Proceedings of the 8th Brazilian Congress of Biotechnology

SOYBEAN DNA-FREE GENOME EDITING VIA CRISPR/SACAS9 RIBONUCLEOPROTEIN DELIVERY

Published in 21/11/2025 - ISBN: 978-65-272-1865-4

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Paper Title
SOYBEAN DNA-FREE GENOME EDITING VIA CRISPR/SACAS9 RIBONUCLEOPROTEIN DELIVERY
Authors
  • Gustavo Ruffo
  • Nayara Sabrina de Freitas Alves
  • Isabela Tristan Lourenço Tessutti
  • Fabrício Arraes
  • Maria Cristina Silva
  • Leonardo Lima Pepino de Macedo
  • Carolina Morgante
  • Maria Fátima Grossi de Sá
Modality
Abstract
Subject area
Applications of Biotechnology in Environmental and Agricultural Areas;
Publishing Date
21/11/2025
Country of Publishing
Brazil | Brasil
Language of Publishing
en-US
Paper Page
https://www.even3.com.br/anais/biotecnologia2025/1256994-soybean-dna-free-genome-editing-via-crisprsacas9-ribonucleoprotein-delivery
ISBN
978-65-272-1865-4
Keywords
Glycine max. Transgene-free. RNP. Biolistic.
Summary
CRISPR/Cas systems have emerged as powerful tools for precise genome editing in molecular breeding. However, conventional delivery methods in plants often involve DNA integration, raising regulatory and consumer concerns in many countries. To address this, DNA-free approaches—such as the delivery of pre-assembled ribonucleoproteins (RNPs) offer a safer alternative that circumvents the use of foreign DNA, enhancing regulatory acceptance. Despite their potential, these strategies remain underexplored in soybean (Glycine max L.). In this work, we developed a DNA-free genome editing system for soybeans using Staphylococcus aureus Cas9 (SaCas9) RNP delivery to embryonic axis via gold particle-assisted bombardment, targeting the shoot apical meristem (SAM). The heterologous expression of SaCas9 was optimized in Escherichia coli BL21 (DE3) pLysS for maximum yield. Single-guide RNAs (sgRNAs) were designed and synthesized in vitro to knock out phytoene desaturase (GmPDS), which leads to an albino phenotype. Firstly, RNP activity was validated through in vitro cleavage assays. RNPs were then introduced into the shoot apical meristem of embryonic axes (cultivar Williams 82) via gold particle bombardment, evaluating variations in shot pressure and number of shots per plate. Also, we introduced an additional sonication step of embryos prior to bombardment. Regenerated plants exhibiting green-white variegated leaves were genotyped by PCR and Sanger sequencing of the target region. Analysis with ICE software (Synthego) revealed an editing efficiency of 8%. Encouraged by these results, we extended the approach to target GmAIP10 (ABAP1 Interaction Protein 10), a gene associated with increased biomass. Editing of GmAIP10 was detected in 20% of regenerated plants, and preliminary findings suggest heritability of the CRISPR-induced mutations in the T1 generation. In conclusion, the RNP-based genome editing system developed here offers an efficient, DNA-free method for targeted mutagenesis in soybean, avoiding genetically modified organism regulations and supporting crop improvement. Future work will aim to optimize editing efficiency and expand the approach to other key crops, such as cotton (Gossypium hirsutum), broadening its impact on sustainable and accepted agricultural innovation.
Title of the Event
8th Brazilian Biotechnology Congress.
City of the Event
Natal
Title of the Proceedings of the event
Proceedings of the 8th Brazilian Congress of Biotechnology
Name of the Publisher
Even3
Means of Dissemination
Meio Digital

How to cite

RUFFO, Gustavo et al.. SOYBEAN DNA-FREE GENOME EDITING VIA CRISPR/SACAS9 RIBONUCLEOPROTEIN DELIVERY.. In: Proceedings of the 8th Brazilian Congress of Biotechnology. Anais...Natal(RN) Hotel Praia Mar, 2025. Available in: https//www.even3.com.br/anais/biotecnologia2025/1256994-SOYBEAN-DNA-FREE-GENOME-EDITING-VIA-CRISPRSACAS9-RIBONUCLEOPROTEIN-DELIVERY. Access in: 22/05/2026

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