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Related Experiment Video

Updated: Jun 27, 2026

A Customizable Protocol for String Assembly gRNA Cloning (STAgR)

A Customizable Protocol for String Assembly gRNA Cloning (STAgR)

Published on: December 26, 2018

An infinitely expandable cloning strategy plus repeat-proof PCR for working with multiple shRNA.

Glen John McIntyre¹, Jennifer Lynne Groneman, Anna Tran

¹Johnson and Johnson Research Pty Ltd, Australian Technology Park, Eveleigh, New South Wales, Australia. glen@madebyglen.com

|December 2, 2008

Summary

This study presents an infinitely expandable cloning strategy using restriction enzymes for constructing complex DNA vectors. This method simplifies repeated insertions, enabling efficient assembly of multiple genetic elements like short hairpin RNA expression vectors.

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CRISPR-based Shuttle Cloning: A High-throughput Cloning Method

CRISPR-based Shuttle Cloning: A High-throughput Cloning Method

Published on: June 13, 2025

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Last Updated: Jun 27, 2026

A Customizable Protocol for String Assembly gRNA Cloning (STAgR)

A Customizable Protocol for String Assembly gRNA Cloning (STAgR)

Published on: December 26, 2018

CRISPR-based Shuttle Cloning: A High-throughput Cloning Method

CRISPR-based Shuttle Cloning: A High-throughput Cloning Method

Published on: June 13, 2025

Area of Science:

Molecular Biology
Synthetic Biology
Biotechnology

Background:

Traditional restriction enzyme (RE)-based cloning faces challenges with complex strategies requiring multiple insertions, such as constructing short hairpin RNA (shRNA) expression vectors for RNA interference (RNAi).
Difficulty arises from the limited availability of unique RE recognition sites as vector size and the number of cloning events increase.

Purpose of the Study:

To develop a technically simple, directional cloning solution for efficiently constructing complex DNA vectors with repeated insertions.
To address the limitations of traditional cloning methods by enabling the insertion of multiple DNA fragments ad infinitum.

Main Methods:

Utilized restriction enzymes with compatible cohesive ends that are repeatedly destroyed and reintroduced during sequential cloning steps.
Developed a strategy incorporating multiple cloning cores for broad compatibility with various donor sequences.
Employed parallel assembly of sub-combinations for time-efficiency in constructing multi-insertion vectors.
Optimized PCR screening using Pfu polymerase for successful amplification of constructs with multiple repeated elements.

Main Results:

Demonstrated a cloning strategy allowing ad infinitum insertion of donor fragments in any combination.
Showed that parallel construction of sub-combinations is more time-efficient than sequential assembly for four or more insertions.
Successfully amplified constructs with up to eleven consecutive hairpin expression cassettes using Pfu polymerase-based PCR.

Conclusions:

The developed cloning strategy offers a general and infinitely expandable solution for various molecular cloning scenarios, particularly for complex vector construction.
The optimized PCR conditions using Pfu polymerase are beneficial for screening constructs with multiple repeated sequences, including multiple shRNA expression cassettes.