Studies Using Genome-directed RNA-coupled Endonucleases In Plants For Genome Editing

Genome editing is a recently developed technique that can generate mutants in target DNA sequences,which is a major methodis to modify target genome for the purpose of studying the gene with unknown functions. Precise genome editing is a powerful tool for elucidating gene function and applicable gene therapy. The genome editing approaches of engineered nucleases with sequence-specific DNA binding proteins, such as zinc-finger nuclease(ZFN)or transcription activator-like effector nuclease(TALEN), have been developed to cause genome alterations in diverse cell lines or organisms. Both ZFN and TALEN methods require to assemble two arrays of repetitive DNA binding proteins in reverse orientation, which is time-consuming and labor-intensive.More recently, a programmable RNA-guided nuclease termed clustered regularly interspaced short palindromic repeat(CRISPR) /CRISPR–associated(Cas), derived from bacterial type II adaptive immune system, was harnessed to generate site-specific genome modifications.Compared with ZFN and TALEN, CRISPR/Cas system is straightforward for constructing DNA recognition module, a short guide-RNA(gRNA) complementary to target sequence.Whereas for CRISPR/Cas system implemented in genome editing, the selection of target sequence is limited by initiating with G for its transcription driven by U6 polymerase III promoter, interspaced 20-basepair(bp) target sequence and terminating with NGG, a protospacer-adjacent motif(PAM) essential for the Cas9 endonuclease to recognize and cleave target site.Additionally, recent studies revealed that this system, like ZFN and TALEN,could generate undesirable off-target effects.Consequently, a novel genome editing methodology remains to be contrived for simple customizable design, high flexibility for selecting target sequence and site-specific mutagenesis. Guided RNA and Tethered Endonuclease-( GRATEN) system contains single chimeric RNA comprising guided RNA for target recognition and adapters for bacteriophage MS2 coat protein(MCP) to tether engineered endonuclease. We explored the possibility of site-directed genome mutagenesis by GRATEN system in the plant and then transiently expressed in tobacco leaves, and conducted a preliminary study of its mutagenic effect. These ribonucleoprotein complexes efficiently facilitate to disrupt targeted genes in Nicotiana benthamiana, which is characterized by simplecustomizable design, no constraint for selecting target sequence and site-specific mutagenesis.The objective of this research will establish a novel system for RNA-programmable genome editing in plant featured with simple customer design, precision and high efficiency.In this study, we cloned multiple gene fragments by DNA shuffling. We also constructed an expression vector using DNA shuffling technology. We constructed two sets of GFP tagged protein expression plasmids,in which the N and C terminals of GFP fragments contained multiple cloning sites for inserting genes of interesting. By utilizing these constructs, we cloned endonuclease FokI cleavage domain fused with two MS2 bacteriophage coat protein(MCP) forming fusion protein of MCP:FokI:MCP(FMF), in which the cellular nuclear localization signals and GFP were linked to N and C terminals of FMF respectively.The expression of cloned genes are expected to create fusion proteins of GFP:FMF or FMF:GFP respectively. The resulting constructs were transformed into agro-bacterium and transient expressed both in the cells of Nicotiana benthamiana and onion leaf epidermic cells via agrobactereal infiltration.The observation under confocal microscope showed that the localization of GFP protein present both in nuclear and cytoplasm, while GFP:FMF and FMF:GFP only in the nuclear. Adapters for bacteriophage MS2 coat protein(MCP) to tether engineered endonuclease could be worked in the nucleus.