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Medicago truncatula is the model plant species for studying symbioses with nitrogen-fixing rhizobia and arbuscular mycorrhizae, where edited mutants are invaluable for elucidating the contributions of known genes in these processes. Streptococcus pyogenes Cas9 (SpCas9)-based genome editing is a facile means of achieving loss of function, including where multiple gene knockouts are desired in a single generation. We describe how the user can customize our vector to target single or multiple genes, then how the vector is used to make M. truncatula transgenic plants containing target site mutations. Finally, obtaining transgene-free homozygous mutants is covered., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)