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

Abstract

BackgroundWhen 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 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.ResultsIn 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.ConclusionsThis 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 materialThe online version of this article (10.1186/s13007-018-0353-0) contains supplementary material, which is available to authorized users.

Insights into Drought Stress Signaling in Plants and the Molecular Genetic Basis of Cotton Drought Tolerance

“…Genome modification technologies and transgenic approaches have been employed to develop drought-tolerant crops overexpressing transgenes that are important for plant physiology. Targeted genome editing with the CRISPR/Cas9 system has been utilized to modify the genome to obtain more stable and heritable mutations [13]. Genome-wide studies have been performed to explore stress-related candidate regions and genes for drought tolerance.…”

Identification of NHXs in Gossypium species and the positive role of GhNHX1 in salt tolerance

“…Plant NHX family plays an important role in salt resistance; however, research involving NHXs is still rare in cotton. The rapid development of biotechnology accelerated the molecular breeding process in cotton that allows us to study cotton NHXs in the genome-wide level [23][24][25][26][27]. Recently, GhSOS1, homologous to AtSOS1, was isolated from a salt-tolerant genotype of G. hirsutum.…”

Introductory Chapter: Recent Trends in “Cotton Research”

“…Efficient CRISPR/Cas9 cassettes can be selected to get the better mutagenesis rate by the use of GhU6 promoter instead of Arabidopsis ATU6-29 promoter and GhU6 promoter. When CRISPR/cas9 expressed the sgRNA under the GhU6 promoter, CRISPR/cas9 mutagenesis rate was increased four to six times and expression level of sgRNA was increased from six to seven times, which was a great achievement in the targeted mutagenesis of cotton by the CRISPR/cas9 system [31]. CRISPR/cas9 system has been used to generate multiple sites in Gossypium hirsutum.…”