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Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms
Published on: May 25, 2018
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An Efficient CRISPR/Cas9 Genome Editing System for a Ganoderma lucidum Cultivated Strain by Ribonucleoprotein Method
Yi Tan1, Xianglin Yu2, Zhigang Zhang3
1National Engineering Research Center of Edible Fungi, Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, Shanghai 201403, China.
Journal of Fungi (Basel, Switzerland)
|December 22, 2023
Summary
This study optimized a CRISPR/Cas9 ribonucleoprotein (RNP) genome editing system for Ganoderma lucidum, achieving 100% targeting efficiency and high editing rates for the ura3 gene. The system enables targeted breeding and metabolic regulation.
Area of Science:
- Molecular Biology
- Mycology
- Biotechnology
Background:
- CRISPR/Cas9 is a popular genome editing tool.
- Ribonucleoprotein (RNP) complexes offer advantages over plasmid-based systems but have lower reported efficiency.
- Efficient genome editing systems are needed for Ganoderma lucidum.
Purpose of the Study:
- To develop and optimize an RNP-mediated CRISPR/Cas9 genome editing system for Ganoderma lucidum.
- To improve editing efficiency and targeting in G. lucidum.
- To investigate DNA repair mechanisms after CRISPR/Cas9-induced double-strand breaks.
Main Methods:
- Developed an RNP-mediated CRISPR/Cas9 system for Ganoderma lucidum L1 strain.
- Utilized selective media containing 5-fluoroorotic acid (5-FOA) for selection.
- Analyzed gene editing outcomes, including insertions, substitutions, and DNA repair pathways (NHEJ and MMEJ).
Main Results:
- Achieved 100% targeting efficiency for genomic editing.
- Obtained high editing efficiency for the orotidine 5'-monophosphate decarboxylase (ura3) gene (>35 mutants/10^7 protoplasts).
- Characterized DNA repair, with 94.4% of mutants using non-homologous end joining (NHEJ) and 5.6% using microhomology-mediated end joining (MMEJ).
- Successfully edited other G. lucidum genes (cyp512a3, cyp5359n1).
Conclusions:
- The optimized RNP-CRISPR/Cas9 system is highly efficient for Ganoderma lucidum genome editing.
- This system facilitates targeted breeding and synthetic metabolic regulation in G. lucidum.
- The study provides insights into DNA double-strand break repair mechanisms in G. lucidum.
