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Updated: Jun 6, 2026
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CRISPR Guide RNA Cloning for Mammalian Systems
Published on: October 2, 2018
A high-throughput, streamlined cloning protocol to generate guide RNAs for CRISPR activation.
Shusen Zhu1,2, Aura A Tamez González1,2, Abdelrahman Alokda1,2
1Department of Neurology and Neurosurgery, McGill University, Montreal, QC, Canada.
Frontiers in Genome Editing
|June 5, 2026
Summary
We developed a streamlined CRISPR activation (CRISPRa) cloning method for Caenorhabditis elegans. This efficient protocol accelerates high-throughput gene screening and functional genomics studies in this model organism.
Area of Science:
- Genomics
- Molecular Biology
- Model Organisms
Background:
- Caenorhabditis elegans is a key model for gene function and disease research.
- CRISPR activation (CRISPRa) enables gene upregulation, complementing RNA interference.
- Traditional cloning methods hinder high-throughput CRISPRa screening in C. elegans.
Purpose of the Study:
- To develop a rapid, efficient, and scalable cloning protocol for CRISPRa in C. elegans.
- To overcome the bottleneck of traditional cloning workflows for high-throughput screening.
- To advance functional genomics studies using CRISPRa in C. elegans.
Main Methods:
- A pooled, one-step dual guide RNA (gRNA) cloning protocol was designed.
- Primers containing gRNA sequences were used to amplify and pool multiple gRNA inserts.
- A single reaction pipeline was established for constructing gRNA expression vectors.
Main Results:
- 126 gRNA inserts were successfully amplified and pooled in a single pipeline.
- Pooled cloning achieved 42%-56% coverage per round, with individual processing for remaining clones.
- The protocol significantly reduced time, labor, and reagent costs.
Conclusions:
- The new protocol enables rapid and efficient construction of gRNA expression vectors for CRISPRa.
- This method is highly scalable and reproducible, suitable for high-throughput screening.
- It facilitates large-scale gene activation studies and downstream applications like phenotypic screening in C. elegans.
