Genome Editing in Cotton with the CRISPR/Cas9 System

Abstract

Genome editing is an important tool for gene functional studies as well as crop improvement. The recent development of the CRISPR/Cas9 system using single guide RNA molecules (sgRNAs) to direct precise double strand breaks in the genome has the potential to revolutionize agriculture. Unfortunately, not all sgRNAs are equally efficient and it is difficult to predict their efficiency by bioinformatics. In crops such as cotton (Gossypium hirsutum L.), with labor-intensive and lengthy transformation procedures, it is essential to minimize the risk of using an ineffective sgRNA that could result in the production of transgenic plants without the desired CRISPR-induced mutations. In this study, we have developed a fast and efficient method to validate the functionality of sgRNAs in cotton using a transient expression system. We have used this method to validate target sites for three different genes GhPDS, GhCLA1, and GhEF1 and analyzed the nature of the CRISPR/Cas9-induced mutations. In our experiments, the most frequent type of mutations observed in cotton cotyledons were deletions (∼64%). We prove that the CRISPR/Cas9 system can effectively produce mutations in homeologous cotton genes, an important requisite in this allotetraploid crop. We also show that multiple gene targeting can be achieved in cotton with the simultaneous expression of several sgRNAs and have generated mutations in GhPDS and GhEF1 at two target sites. Additionally, we have used the CRISPR/Cas9 system to produce targeted gene fragment deletions in the GhPDS locus. Finally, we obtained transgenic cotton plants containing CRISPR/Cas9-induced gene editing mutations in the GhCLA1 gene. The mutation efficiency was very high, with 80.6% of the transgenic lines containing mutations in the GhCLA1 target site resulting in an intense albino phenotype due to interference with chloroplast biogenesis.

Light‐ and temperature‐entrainable circadian clock in soybean development

“…CRISPR‐associated editing technology has been widely used in the study of functional genomes of many species (Ma et al ., ; Gao et al ., ). In recent years, it has been applied to soybean to obtain a direct mutation of targeted acetolactate synthase1 gene (Li et al ., ).…”

Targeted Genome Editing for Cotton Improvement

“…Using this robust method, activity of sgRNAs can be validated in 3 days which will be helpful in selection of potential sgRNAs for stable transformation in cotton. Individual genes (GhPDS, GhCLA1, and GhEF1) were targeted resulting in typical albino phenotypes by inducing mutation in GhCLA1, simultaneous editing of homoeologous genes, and genomic fragment deletions [129]. This kind of studies made a foundation stone for undertaking functional genomics studies in cotton.…”

Edição genética associada ao uso da nova técnica CRISPR/Cas9, ferramenta de defesa utilizada pelas bactérias contra DNA invasor.

“…Sim, é possível, e o estudo da edição genética traz cada vez mais novas técnicas que podem nos ajudar muito, pois é uma ferramenta importante para estudos funcionais de genes, bem como a melhoria de uma determinada situação (GAO et al, 2017). A edição genética tem um proveitoso uso de novas tecnologias que podem ser introduzidas na quebra de uma determinada região da dupla fita de DNA, sendo que essa quebra do DNA é bastante útil, pois ativa o sistema de reparo de DNA natural da célula, que pode ser explorado para melhorar significativamente a eficiência de introduzir alterações da qual o indivíduo seja favorecido com a mutação realizada no genoma, e não uma mutação indesejável levando a complicações.…”