Full text
| |
| Author(s): |
Danyel Fernandes Contiliani
Total Authors: 1
|
| Document type: | Doctoral Thesis |
| Press: | Ribeirão Preto. |
| Institution: | Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC) |
| Defense date: | 2025-06-05 |
| Examining board members: |
Silvana Aparecida Creste Dias de Souza;
Fernando Bonifacio Anacleto;
Perla Novais de Oliveira;
Zila Luz Paulino Simoes
|
| Advisor: | Silvana Aparecida Creste Dias de Souza; Tiago Campos Pereira |
| Abstract | |
For over a decade, the advent of CRISPR-Cas9 genome engineering technology has revolutionized various fields of knowledge, including plant breeding, by providing unprecedented solutions and biotechnological strategies to intricate real-world problems, including climate change. Within this major topic, this thesis aimed to explore two main themes: (i) the functional characterization of drought-associated genes in sugarcane (Saccharum spp.) and rice (Oryza sativa) and (ii) the development of novel cytosine base editors. In Chapter I, the sugarcane phytocyanin family was investigated for the first time, revealing 97 members categorized according to their protein domain architectures and copper ion-binding sites. Some were identified as differentially expressed in sugarcane under moderate and severe drought stresses. Notably, a genotype-dependent transcriptional regulation of the uclacyanin-encoding gene SCQGHR1013B08.g was observed with opposite gene expression profiles between drought-sensitive (\'IACSP97-7065\') and -resistant (\'IACSP94-2094\') genotypes. The putative rice ortholog of SCQGHR1013B08.g, OsUCL23, was found in silico and subsequently knocked out by the CRISPR-Cas9. The Osucl23 knockout rice plants were subjected to biometric and physiological assays. Compared with the wild-type plants, Osucl23 plants displayed shorter shoots in the mature phase. Under drought conditions, these plants exhibited improved water status and higher photochemical efficiency, resulting in greater carboxylation and water use efficiencies. These findings suggest the overarching molecular activity of sugarcane phytocyanins and their drought responsiveness, further offering evidence of the protective role of the negative regulation of OsUCL23 on drought stress. In Chapter II, 66 hitherto uncharacterized cytidine deaminases from the animal kingdom were screened and benchmarked in rice protoplasts aiming at the OsCGRS55 target, and their performances were compared against well-established base editors like hA3A-Y130F, hAID, and PmCDA1. The most promising candidates were subsequently tested again in rice protoplasts to edit four different target genes, OsALS, OsGN1a, OsGS3, and OsGW2, to assess their base-editing potential. The cytidine deaminase OoA3GX2 consistently demonstrated high efficiency in both protoplasts and rice plants transformed via Agrobacterium tumefaciens. Additionally, OoA3GX2 displayed comparable editing activity at GC/CC/TC/AC sites - suggesting reduced genomic context dependency - lower indel rates, minimal C-to-R (R = A/G) conversions, and fewer off-target effects, indicating higher purity of gene-editing products. Furthermore, its wide base editing window enabled the conversion of cytosines that are typically inaccessible for traditional base editors. This work not only expands the repertoire of cytosine base editors but also enhances the robustness of this technology, enabling sophisticated genome engineering strategies in rice and other species. (AU) | |
| FAPESP's process: | 21/13478-2 - Genome editing of grass species via CRISPR/Cas9 |
| Grantee: | Danyel Fernandes Contiliani |
| Support Opportunities: | Scholarships in Brazil - Doctorate |