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Updated: Aug 6, 2026
12:02
Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun
Published on: April 18, 2014
RNA-Guided Engineering of the Chloroplast Genome Enabled by Plastid-Expressed Guide RNAs
Malihe Mirzaee1, Corinne Best1, Evelyn V Wachowski1
1Waksman Institute of Microbiology, Rutgers University, Piscataway, New Jersey, USA.
Plant Biotechnology Journal
|July 18, 2026
Summary
Researchers engineered the chloroplast genome using CRISPR/Cas9 technology. This study demonstrates the first step toward RNA-guided genome editing in crops, enabling targeted DNA modifications in plant chloroplasts.
Area of Science:
- Plant Biotechnology
- Molecular Biology
- Genetics
Background:
- The CRISPR/Cas9 system offers powerful tools for genome engineering.
- Chloroplast genomes in plants are crucial for photosynthesis but are challenging to modify genetically.
Purpose of the Study:
- To develop an RNA-guided system for engineering the chloroplast genome using CRISPR/Cas9.
- To investigate methods for delivering CRISPR/Cas9 components into chloroplasts for targeted DNA modification.
Main Methods:
- Designed chloroplast minigenes for single guide RNA (sgRNA) production in tobacco.
- Expressed Cas9 in the nucleus, targeted it to chloroplasts via a transit peptide.
- Utilized microhomology-mediated end joining (MMEJ) for DNA repair after Cas9-induced breaks.
- Explored viroid RNA fusion for nuclear-expressed sgRNA delivery into chloroplasts.
Main Results:
- Successfully generated double-strand breaks in tobacco plastid DNA (ptDNA) at specific gene loci (ndhA, rpoC1).
- Observed MMEJ-mediated repair of these breaks, leading to deletions in the ptDNA.
- Demonstrated a method for delivering sgRNAs into chloroplasts without direct genome transformation, using viroid RNA fusion.
Conclusions:
- Established the foundation for RNA-guided genome editing in crop chloroplasts.
- Showcased the potential of CRISPR/Cas9 for targeted modification of the chloroplast genome.
- Paved the way for future advancements in chloroplast genetic engineering and crop improvement.
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