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Related Concept Videos

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Updated: Dec 6, 2025

Using Next Generation Sequencing to Identify Mutations Associated with Repair of a CAS9-induced Double Strand Break Near the CD4 Promoter
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Versatile in vitro assay to recognize Cas9-induced mutations.

Heinrich Bente1, Ortrun Mittelsten Scheid1, Mattia Donà1

  • 1Gregor Mendel Institute of Molecular Plant Biology Austrian Academy of Sciences Vienna BioCenter (VBC) Vienna Austria.

Plant Direct
|October 5, 2020
PubMed
Summary

This study introduces a modified in vitro assay to quickly identify effective single-guide RNAs (sgRNAs) for CRISPR/Cas9 gene editing in plants. This method accelerates the screening of Cas9-induced mutations, reducing workload and costs in plant biotechnology.

Keywords:
CRISPR/Cas9cost and labor‐saving protocolgenotyping protocolin vitro cleavagemutagenesissgRNA cleavage efficiency

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Area of Science:

  • Molecular Biology
  • Plant Biotechnology
  • Genetics

Background:

  • CRISPR/Cas9 technology has transformed plant biotechnology and breeding.
  • Single-guide RNA (sgRNA) efficiency in CRISPR/Cas9 gene editing varies and is not fully understood, impacting plant mutagenesis success.
  • Existing sgRNA design tools and databases often lack plant-specific data, leading to potential failures in gene editing.

Purpose of the Study:

  • To present a modified in vitro assay for initial testing of sgRNAs.
  • To accelerate the genotyping of Cas9-induced mutations in plants.
  • To provide a simple, low-cost, and rapid method for identifying edited genes in plants and other organisms.

Main Methods:

  • A modified in vitro assay was developed to test sgRNA suitability.
  • The protocol was applied to mutagenesis and mutation screening for specific genes in Arabidopsis.
  • The method focuses on accelerating the identification of effective sgRNAs before laborious plant transformation.

Main Results:

  • The modified in vitro assay demonstrated applicability for accelerating mutagenesis and mutation screening in Arabidopsis.
  • The protocol offers a way to pre-screen sgRNAs, reducing the risk of failure in plant gene editing.
  • The method is designed to be universally suitable for various plant species and other organisms.

Conclusions:

  • The developed protocol provides a simple, low-cost, and rapid method for identifying effective sgRNAs for CRISPR/Cas9 gene editing.
  • This approach can significantly reduce the workload and costs associated with plant breeding and biotechnology.
  • The universally applicable principle aids in accelerating the discovery of edited genes across different species.