Guided RNA And Tethered Regulator(GRATR)for Down-regulating Gene Expression And DNA Recombination Caused By Double Strand Breacks

This study established a simple,accurate and efficient technology for inhibiting plant gene expression.The genes of green fluorescent protein,guide RNA and fusion protein of transcription inhibitory factor fused with MS2 phage coat protein were constructed into the plant expression vector.Transient expression was implemented by using agrobaterium infiltration on the leaves of Nicotiana benthamiana.The results showed that the expression of GFP gene in the leaves were significantly suppressed and the inhibition rate was 41.5%.This system is expected to be applied in regulating other genes' transcription.Because only gRNA is needed to change,while other component remains the same,our system possesses the characteristics of simple operation and extensive adaptability.It provides an effective tool for inhibiting gene expression in plant.In recent years,the research of gene editing technology has gradually developed,which is a method of DNA site mutation.A new and efficient method of genome editing technology can realize the mutation of specific sites,and it is simple and convenient to design.In recent years,zinc finger nuclease technology(ZFN),clustered regularly interspaced short palindromic repeats(CRISPR)and gene transcription-activating factor-like effector nuclease(TALEN)and other gene editing techniques have been widely used.They can produce incision accurately in the DNA double-stranded specific sites,and thus achieve gene insertion,deletion,base replacement and other mutations.Compared to traditional genomic editing techniques,these new gene editing techniques can not only achieve a fixed point of DNA modification,and also be applied to more species,and can greatly improve the efficiency of gene mutation.In addition,the cost of vector construction is relatively low,and the time is short,so that the target mutation of genome becomes possible.However,recent studies have found that off-target effects are prevalent in these systems.In the study of GRATEN system,we found that RNAs that are complementary to double-stranded DNA bases can cause DNA fragmentation.Two novel fragments of green fluorescent protein(GFP)and guide RNA(gRNA)were constructed into plant expression vector.Transient expression was implemented by using agrobaterium infiltration on the leaves of Nicotiana benthamiana.The results showed that the expression of GFP gene in the leaves were significantly suppressed and the inhibition rate was 36.4%.Only need to change the gRNA,the system can be used for other genes targeted mutation.