Homologous recombination mediated by the CRISPR/Cas system and insertion of a resistance gene in the development of Huanglongbing-resistant sweet orange varieties

Abstract

CRISPR/Cas system as genome editing technology has revolutionized researches related to animals, plants and humans. This system has been widely used in studies about gene functions and biological processes, as well as in genetic plant breeding. As a genome editing tool, the use of Cas-type endonucleases with their intact catalytic sites results in the break of DNA strand. There are two repair mechanisms that can act: Non-Homologous End Joining (NHEJ) and Homology-Directed Repair (HDR). Although it is a great challenge to change genomes via HDR, the precise replacement of an existing allele by an "elite" or gene insertions in commercial cultivars is an important objective in crops breeding. Therefore, this project aims to establish the CRISRP/Cas technology by using the sgRNA/Cas Ribonucleoprotein (RNP) complex to promote HDR. The combined use of the RNP complex and HDR as the repair mechanism makes it possible to improve crops of economic interest without leaving signs of transgenics that may difficult their commercialization. Due to the challenge of establishing the technique, the project proposes to start the experiments in a model plant and induce HDR by CRISPR/Cas using the RUBY gene as a DNA donor template. Its genetic insertion will lead to the development of altered phenotypes. After the success of these steps, commercial varieties of citrus and Huanglongbing (HLB) resistance genes will become the targets of this study, due to the economic damage this disease has been causing in citrus. However, because we are aware of the difficulty in establishing CRISPR/Cas - HDR technology for plant species, the project also proposes the development of varieties resistant to HLB through the use of conventional genetic engineering techniques. That strategy will be used to promote overexpression of the Endochitinase B gene, which was associated with resistance to HLB. In the end, the project will contribute to establish an innovative technology that can be used in the future to improve other agricultural crops and also to develop varieties of citrus resistant to HLB. (AU)