A method using electroporation for the protein delivery of Cre recombinase into cultured Arabidopsis cells with an intact cell wall

Yuichi Furuhata,Mone Morikawa,Takeshi Yoshizumi,Tomi Murakami,Ushio Fujikura,Keiji Nishida,Chikashi Nakamura,Ayako Sakai,Yoshio Kato

doi:10.1038/s41598-018-38119-9

Abstract

Genome engineering in plants is highly dependent on the availability of effective molecular techniques. Despite vast quantities of research, genome engineering in plants is still limited in terms of gene delivery, which requires the use of infectious bacteria or harsh conditions owing to the difficulty delivering biomaterial into plant cells through the cell wall. Here, we describe a method that uses electroporation-mediated protein delivery into cultured Arabidopsis thaliana cells possessing an intact cell wall, and demonstrate Cre-mediated site-specific recombination. By optimizing conditions for the electric pulse, protein concentration, and electroporation buffer, we were able to achieve efficient and less-toxic protein delivery into Arabidopsis thaliana cells with 83% efficiency despite the cell wall. To the best of our knowledge, this is the first report demonstrating the electroporation-mediated protein delivery of Cre recombinase to achieve nucleic acid-free genome engineering in plant cells possessing an intact cell wall.

Highlights

Genome engineering is a powerful molecular tool that has been extensively used in numerous areas of the life sciences
These methods enable the efficient expression of genome engineering proteins, including Cre recombinase, exogenous DNA fragments that are introduced using a particle gun or Agrobacterium are often or invariably incorporated into the genomic DNA, and may induce unexpected effects that interfere with subsequent analyses
The number of poring pulses did not affect normalized GUS activity at more than 2 pulses (Fig. 3c), while cytotoxicity did not increase at greater than 1 pulse (Supplementary Figure 5c). These results indicate that the field strength and duration of poring pulse are important factors for protein electroporation into Arabidopsis thaliana cells, and that more protein can be electroporated by increasing the field strength or/and duration of poring pulse

Summary

Introduction

Genome engineering is a powerful molecular tool that has been extensively used in numerous areas of the life sciences. In order to introduce genetic materials into plant cells through the cell wall, the delivery of DNA using a particle gun or Agrobacterium (Rhizobium)-mediated techniques are widely used in plant research[25,26] These methods enable the efficient expression of genome engineering proteins, including Cre recombinase, exogenous DNA fragments that are introduced using a particle gun or Agrobacterium are often or invariably incorporated into the genomic DNA, and may induce unexpected effects that interfere with subsequent analyses. To the best of our knowledge, this is the first report to demonstrate the electroporation-mediated protein delivery of Cre recombinase to achieve nucleic acid-free genome engineering in plant cells possessing a cell wall

Methods

Results

Conclusion