NgAgo/gDNA System Directed Insertion Of GFP Into The Human Genome

Targeted nucleases are powerfu L tools for mediating genome alteration with high precision.We recently discovered that DNA-guided NgAgo nuclease system has become the latest genome editing tool,which can be used to facilitate efficient genome engineering in eukaryotic cells.The purpose of this experiment is to construct a DNA double-strand break(DSB)on the human genome by NgAgo / g DNA system,and then to knock the enhanced green fluorescent protein(EGFP)gene into the last exon of the endogenous DYRK1 A gene by using the intracellular non-homologous end joining(NHEJ)repair mechanism,whose c DNA is driven by the promoter of DYRK1 A gene to express the green fluorescent protein.At last acquiring more accurate molecule tracing about human genome expression after knocking in(KI)is achieved by fluorescence inverted microscope to complete genome editing experiment.First we construct the NgAgo gene sequence into the eukaryotic expression vector,and make the NgAgo protein express successfully in human cells,then construct the recombinant vector NLS-NgAgo-Red by standard molecular cloning method.We choose enhance green fluorescent protein as a fragment of KI,which can be observed the most directly by a fluorescence inverted microscope.The donor contain the entire EGFP reading frame and the SV40 poly A signal sequence.We find DYRK1 A gene and design a target site on its last exon as g DNA,which is expressed widely in human cells.After a number of cell cultures,observations,detections,finally we get a batch of clean and non-pollution Hela cell.We co-transfect prepared plasmid,g DNA and donor into Hela cells,detecting production of EGFP protein under fluorescence inverted microscope after culturing 48 h.Whereas the control group without the NgAgo expression plasmid dose not display green fluorescence.Thus the KI of the experimental group is presumed to be successful.After the extraction of genome,PCR amplification and sequencing analysis,we finally determin that we successfully accomplish directed KI in human genome under the action of g DNA and NgAgo.