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

Author(s)

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

• Document type: Journal article
• Source: Plant Biotechnology Journal; v. N/A, p. 11-pg., 2025-02-14.

Abstract

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)

• FAPESP's process: 20/07045-3 - Biotechnological and genomic strategies for quality, productivity and sustainable management of citrus, coffee and sugar cane in the State of São Paulo
• Grantee: Mariângela Cristofani-Yaly
• Support Opportunities: Research Grants - Problem-Oriented Research Centers in São Paulo
• FAPESP's process: 21/13478-2 - Genome editing of grass species via CRISPR/Cas9
• Grantee: Danyel Fernandes Contiliani
• Support Opportunities: Scholarships in Brazil - Doctorate
• FAPESP's process: 22/11738-0 - Plant genome engineering with CRISPR-dCas12b system
• Grantee: Danyel Fernandes Contiliani
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate