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

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 taking between 8 and 12 months to generate T 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 promoter that increases sgRNA expression levels over the - promoter. The 300 bp promoter was cloned and validated using the transient expression system. When sgRNAs were expressed under the control of the promoter in CRISPR/Cas9 cassettes, expression levels were 6-7 times higher than those provided by the - 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.