Abstract
Clustered regularly interspaced short palindromic repeat/CRISPR-associated protein (CRISPR-Cas) systems were efficiently used for precise genome editing. 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. Among Cas nuclease proteins, Cas9 from Streptococcus pyogenes (SpCas9) is well-studied, and the CRISPR-Cas9 is the most widely used genome editing tool for basic research and crop improvements. It is suitable not only for targeted mutagenesis but also for creating large fragment deletions in plants. In this protocol, we present a step-by-step guide to the CRISPR-Cas9-mediated targeted mutagenesis or large fragment deletions in soybean. Detailed procedures will guide through the essential steps including the design of sgRNAs, construction of CRISPR-Cas9 vectors, Agrobacterium-mediated soybean transformation, and identification of mutant lines.
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Acknowledgments
This work was supported by the Major Science and Technology Projects of China (2016ZX08010-004), the Ministry of Science and Technology of China (2016YFD0100504), the National Natural Science Foundation of China (31871644), and the CAAS (Chinese Academy of Agriculture Sciences) Agricultural Science and Technology Innovation Project.
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Chen, L., Cai, Y., Hou, W. (2021). Generation of Knockout and Fragment Deletion Mutants in Soybean by CRISPR-Cas9. In: Islam, M.T., Molla, K.A. (eds) CRISPR-Cas Methods. Springer Protocols Handbooks. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1657-4_9
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DOI: https://doi.org/10.1007/978-1-0716-1657-4_9
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