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Updated: Jan 30, 2026

A Customizable Protocol for String Assembly gRNA Cloning STAgR
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A Customizable Protocol for String Assembly gRNA Cloning (STAgR).

Christopher T Breunig1, Andrea M Neuner1, Jessica Giehrl-Schwab2

  • 1MCN Junior Research Group, Munich Center for Neurosciences, Ludwig Maximilian Universitat, BioMedical Center; Institute of Stem Cell Research, Helmholtz Zentrum, German Research Center for Environmental Health.

Journal of Visualized Experiments : Jove
|January 15, 2019
PubMed
Summary
This summary is machine-generated.

The CRISPR/Cas9 system enables genetic engineering, but delivering multiple guide RNAs (gRNAs) is challenging. STAgR is a new method for fast, efficient, and cost-effective cloning of multiplexed gRNA expression vectors in a single step.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • The CRISPR/Cas9 system has revolutionized life sciences, enabling genetic and genomic engineering across diverse systems.
  • CRISPR's broad applicability stems from its bipartite nature, with gRNAs directing Cas9 to specific genomic targets.
  • Current CRISPR methods often require simultaneous delivery of multiple gRNAs, posing a technical challenge.

Purpose of the Study:

  • To develop a simple, fast, and efficient method for generating multiplexed gRNA expression vectors.
  • To present a customizable protocol for string assembly gRNA cloning (STAgR).
  • To enable the cost-effective incorporation of multiple gRNAs in a single cloning step.

Main Methods:

  • Developed the string assembly gRNA cloning (STAgR) protocol.
  • Utilized short overhang primers to introduce individual targeting sequences.
  • Employed reusable long DNA templates for gRNA expression cassettes.

Main Results:

  • STAgR allows for the simple, fast, and efficient generation of multiplexed gRNA expression vectors.
  • The method is cost-effective due to reusable DNA templates and primer-based targeting sequence introduction.
  • STAgR enables the single-step incorporation of a large number of gRNAs and combinations of different gRNA variants, vectors, and promoters.

Conclusions:

  • STAgR provides a versatile and efficient solution for constructing multiplexed gRNA expression vectors.
  • This method simplifies and accelerates CRISPR-based genetic, transcriptional, and epigenomic engineering.
  • STAgR enhances the applicability and cost-effectiveness of complex CRISPR workflows.