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ARS Home » Southeast Area » Gainesville, Florida » Center for Medical, Agricultural and Veterinary Entomology » Chemistry Research » Research » Publications at this Location » Publication #364520

Research Project: Molecular and Biochemical Characterization of Biotic and Abiotic Stress on Plant Defense Responses in Maize

Location: Chemistry Research

Title: Considerations for using CRISPR/Cas9 in targeted mutagenesis for functional genetics in plants

item Hunter, Charles

Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/7/2021
Publication Date: 4/8/2021
Citation: Hunter, C.T. 2021. Considerations for using CRISPR/Cas9 in targeted mutagenesis for functional genetics in plants. Plants. 10(4): 723.

Interpretive Summary: The CRISPR/Cas9 system represents a powerful new technology for genetic modification. Genetic studies to understand the function of genes rely on loss-of-function mutants in those genes. CRISPR/Cas9 is the most direct and affordable system for site-directed mutagenesis, greatly facilitating the production of such mutants. While the CRISPR/Cas9 system is relatively simple and accessible, there are numerous points to consider when developing experimental designs for using CRISPR that have not received adequate attention in the current literature. In this invited Perspective, important considerations for using CRISPR in time- and resource-efficient manner is presented. A novel vector for utilizing CRISPR in maize is also presented, which allows multiple genes to be targeting simultaneously with a simple vector assembly. This information will help readers design experiments and avoid common pitfalls and misconceptions regarding expected products and timelines while utilizing the power of CRISPR for gene disruption.

Technical Abstract: Utilization of the CRISPR/Cas9 system for targeted mutagenesis has become an indispensable tool for functional genetics in plants. CRISPR/Cas9 allows users to generate loss-of-function alleles in genes of interest with precision and in a simple-to-use system. This Perspective outlines important points to consider for experimental design and utilization of CRISR/Cas9 in targeted mutagenesis in plants. Vector selection, gRNA design, genetic strategies, and genotyping approaches are discussed, with an emphasis on achieving isolation of homozygous mutant plants in a time- and cost-efficient manner.