Analysis of the Effectiveness of Crispr-Editing of the GEX2 Gene by Ribonucleoprotein Complexses in Maize Protoplasts

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Abstract

The GEX2 protein is expressed in the maize gamete membranes and necessary for gamete membranes contact (adhesion). Knockout of GEX2 gene, presumably, can lead to impaired fertilization and, as a result, to the haploid embryo formation. The aim of the study is to analyze the efficiency of CRISPR/Cas9 editing of the GEX2 gene after PEG-mediated transfection of maize protoplasts by ribonucleoprotein (RNP) complexes with different sgRNA. For the first time, the RNP complexes with different sgRNA to the GEX2 gene have been created. The effectiveness of CRISPR/Cas9 editing of the GEX2 gene have been proven on protoplasts and reaches 10.7%, depending on the sgRNA, level and thesgRNA:Cas9 ratio in the RNP complex.

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About the authors

E. M. Moiseeva

Institute of Biochemistry and Physiology of Plants and Microorganisms – Subdivision of the Federal Research Center, Saratov Scientific Centre of the Russian Academy of Sciences

Email: chumakov_m@ibppm.ru
Russian Federation, Saratov, 410049

V. V. Fadeev

Institute of Biochemistry and Physiology of Plants and Microorganisms – Subdivision of the Federal Research Center, Saratov Scientific Centre of the Russian Academy of Sciences; Chernyshevsky Saratov National Research State University

Email: chumakov_m@ibppm.ru
Russian Federation, Saratov, 410049; Saratov, 410012

Y. V. Fadeeva

Institute of Biochemistry and Physiology of Plants and Microorganisms – Subdivision of the Federal Research Center, Saratov Scientific Centre of the Russian Academy of Sciences; Chernyshevsky Saratov National Research State University

Email: chumakov_m@ibppm.ru
Russian Federation, Saratov, 410049; Saratov, 410012

Y. S. Gusev

Institute of Biochemistry and Physiology of Plants and Microorganisms – Subdivision of the Federal Research Center, Saratov Scientific Centre of the Russian Academy of Sciences; Chernyshevsky Saratov National Research State University

Email: chumakov_m@ibppm.ru
Russian Federation, Saratov, 410049; Saratov, 410012

M. I. Chumakov

Institute of Biochemistry and Physiology of Plants and Microorganisms – Subdivision of the Federal Research Center, Saratov Scientific Centre of the Russian Academy of Sciences

Author for correspondence.
Email: chumakov_m@ibppm.ru
Russian Federation, Saratov, 410049

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2. Fig. 1. Evaluation of the effectiveness of RNP complexes in vitro and in corn protoplasts. a – nucleotide sequences of sections of the GEX2 gene containing protospacers for hydRNA 1, 2; target sites for hydRNA are highlighted in gray, FRAME sequences are black. b – electrophoresis of PCR products from a fragment of the GEX2 gene after treatment with RNP complexes in vitro; tracks: 1 - PCR product with a target locus for hydRNA 1 after incubation with an RNP complex; 2 – PCR product with a target locus for hydRNA 1 (without treatment); 3 - DNA molecular weight marker; 4 – PCR product with target locus for hydRNA 2 after incubation with RNP complex; 5 - PCR product with target locus for hydRNA 2 (without treatment). b – electrophoresis of PCR products with a target locus for hydRNA 2. Tracks: 1 – PCR product from a fragment of the GEX2 gene obtained from the DNA of corn protoplasts after transformation with RNP complexes (45 mg nuclease/15 mg hydRNA) and treated with BstMAI restrictase (expected band sizes after hydrolysis are 324 and 183

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