An Agrobacterium-Mediated CRISPR/Cas9 Platform for Genome Editing in Maize
Precise genome engineering can be efficiently made using the revolutionary tool named CRISPR/Cas (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein) systems. Adapted from the bacterial immune system, CRISPR/Cas systems can generate highly specific double-strand breaks (DSBs) at the target site, and desired sequence modifications can be introduced during the DSB repair process, such as nonhomologous end-joining (NHEJ) or homology-directed repair (HDR) pathways. CRISPR/Cas9 is the most widely used genome editing tool for targeted mutagenesis, precise sequence modification, transcriptional reprogramming, epigenome editing, disease treatment, and many more. The ease of use and high specificity make CRISPR/Cas9 a great tool not only for basic researches but also for crop trait improvements, such as higher grain yield, better tolerance to abiotic stresses, enhanced disease resistance, and better nutritional contents. In this protocol, we present a step-by-step guide to the CRISPR/Cas9-mediated targeted mutagenesis in maize Hi II genotype. Detailed procedures will guide through the essential steps including gRNA design, CRISPR/Cas9 vector construction, Agrobacterium-mediated maize immature embryo transformation, and molecular analysis of the transgenic plants to identify desired mutant lines.
Key wordsAgrobacterium-mediated transformation CRISPR/Cas9 Genome editing Maize Targeted mutagenesis
The authors wish to thank Marcy Main, Daniel Little, and Minjeong Kang for their contributions to this work. This project was partially supported by the US National Science Foundation (BREAD #1543888 to K.W. and B.Y.); by the USDA National Institute of Food and Agriculture, Hatch project number # IOW05162; by the State of Iowa funds; and by the Crop Bioengineering Center of Iowa State University.
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