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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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Dissection of Enhancer Function Using Multiplex CRISPR-based Enhancer Interference in Cell Lines

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RAPID-DASH: Fast and Efficient Assembly of Guide RNA Arrays for Multiplexed CRISPR-Cas9 Applications.

Asfar Lathif Salaudeen¹, Nicholas Mateyko¹, Carl G de Boer²

¹Genome Science and Technology Graduate Program, University of British Columbia, Vancouver, BC, Canada, V6T 1Z4.

Biorxiv : the Preprint Server for Biology

|April 28, 2025

Summary

Researchers developed a fast CRISPR-Cas9 method to create guide RNA (gRNA) arrays for simultaneous multi-locus targeting. This technique enables efficient combinatorial perturbation studies by rapidly constructing and testing gRNA libraries.

Keywords:

CRISPR-Cas9 Golden Gate Assembly gRNA Array genome engineering multiplexing polymerase cycling assembly

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A Customizable Protocol for String Assembly gRNA Cloning STAgR

A Customizable Protocol for String Assembly gRNA Cloning STAgR

Published on: December 26, 2018

Area of Science:

Molecular Biology
Genetics
Biotechnology

Background:

CRISPR-Cas9 technology allows for precise genome editing.
Simultaneous targeting of multiple genomic loci is crucial for complex genetic studies.
Current methods for constructing multiplexed guide RNA (gRNA) arrays can be time-consuming and inefficient.

Purpose of the Study:

To develop a streamlined and efficient method for constructing guide RNA (gRNA) arrays.
To enable rapid assembly of gRNA arrays with up to 10 gRNA units.
To facilitate combinatorial perturbation research through scalable and multiplexed gRNA array construction.

Main Methods:

A novel, rapid protocol for synthesizing gRNA arrays was established.
The method allows for the incorporation of up to 10 distinct gRNA units within a single array.
Functional activity of gRNAs was assessed across all positions within the constructed arrays.

Main Results:

The developed method enables the construction of functional gRNA arrays in a single day.
All gRNA units within the arrays demonstrated robust functional activity, irrespective of their position.
The approach supports the incorporation of diverse gRNA libraries, offering scalability and multiplexing capabilities.

Conclusions:

The new method provides an economical and rapid solution for constructing gRNA arrays.
This technique significantly streamlines the process of designing, testing, and iterating gRNA arrays for combinatorial studies.
The findings will accelerate research in areas requiring simultaneous targeting of multiple genomic sites.