Creation of a genetic vector carrying a synthesis bacterial protein gene CAS9 for plant genome editing

  • I. S. Hnatiuk
  • O. I. Varchenko
  • M. F. Parii
  • Yu. V. Symonenko
DOI: https://doi.org/10.7124/FEEO.v26.1263

Abstract

Aim. To create a genetic construct carrying the bacterial protein Cas9 gene, the reporter β-glucuronidase gus gene, as well as the marker phosphinotricin-N-acetyltransferase bar gene for plant genome editing. Methods. Molecular-biological, biotechnological, microbiological and bioinformatic methods were used in the study; Golden Gate molecular cloning method was used to create genetic constructs. Results. The genetic construct pSPE2053 which carries the Cas9 endonuclease gene, the gus and bar genes was created; the assembly correctness of all vector elements was confirmed by polymerase chain reaction; the construct was transferred to Escherichia coli and Agrobacterium tumefaciens cells; β-glucuronidase gene expression was verified by histochemical analysis after Nicotiana rustica L transient genetic transformation. Conclusions. The created genetic construct can be used to edit the plant genome for both stable and transient genetic transformation to accumulate recombinant Cas9 protein. The guide RNA sequences may be subsequently transferred into such plants using either stable or transient genetic transformation or traditional crossing methods.

Keywords: cloning, genetic construction, gus and bar genes, Cas9 endonuclease protein, transient expression. 

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