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A Robotic Platform for High-throughput Protoplast Isolation and Transformation
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A Streamlined Protocol for Wheat (Triticum aestivum) Protoplast Isolation and Transformation With CRISPR-Cas
Kali M Brandt1, Hilary Gunn1, Nathalia Moretti1
1Wheat Breeding and Genetics, Crop and Soil Science, Oregon State University, Corvallis, OR, United States.
Frontiers in Plant Science
|June 27, 2020
Summary
A new method streamlines CRISPR gene editing in wheat by testing sgRNA accessibility in protoplasts. This allows researchers to quickly assess target site accessibility before costly transformations, improving wheat genetic engineering efficiency.
Area of Science:
- Agricultural biotechnology
- Molecular biology
- Plant genetics
Background:
- CRISPR technology offers efficient genetic modification for agriculture.
- A key challenge is predicting sgRNA target site accessibility due to chromatin state.
- Currently, accessibility is difficult to predict during sgRNA design and requires experimental validation.
Purpose of the Study:
- To develop a streamlined protocol for testing sgRNA target accessibility in wheat.
- To enable efficient validation of CRISPR-Cas9 editing efficiency in vivo.
- To reduce time and resources spent on wheat genetic transformation projects.
Main Methods:
- Developed a protocol for wheat protoplast isolation from leaf tissue using enzymatic digestion and sucrose gradients.
- Transformed isolated protoplasts with Cas9 ribonucleoprotein complexes via PEG-mediated transformation.
- Assessed sgRNA editing efficiency by isolating DNA, performing PCR, and Sanger sequencing of the target region.
Main Results:
- Successfully established a simplified protocol for wheat protoplast isolation and transformation.
- Demonstrated the protocol's utility in testing sgRNA accessibility and editing efficiency in vivo.
- The method allows for rapid assessment of CRISPR target site accessibility in wheat.
Conclusions:
- The developed protocol provides a fast and easy method to test sgRNA accessibility in wheat protoplasts.
- This streamlines CRISPR transformation projects by enabling early detection of inaccessible targets.
- Facilitates more efficient and resource-effective genetic engineering in wheat.
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