CRISPR/Cas9‐Mediated Gene Knockout in Cereal Crops

Sep 25, 2025Current protocols

Using CRISPR to Remove Specific Genes in Cereal Crops

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CRISPR Gene Editing on OpenScience ↗PubMed ↗DOI ↗OA ↗

Abstract

CRISPR/Cas9 genome editing tools are significantly impacting crop breeding and research.

CRISPR/Cas9 introduces targeted, site-specific double-stranded breaks in DNA, often leading to gene knockout through frame-shift mutations.
This system has been widely adopted for modifying genes in important cereal crops such as rice, maize, wheat, and sorghum.
Cereal crops play a crucial role in global food security and provide essential nutrition.
These crops also support economic stability and are integral to biofuel production and livestock feed.
The article outlines strategies for implementing CRISPR/Cas9, including target selection and genome edit analysis.

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High-precision genome editing tools, such as programmable nucleases, are poised to transform crop breeding and significantly impact fundamental plant research. Among these tools, the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 (CRISPR-associated 9) system is a programmable, RNA-guided nuclease that introduces targeted, site-specific double-stranded breaks in the target DNA loci. When these breaks are repaired, it often results in a frame-shift mutation via short insertion/deletion (indel), leading to gene knockout. Since its first successful use in plants, CRISPR/Cas9 has been widely adopted for targeting genes of agronomic and scientific importance in multiple crops, including rice, maize, wheat, and sorghum. These cereal crops ensure global food security, provide essential nutrition, and support economic stability. Additionally, such crops support biofuel production, livestock feed, and sustainable farming practices through crop rotation. This article outlines the strategies for implementing CRISPR/Cas9 genome editing in plants, including a step-by-step process of guide RNA target selection, oligonucleotide design, construct development, assembly, and analysis of genome edits. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: CRISPR/Cas9 guide RNA target selection Support Protocol 1: Genomic DNA extraction in-house protocol Basic Protocol 2: Construction of a binary plasmid vector Support Protocol 2: Agrobacterium transformation with a binary vector construct and stability check Support Protocol 3: Plant transformation Basic Protocol 3: Genotyping of edited events.

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CRISPR/Cas9‐Mediated Gene Knockout in Cereal Crops

Abstract

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