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Abstract Until recently, precise genome editing has been limited to a few organisms. The ability of Cas9 to generate double stranded DNA breaks at specific genomic sites has greatly expanded molecular toolkits in many organisms and cell types. Before CRISPR‐Cas9 mediated genome editing,P. patenswas unique among plants in its ability to integrate DNA via homologous recombination. However, selection for homologous recombination events was required to obtain edited plants, limiting the types of editing that were possible. Now with CRISPR‐Cas9, molecular manipulations inP. patenshave greatly expanded. This protocol describes a method to generate a variety of different genome edits. The protocol describes a streamlined method to generate the Cas9/sgRNA expression constructs, design homology templates, transform, and quickly genotype plants. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Constructing the Cas9/sgRNA transient expression vector Alternate Protocol 1: Shortcut to generating single and pooled Cas9/sgRNA expression vectors Basic Protocol 2: Designing the oligonucleotide‐based homology‐directed repair (HDR) template Alternate Protocol 2: Designing the plasmid‐based HDR template Basic Protocol 3: Inducing genome editing by transforming CRISPR vector intoP. patensprotoplasts Basic Protocol 4: Identifying edited plants.
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This content will become publicly available on September 1, 2026
CRISPR/Cas9‐Mediated Gene Knockout in Cereal Crops
Abstract 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:Agrobacteriumtransformation with a binary vector construct and stability check Support Protocol 3: Plant transformation Basic Protocol 3: Genotyping of edited events
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- Award ID(s):
- 1917138
- PAR ID:
- 10655334
- Publisher / Repository:
- Current Protocols
- Date Published:
- Journal Name:
- Current Protocols
- Volume:
- 5
- Issue:
- 9
- ISSN:
- 2691-1299
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR‐associated protein)‐mediated genome editing has revolutionized fundamental research and plant breeding. Beyond gene editing, CRISPR/Cas systems have been repurposed as a platform for programmable transcriptional regulation. Catalytically inactive Cas variants (dCas), when fused with transcriptional activation domains, allow for specific activation of any target gene in the genome without inducing DNA double‐strand breaks. CRISPR activation enables simultaneous activation of multiple genes, holding great promise in the identification of gene regulatory networks and rewiring of metabolic pathways. Here, we describe a simple protocol for constructing a dCas9‐mediated multiplexed gene activation system based on the CRISPR‐Act3.0 system. The resulting vectors are tested in rice protoplasts. © 2022 Wiley Periodicals LLC. Basic Protocol 1: sgRNA design and construction of CRISPR‐Act3.0 vectors for multiplexed gene activation Basic Protocol 2: Determining the activation efficiency of CRISPR‐Act3.0 vectors using rice protoplastsmore » « less
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