A. Koltun, Nathalia Volpi e Silva, Jéssika Angelotti-Mendonça, S. R. Marin, L. Gonçalves, A. Nepomuceno, L. M. Mertz-Henning
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CRISPR-transient expression in soybean for simplified gRNA screening in planta
Abstract The objective of this work was to develop a method to create and validate CRISPR-Cas systems and different gRNAs in soybean (Glycine max) embryos. Two model genes were used for simple mutation with one gRNA or partial gene deletion with two guides. The gRNAs were inserted into the CRISPR transformation vectors by a type IIS restriction enzyme or by subcloning and inserting the promoter + gRNA2 in the final transformation vector using the classic restriction enzyme cloning method. The vectors were successfully constructed for one and two gRNAs. Agrobacterium-mediated transient transformation in soybean was carried out to test the quality of gRNAs and of the system itself (expression cassette). Simple mutation and gene deletion were detected in the embryos transformed after DNA enrichment by enzyme digestion followed by polymerase chain reaction and sequencing, which indicates that the CRISPR-Cas system and guides were working. This protocol can be used to accelerate CRISPR-based genome editing strategies for genetic transformation in soybean.
期刊介绍:
Pesquisa Agropecuária Brasileira – PAB – is issued monthly by Empresa Brasileira de Pesquisa Agropecuária – EMBRAPA, affiliated to Ministry of Agriculture, Livestock and Food Supply. PAB publishes original scientific-technological articles on Plant Physiology, Plant Pathology, Crop Science, Genetics, Soil Science, Food Technology and Animal Science.
Its abbreviated title is Pesq. agropec. bras., and it should be used in bibliographies, footnotes, references and bibliographic strips.