Uso da tecnologia CRISPR/Cas9 na edição de genes e validação de vetores para tolerância a estresses bióticos em bananeira (Musa spp.)

Abstract

The various diseases caused by fungi, oomycetes, bacteria and viral pathogens compromise the development of plants affecting their final yield. To overcome these challenges, breeding programs seek methods and techniques to improve crop performance under stress scenarios. Gene editing via CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) appears as a versatile tool with the potential to develop crops tolerant to multiple stresses. The first chapter of this work aimed to develop a systematic review of the literature generated in the last twelve years on the use of CRISPR technology in gene editing for tolerance to biotic stresses. We sought to evaluate articles deposited in different electronic databases, using search strings and predefined inclusion and exclusion criteria. This review demonstrated that the CRISPR/Cas system is applied to several plant species in order to promote tolerance to the main biotic stresses. Most studies were developed in the Asian continent, specifically in China. The Cas9 enzyme is used in most articles, enzymes such as Cas12 (Cpf1) and Cas13 can also be used as an additional tool for genome editing. The review also reveals several genes edited by CRISPR and that plant responses to stress factors are mediated by many complex signaling pathways. Furthermore, the quality of the articles included in this review was confirmed through a risk of bias analysis. In the second chapter, it was proposed to develop at least one construct/cassette as a biotechnological product containing or not a tissue-specific promoter from banana root, in addition to using CRISPR/Cas9 technology to knockout the PDS (Phytoene desaturase) gene in the Prata-Anã cultivar. For this, two guide RNAs (gRNA) were designed and two vectors/constructs were made, their parts were isolated, purified and transformed into Agrobacterium tumefaciens. Embryogenic cells of Prata-anã banana were used as explants for transformation via A. tumefaciens. From these methods it was possible to develop a protocol for construction and validation of CRISPR/Cas vectors for gene knockout