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CRISPR/Cas9 and Cytidine Base-Editing Systems for Efficient Genome Engineering in Oleaginous Rhodococcus
Martín A Duhalde1, Román A Martino2,3, Andrea Smania2,3
1Instituto de Biociencias de la Patagonia (INBIOP), CONICET, Universidad Nacional de la Patagonia San Juan Bosco, Chubut 9000, Argentina.
ACS Synthetic Biology
|April 10, 2026
Summary
New CRISPR-based genome editing tools for oleaginous Rhodococcus strains have been developed. These systems enable efficient DNA modification, advancing biotechnological applications and the study of Rhodococcus metabolism.
Area of Science:
- Microbiology
- Molecular Biology
- Biotechnology
Background:
- Oleaginous *Rhodococcus* strains are valuable for biotechnology due to their lipid synthesis capabilities.
- Efficient genome editing is crucial for understanding *Rhodococcus* metabolism and physiology but remains challenging.
- Existing genome editing methods in *Rhodococcus* are often inefficient or complex.
Purpose of the Study:
- To develop novel and efficient genome editing tools for oleaginous *Rhodococcus* strains.
- To enable precise genetic modifications for studying microbial metabolism and enhancing biotechnological applications.
- To establish CRISPR-based systems for both gene knockout and precise base editing in *Rhodococcus*.
Main Methods:
- Implementation of a CRISPR/Cas9 system utilizing the nonhomologous end joining (NHEJ) repair pathway for gene editing.
- Development and application of a cytidine base editor (CBE) for precise C•G to T•A nucleotide substitutions.
- Utilization of a dual-plasmid CRISPR platform (pTipCas9/pCA71sgRNA and pTipBE/pCA71sgRNA).
- Integration of a Csy4-mediated sgRNA processing module for multiplex genome editing with the CBE system.
Main Results:
- Achieved high rates of INDEL formation (70-80% efficiency) using the CRISPR/Cas9 NHEJ system.
- Demonstrated high efficiency (75-85%) for C•G to T•A base conversions using the cytidine base editor.
- Successfully implemented multiplex genome editing for simultaneous modification of multiple genetic loci.
- The developed CRISPR tools outperform traditional recombination-based approaches in *Rhodococcus*.
Conclusions:
- Novel CRISPR/Cas9 and cytidine base editing systems provide efficient genome editing capabilities in oleaginous *Rhodococcus* strains.
- These tools facilitate in-depth study of microbial metabolism, stress responses, and cellular physiology.
- The developed systems are transferable to other actinobacteria and hold significant potential for advancing genetic engineering in biotechnology.
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