GRATEN System For Detecting DNA Double Strand Breaks And Genome Editing Research

Genome editing technologies by targeting-site-specific loci in the genomes of certain species have rapidly evolved into powerful tools that can decipher gene function and be applied to gene therapy.The principle of genome editing technology is to cleave DNA at a predetermined genome location by constructing an endonuclease,and the resulted double strand breaks will be repaired by the intracellular DNA repair system to achieve the site-directed genome remodeling.It is very important to detect DNA double-strand breaks(DSBs).In this study,a convenient and quick method for detecting gene recombination caused by DSBs was established.DSBs can cause site-specific homology-directed repair(HDR).Therefore,the detection of DSBs is the key step for the study of GE production and its efficiency.The traditional methods for detecting DSBs are ?-H2 AX detection methods and fluorescent reporter gene detection.?-H2 AX is a DNA damage marker and used to detect ?-H2 AX caused DSBs.However,the method is complicated and the operation is cumbersome.Even some of the cell activity does not lead to the production of DSBs,it is still possible to produce ?-H2 AX.The fluorescence reporter gene detection method is realized by constructing a mutant fluorescent protein gene.However,this method detects only the mutations that generates recovered open reading frame of fluorescent protein,while the rest are undetectable and mainly used in transgenic plants.The real operation is not simple.In general,the traditional methods of detecting DSBs are complicated and cumbersome.Consequently,it is necessary to develope a simple and fast detection technique.This study was conducted by using the direct repeated GUS gene and guide RNA combined with phage MS2 coat protein coupled with restriction endonuclease genome editing system,in which gRNA is complementary with GUS repeat sequences.Based on producing DSBs,functional GUS gene can be restored via HDR.Our material is tobacco leaves,in which the gene were transiently expressed.Using direct reapeated report gene of GUS makes observation easier.The whole system is characterized by its simple design,no specific requirements for the target sequence,and a simple,accurate and efficient for plant genome editing technology system.By using the staining reaction of GUS with its substrate,the results can be achieved and quantitatively detected for DSBs.This method can be used quickly to detect site specific DSBs and intuitively observe the results after GUS staining.Currently,the most utilized GE technology is Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR).In addition there have been developed zinc finger nuclease(ZFN)and transcription activator-like effector nucleases(TALEN).All of these techniques cause DSBs at specific DNA sites.However,the ZFN,TALEN or CRISPR systems can generate off-target effects.At the same time,we used this system to perform site-directed mutagenesis of the GUS gene,and the results indicated that a large deletion of the DNA fragment within the GUS gene was obtained,indicating that this technique is feasible in genome editing.