I-SceI Endonuclease-Mediated Plant Genome Editing by Protein Transport through a Bacterial Type III Secretion System.

Yuki Yanagawa,Kasumi Takeuchi,Ichiro Mitsuhara,Seiichi Toki,Masaki Endo,Ayako Furutani,Hirokazu Ochiai

doi:10.3390/plants9091070

Yuki Yanagawa, Kasumi Takeuchi + Show 5 more

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https://doi.org/10.3390/plants9091070

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Abstract

Xanthomonas campestris is one of bacteria carrying a type III secretion system which transports their effector proteins into host plant cells to disturb host defense system for their infection. To establish a genome editing system without introducing any foreign gene, we attempted to introduce genome editing enzymes through the type III secretion system. In a test of protein transfer, X. campestris pv. campestris (Xcc) transported a considerable amount of a reporter protein sGFP-CyaA into tobacco plant cells under the control of the type III secretion system while maintaining cell viability. For proof of concept for genome editing, we used a reporter tobacco plant containing a luciferase (LUC) gene interrupted by a meganuclease I-SceI recognition sequence; this plant exhibits chemiluminescence of LUC only when a frameshift mutation is introduced at the I-SceI recognition site. Luciferase signal was observed in tobacco leaves infected by Xcc carrying an I-SceI gene which secretes I-SceI protein through the type III system, but not leaves infected by Xcc carrying a vector control. Genome-edited tobacco plant could be regenerated from a piece of infected leaf piece by repeated selection of LUC positive calli. Sequence analysis revealed that the regenerated tobacco plant possessed a base deletion in the I-SceI recognition sequence that activated the LUC gene, indicating genome editing by I-SceI protein transferred through the type III secretion system of Xcc.

Highlights

Genome editing technology offers an attractive new approach to plant breeding
We first examined whether the type III secretion system of X. campestris pv. campestris (Xcc) could be used to transfer proteins into plant cells
In this work we demonstrated the feasibility of plant genome editing without introduction of any foreign DNA or RNA by transferring genome editing enzymes into living cells in tobacco leaves any foreign DNA or RNA by transferring genome editing enzymes into living cells in tobacco leaves through the type III secretion system of the bacterium X. campestris

Summary

Introduction

Genome editing technology offers an attractive new approach to plant breeding. Genome editing involves making functional changes in endogenous genes by mutation and deletion with genome editing enzymes composed of protein or protein/RNA complex such as Zinc finger nucleases (ZFNs), Transcription activator-like effector nucleases (TALENs) and Clustered regularly interspaced short palindromic repeats (CRISPR)/Crispr associated protein 9 (Cas9) [1,2,3,4].In mammalian cells, genome editing has been achieved by introducing Cas and guide RNA (gRNA) ribonucleoprotein complex by lipofection or electroporation [5]. Genome editing technology offers an attractive new approach to plant breeding. Genome editing involves making functional changes in endogenous genes by mutation and deletion with genome editing enzymes composed of protein or protein/RNA complex such as Zinc finger nucleases (ZFNs), Transcription activator-like effector nucleases (TALENs) and Clustered regularly interspaced short palindromic repeats (CRISPR)/Crispr associated protein 9 (Cas9) [1,2,3,4]. Genome editing has been achieved by introducing Cas and guide RNA (gRNA) ribonucleoprotein complex by lipofection or electroporation [5]. Microinjection was used for CRISPR/Cas9-mediated genome editing in murine zygotes [5]. Nanoparticles worked to deliver the Cas9/gRNA complex for correcting gene mutation of genetic disease [6]. The foreign protein and RNA are degraded in the cells; genome editing is expected to accomplish functional changes without introducing any foreign gene.

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