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Related Experiment Video

Updated: Feb 7, 2026

Author Spotlight: Simplifying Genome-Wide Plasmid Library Construction Using CRISPRmass

Author Spotlight: Simplifying Genome-Wide Plasmid Library Construction Using CRISPRmass

Published on: May 17, 2024

Optimized Plasmid Construction Strategy for Cas9.

Jianyong Xu^1,2, Wenlei Li³, Md Munnaf Hossen^1,2

¹Department of Immunology, School of Medicine, Shenzhen University, Shenzhen, China.

Cellular Physiology and Biochemistry : International Journal of Experimental Cellular Physiology, Biochemistry, and Pharmacology

|July 13, 2018

Summary

We optimized Cas9-guide RNA (gRNA) plasmid construction for genome editing. A new strategy using Golden-gate assembly and blue-white screening is cost-effective and time-saving, enhancing research efficiency.

Keywords:

Cas9 Genome editing Guide RNA RNA guided endonuclease gRNA

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Use of In Vivo Assembly for High-efficiency Plasmid Construction

Use of In Vivo Assembly for High-efficiency Plasmid Construction

Published on: February 7, 2025

Area of Science:

Molecular Biology
Genome Editing Technologies

Background:

Genome editing technologies are crucial for biological research and clinical applications.
RNA Guided EndoNuclease (RGEN) systems, like Cas9, offer easier construction and modification.
Optimizing plasmid strategies for Cas9-guide RNA (gRNA) is essential for efficient genome editing.

Purpose of the Study:

To compare different plasmid construction strategies for Cas9-gRNA.
To develop a more efficient, cost-effective, and time-saving method for Cas9-gRNA plasmid construction.

Main Methods:

Comparison of single-plasmid versus two-plasmid systems for Cas9 and gRNA expression.
Integration of Golden-gate assembly and blue-white screening into the Cas9-gRNA construction process.
Elimination of vector digestion and gel purification steps.

Main Results:

Single-plasmid Cas9-gRNA expression showed lower construction efficiency than two-plasmid systems.
Both single and two-plasmid systems demonstrated comparable genome editing efficiency.
The optimized strategy, incorporating Golden-gate assembly and blue-white screening, streamlined the construction process.

Conclusions:

A novel, efficient plasmid construction strategy for Cas9-gRNA was established.
This optimized method is cost-effective and time-saving.
The strategy is enhanced by combining with a previously identified optimized gRNA structure (gRNA-BL).