Development of approaches for genome editing of pea plants using CRISPR/Cas9 prime-editing technique

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Abstract

Mitogen-activated protein kinases (MAPKs) play an important role as intracellular regulators of signal pathways in plants. Some MAPKs have been shown to be induced in the roots of legume plants during symbiosis with nitrogen-fixing rhizobial bacteria. One of such signal regulators, MAPK6, was shown to be involved in the development of symbiosis between Pisum sativum L. pea plants and rhizobia. Using genetic engineering approaches, we overexpressed the MAPK6 gene in transgenic roots, that resulted in an increase in the number of nodules and the biomass of pea plants. New approaches for genome editing of pea plants have been designed using the CRISPR/Cas9 prime-editing technique, when the MAPK6 gene was used as a target. We have analyzed the transgenic roots of pea transformants and observed the presence of the gene encoding Cas9 the pegRNA sequence in the genome of transformants. Therefore, the possibility of using genetic engineering methods to obtain plants with increased efficiency of symbiosis will be investigated in future experiments.

About the authors

Elizaveta S. Kantsurova

All-Russia Research Institute for Agricultural Microbiology

Email: rudaya.s.e@gmail.com
SPIN-code: 4752-1910

Junior Researcher, Signal Regulation Laboratory

Russian Federation, 3 Podbelskogo highway, Pushkin-8, Saint Petersburg, 196608

Nikolay V. Kozlov

All-Russia Research Institute for Agricultural Microbiology

Email: bionkbio@gmail.com
Russian Federation, 3 Podbelskogo highway, Pushkin-8, Saint Petersburg, 196608

Elena A. Dolgikh

All-Russia Research Institute for Agricultural Microbiology

Author for correspondence.
Email: dol2helen@yahoo.com
SPIN-code: 4453-2060

Dr. Sci. (Biology)

Russian Federation, 3 Podbelskogo highway, Pushkin-8, Saint Petersburg, 196608

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2. Fig. 1. Schematic representation of MAPK6 gene structure and the target site TXY for CRISPR/Cas prime-editing: a, MAPK6 gene structure; b, MAPK6 protein and site for replacement TXY amino acid residues to DXD; c, location of pegRNA including gRNA, RNA scaffold and template for reverse transcriptase (RT template) as well as PAM site for Cas9

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3. Fig. 2. Scheme illustrating the pGGK-A-G system for CRISPR/Cas9 genome editing. The pGG-C-D vector contains the sequences encoding Cas9 (H840A) nickase and reverse transcriptase of murine leukemia virus (codon-optimized for expression in plants). Replacement of gRNA with pegRNA was performed in the pGG-F-G vector. A modified sequence consists of the gRNA, RNA-scaffold and RT template with the replacements in the gene encoding MAPK6. tRNA — target RNA

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4. Fig. 3. Scheme illustrating the modifications in the pGG-C-D vector (a) and pGG-F-G vector (b) applied in this study for CRISPR/Cas9 genome editing. tRNA — target RNA

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5. Fig. 4. PCR analysis of pea transgenic roots from composite plants of cv. Frisson created using Agrobacterium rhizogenes. DNA was extracted from the roots of transformants (T). Plant transformed with construct with gene encoding bacterial β-glucuronidase (GUS) were used as a control. a, analysis revealed the presence of the Cas9 gene in the genome of transformants (T), but not in the GUS-control. The primers to promoter pPcUBI (F) and Cas9 gene (R) have been used; b, the presence of pegRNA sequence was shown in the genome of transformants, but not in the GUS-control. The primers to promoter pAtU6 (F) and gRNA-MK6 (R) have been used

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