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CRISPR/Cas9 Genome Editing

CRISPR/Cas9 Genome Editing

The CRISPR-Cas system serves as a bacterial defense mechanism against invading genetic elements such as viruses and plasmids, forming the foundation for its adaptation as a powerful genome-editing tool. Originally discovered in prokaryotes, this system has been repurposed to revolutionize genetic engineering across a wide range of organisms, including plants, animals, and humans. The core component, Cas9, is an endonuclease derived from Streptococcus pyogenes, capable of introducing...

CRISPR

CRISPR

Genome editing technologies allow scientists to modify an organism’s DNA via the addition, removal, or rearrangement of genetic material at specific genomic locations. These types of techniques could potentially be used to cure genetic disorders such as hemophilia and sickle cell anemia. One popular and widely used DNA-editing research tool that could lead to safe and effective cures for genetic disorders is the CRISPR-Cas9 system. CRISPR-Cas9 stands for Clustered Regularly Interspaced...

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Related Experiment Video

Updated: Jul 15, 2025

A Universal Protocol for Large-scale gRNA Library Production from any DNA Source

A Universal Protocol for Large-scale gRNA Library Production from any DNA Source

Published on: December 6, 2017

FLASH Genome Editing Pipeline: An Efficient and High-Throughput Method to Construct Arrayed CRISPR Library for Plant

Lu Yao¹, Xiaochun Wang^1,2, Runnan Ke¹

¹National Key Laboratory of Crop Genetic Improvement and Hubei Hongshan Laboratory, Huazhong Agricultural University, Wuhan, People's Republic of China.

Current Protocols

|September 27, 2023

Summary

We developed the FLASH genome editing pipeline for high-throughput CRISPR screening in plants. This method uses unique FLASH tags to link gene targets with Cas9/gRNA vectors, enabling efficient gene function discovery.

Keywords:

CRISPR/Cas9 arrayed library genome editing high throughput plant

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Last Updated: Jul 15, 2025

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Area of Science:

Plant Genomics
Genome Editing Technologies
Crop Breeding

Background:

CRISPR/Cas9 is a powerful tool for gene editing in various organisms.
High-throughput genetic screening using CRISPR/Cas9 is challenging in plants.
Efficient methods are needed for plant functional genomics and crop improvement.

Purpose of the Study:

To establish a novel pipeline for constructing arrayed CRISPR libraries in plants.
To enable high-throughput genetic screening and gene function discovery in plants.
To develop a method for linking target genes to Cas9/gRNA vectors using unique identifiers.

Main Methods:

Development of the FLASH genome editing pipeline using 12 distinct PCR fragments (FLASH tags) for indexing Cas9/gRNA vectors.
Assembly of FLASH-tag indexed Cas9/gRNA plasmids and preparation of a plasmid library.
Agrobacterium-mediated transformation of rice plants with a mixture of indexed strains.
Utilizing regular PCR and gel electrophoresis for assigning gRNA information in gene-edited plants.

Main Results:

Successfully constructed an arrayed CRISPR library in plants using the FLASH genome editing pipeline.
Established a unique link between target genes/gRNAs and FLASH tags for efficient information retrieval.
Demonstrated the capability of reading gRNA information in both bacterial strains and gene-edited plants.
The protocol allows for fast and comprehensive gene function discovery in plants.

Conclusions:

The FLASH genome editing pipeline facilitates high-throughput genetic screening in plants.
This approach enables efficient gene function discovery and supports crop breeding advancements.
The method provides a robust solution for constructing and analyzing arrayed CRISPR libraries in plant systems.