Harnessing novel cytidine deaminases from the animal kingdom for robust multiplexed base editing in rice

Autor(es)

Contiliani, Danyel Fernandes ; Sretenovic, Simon ; Dailey, Micah ; Zhou, Man ; Cheng, Yanhao ; Creste, Silvana ; Xiao, Shunyuan ; Qi, Yiping

• Tipo de documento: Artigo Científico
• Fonte: Plant Biotechnology Journal; v. N/A, p. 11-pg., 2025-02-14.

Resumo

CRISPR-Cas-based cytosine base editors (CBEs) are prominent tools that perform site-specific and precise C-to-T conversions catalysed by cytidine deaminases. However, their use is often constrained by stringent editing preferences for genomic contexts, off-target effects and restricted editing windows. To expand the repertoire of CBEs, we systematically screened 66 novel cytidine deaminases sourced from various organisms, predominantly from the animal kingdom and benchmarked them in rice protoplasts using the nCas9-BE3 configuration. After selecting candidates in rice protoplasts and further validation in transgenic rice lines, we unveiled a few cytidine deaminases exhibiting high editing efficiencies and wide editing windows. CBEs based on these cytidine deaminases also displayed minimal frequencies of indels and C-to-R (R = A/G) conversions, suggesting high purity in C-to-T base editing. Furthermore, we highlight the highly efficient cytidine deaminase OoA3GX2 derived from Orca (killer whale) for its comparable activity across GC/CC/TC/AC sites, thus broadening the targeting scope of CBEs for robust multiplexed base editing. Finally, the whole-genome sequencing analyses revealed very few sgRNA-dependent and -independent off-target effects in independent T0 lines. This study expands the cytosine base-editing toolkit with many cytidine deaminases sourced from mammals, providing better-performing CBEs that can be further leveraged for sophisticated genome engineering strategies in rice and likely in other plant species. (AU)

• Processo FAPESP: 20/07045-3 - Estratégias biotecnológicas e genômicas para qualidade, produtividade e manejo sustentável de citros, café e cana-de-açúcar no estado de São Paulo
• Beneficiário: Mariângela Cristofani-Yaly
• Modalidade de apoio: Auxílio à Pesquisa - Núcleos de Pesquisa Orientada a Problemas em São Paulo
• Processo FAPESP: 21/13478-2 - Edição de genomas em espécies de gramíneas via CRISPR/Cas9
• Beneficiário: Danyel Fernandes Contiliani
• Modalidade de apoio: Bolsas no Brasil - Doutorado
• Processo FAPESP: 22/11738-0 - Engenharia de genomas de plantas via sistema CRISPR-dCas12b
• Beneficiário: Danyel Fernandes Contiliani
• Modalidade de apoio: Bolsas no Exterior - Estágio de Pesquisa - Doutorado