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Background  When developing CRISPR/Cas9 systems for crops, it is crucial to invest time characterizing the genome editing efficiency of the CRISPR/Cas9 cassettes, especially if the transformation system is difficult or time-consuming. Cotton is an important crop for the production of fiber, oil, and biofuel. However, the cotton stable transformation is usually performed using  Agrobacterium tumefaciens  taking between 8 and 12 months to generate T 0  plants. Furthermore, cotton is a heterotetraploid and targeted mutagenesis is considered to be difficult as many genes are duplicated in this complex genome. The application of CRISPR/Cas9 in cotton is severely hampered by the long and technically challenging genetic transformation process, making it imperative to maximize its efficiency.    Results  In this study, we provide a new system to evaluate and validate the efficiency of CRISPR/Cas9 cassettes in cotton using a transient expression system. By using this system, we could select the most effective CRISPR/Cas9 cassettes before the stable transformation. We have also optimized the existing cotton CRISPR/Cas9 system to achieve vastly improved mutagenesis efficiency by incorporating an endogenous  GhU6  promoter that increases sgRNA expression levels over the  Arabidopsis AtU6 - 29  promoter. The 300 bp  GhU6.3  promoter was cloned and validated using the transient expression system. When sgRNAs were expressed under the control of the  GhU6.3  promoter in CRISPR/Cas9 cassettes, expression levels were 6–7 times higher than those provided by the  AtU6 - 29  promoter and CRISPR/Cas9-mediated mutation efficiency was improved 4–6 times.    Conclusions  This study provides essential improvements to maximize CRISPR/Cas9-mediated mutation efficiency by reducing risk and workload for the application of CRISPR/Cas9 approaches in the targeted mutagenesis of cotton.    Electronic supplementary material  The online version of this article (10.1186/s13007-018-0353-0) contains supplementary material, which is available to authorized users.

Optimization of CRISPR/Cas9 genome editing in cotton by improved sgRNA expression