriate donor templates. However, the effectiveness of multiple sgRNAs varies depending on the target locus and the specific sgRNAs used. The study highlights the importance of sgRNA design and the relationship between sgRNA expression levels, DSB frequency, and GT efficiency. The findings suggest that optimizing sgRNA combinations and expression levels can enhance the precision and efficiency of CRISPR/Cas9-mediated GT in plants. The study also shows that the use of multiple sgRNAs can lead to higher HDR event frequencies, thereby improving GT efficiency. However, the study also notes that excessive sgRNA expression may not always lead to improved outcomes, and that the optimal number of sgRNAs depends on the specific target and experimental conditions. Overall, the study provides new insights into the relationship between sgRNAs, DSBs, and GTs, and the molecular mechanisms of CRISPR/Cas9-mediated GT in plants.