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Updated: Aug 23, 2025
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CRISPR Guide RNA Cloning for Mammalian Systems
Published on: October 2, 2018
71.2K
Efficient expression of multiple guide RNAs for CRISPR/Cas genome editing.
Vicki Hsieh-Feng1,2, Yinong Yang1,2
1Intercollege Graduate Degree Program in Plant Biology, The Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, PA 16802 USA.
Abiotech
|October 28, 2022
Summary
The CRISPR/Cas system enables multiplex genome editing through efficient guide RNA (gRNA) expression strategies. Advances in promoters, RNA processing, and delivery methods are revolutionizing genome engineering.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- The Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein (CRISPR/Cas) system is a leading tool for genome engineering.
- Efficient expression of multiple guide RNAs (gRNAs) is crucial for multiplex genome editing applications.
Purpose of the Study:
- This review discusses current strategies for efficient expression and engineering of gRNAs.
- It aims to facilitate CRISPR/Cas-based multiplex genome editing.
Main Methods:
- Exploration of various CRISPR/Cas expression strategies (e.g., two-component, single transcriptional unit, bidirectional promoters).
- Optimization of gRNA production using diverse promoters and RNA processing techniques (ribozymes, RNases, Csy4).
- Investigation of inducible and spatiotemporal gRNA expression control.
Main Results:
- Development of multiple strategies for efficient gRNA and Cas nuclease expression.
- Optimization of gRNA production through advanced promoter and RNA processing methods.
- Emergence of transgene-free multiplex genome editing via CRISPR/Cas ribonucleoprotein delivery.
Conclusions:
- Current strategies significantly enhance gRNA expression for multiplex genome editing.
- Future perspectives focus on further optimizing gRNA engineering and delivery for advanced applications.
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