CRISPR/Cas9-mediated targeted mutagenesis in upland cotton (Gossypium hirsutum L.)

Abstract

The clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR associated (Cas)9 protein system has emerged as a simple and efficient tool for genome editing in eukaryotic cells. It has been shown to be functional in several crop species, yet there are no reports on the application of this or any other genome editing technologies in the cotton plant. Cotton is an important crop that is grown mainly for its fiber, but its seed also serves as a useful source of edible oil and feed protein. Most of the commercially-grown cotton is tetraploid, thus making it much more difficult to target both sets of homeologous alleles. Therefore, in order to understand the efficacy of the CRISPR/Cas9 system to target a gene within the genome of cotton, we made use of a transgenic cotton line previously generated in our laboratory that had a single copy of the green fluorescent protein (GFP) gene integrated into its genome. We demonstrate, for the first time, the use of this powerful new tool in targeted knockout of a gene residing in the cotton genome. By following the loss of GFP fluorescence, we were able to observe the cells that had undergone targeted mutations as a result of CRISPR/Cas9 activity. In addition, we provide examples of the different types of indels obtained by Cas9-mediated cleavage of the GFP gene, guided by three independent sgRNAs. The results provide useful information that will help us target important native genes in the cotton plant in future.

Genomics-enabled analysis of the emergent disease cotton bacterial blight

“…These include the use of existing effective resistance loci as well as the potential disruption of the induction of susceptibility genes through genome editing. The latter is an attractive strategy in part because of recent progress in genome editing [60, 61]. In summary, within a relatively short time frame, through the deployment of modern molecular and genomic techniques, we were able to identify factors that likely contribute to the re-emergence of cotton bacterial blight and generate data that can now be rapidly translated to effective disease control strategies.…”

Using Biotechnology-Led Approaches to Uplift Cereal and Food Legume Yields in Dryland Environments

“…Targeted modification of specific genes using CRISPR and CRISPR-associated proteinP9 (Cas9) leads to useful genetic variations for improving plants (Feng et al, 2013). Indeed, plant breeding enterprises are beginning to exploit genome editing with CRISP/Cas9 to develop genetically enhanced seed-embedded technology showing new traits.…”

Targeted Genome Editing for Cotton Improvement

“…In addition, CRISPRi was also used for multiplexed control of endogenous genes [52] and stable repression of genes with silencing efficiency typically achieved by RNAi while minimally impacting transcription of nontargeted genes. CRISPR/Cas9 has the efficiency to target the green fluorescent protein (GFP) gene within the genome of transgenic cotton line with single copy of GFP gene incorporated previously [53]. Multiplexing ability of CRISPR/Cas system has given a distinction to this system.…”