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

47Citations
Citations of this article
117Readers
Mendeley users who have this article in their library.

This article is free to access.

Abstract

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 12months to generate T0 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 300bp 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.

Cite

CITATION STYLE

APA

Long, L., Guo, D. D., Gao, W., Yang, W. W., Hou, L. P., Ma, X. N., … Song, C. P. (2018). Optimization of CRISPR/Cas9 genome editing in cotton by improved sgRNA expression. Plant Methods, 14(1). https://doi.org/10.1186/s13007-018-0353-0

Register to see more suggestions

Mendeley helps you to discover research relevant for your work.

Already have an account?

Save time finding and organizing research with Mendeley

Sign up for free